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population health is a relatively new , rather fashionable term in the medical field.1 from its probable origins in canada to its current use in the literature ( kindig and stoddart 2003 ; arah and westert 2005 ) , population health calls up images of non - individual health , at least in its literal meaning . do both represent core descriptions of health with respect to the individuals and societies ? both forms of health are , however , rarely analyzed together in the same papers,2 probably due to the prevailing dichotomy of medicine into clinical medicine ( with its personal or individual health purview ) and public health ( with its celebrated public or collective,3 and thus , population health purview ) ( jamrozik and hobbs 2002 ; arah 2005 ; arah et al . this binary approach to health and medicine has also played an important role in differentiating public health from personal medical care ( acheson 1988 ; arah 2009 ; arah et al . arguably , the birth of public health ethics as distinguishable from clinical ethics also rests on this dichotomization of medicine ( see ( beauchamp 1975 , 1983 ; dawson and verweij 2007 ) . this dichotomization could even be traced back to the polarizing approaches of individualism and collectivism in the social sciences ( oneill 1973 ; weale 1981 ; ball 2001 ) . this if - its - not - individual - its - collective approach begs the question if that is all there is to a possible relationship between individual health and population health . is it possible to study the relationship between individual and population health entirely in terms of the individual or the collective ? and if at all , could the same concept of health be easily mapped onto the population level as at the individual level ? this article will argue that neither individual nor population health is identifiable or even definable without informativecontextualization within the other . for instance , a person s health can not be seen only in isolation but must be placed in the rich contextual web such as the socioeconomic circumstances and other health determinants of where they were conceived , born , bred , and how they shaped and were shaped by their environment and communities , especially given the prevailing population health exposures over their lifetime . what and how much of individual and population health until we know the cumulative trajectories of both , using appropriate causal language . indeed , the complementary relationship between individual and population health evokes important socially relevant causal inferences about both having the duality of being determinants and outcomes over time , and within and between places or societies . the causal interpretations accorded both types of health flow directly from and are foundational to their definitional and measurement concerns . health as a concept is the focus of heated debates in the philosophy and medical literature.4 this literature is overwhelmingly concerned with the health of the individual and the medical or healthcare interpretations and interventions at the level of the diseased individual . in those instances , the term healthcare is often used to imply both personal medical care and public health.5 currently , there are at least two major schools of thought on the concept of health , namely , the naturalist and the normativist theories of health ( boorse 1975 , 1977 , 1997 ; schramme 2007 ; nordenfelt 1986 , 1995 , 2007 ) . within the normativist theory , the naturalist theory of health , which claims to be descriptive , value - free and consistent with evolutionary theory , states that an individual is completely healthy if and only if all her organs function normally , that is , given a statistically normal environment , her organs make at least their statistically normal contribution to her survival or to the human species survival ( boorse 1977 , 1997 ; schramme 2007 ) . thus , a healthy person is easily identified through objective medical investigation . according to normativist criticisms ( nordenfelt 2007 ) , the naturalist theory of health lays too much emphasis on internal processes , biology and the absence of disease , effectively excluding extrabiological considerations such as person , intentional action and cultural standards . on the other hand , the normativist account , which espouses a value - laden evaluative approach holds that an individual is completely healthy if and only if she has the ability , given standard circumstances , to reach all her vital or essential goals in life ( nordenfelt 2007 ) . this latter theory depicts a continuum where health accommodates disease , takes a holistic contextual approach , and instrumentalizes health in the larger scheme of vital life goals . interestingly both theories of health , to some extent , see disease in terms of relevant organ dysfunction . for instance , according to the naturalist account , a person has a disease if and only if at least one of her organs functions subnormally , given a statistically normal environment , while the normativist theory asserts that a disease is a state or process in which the individual has at least one organ involved in any state that tends to reduce the individual s health . although engaging in a debate on the merits of each theory of health is beyond the scope of this article , i want to point out that both theories appear to take the context or circumstances or environment of any health - disease continuum as merely observed or passive , not active , interventional or causal.6 yet , we all know that many diseases7 arise from the complex interplay of the person and her context , be it social , psychological , physical , economic or not ( lalonde 1974 ; evans and stoddart 1990 ; van oers 2002 ; arah et al my argument is that any concept of individual health must emphasize the role of the person s circumstances in health maintenance or even in disease causation , fleshing out the imbalance between the internal and external functionings . this imbalance is reflected in a recent attempt to characterize the origins of human disease ( mackenbach 2006):in all its manifestations , human disease is a reaction of organisms to , and/or a failure to cope with , one or more unbalancing changes in their internal environments . these are caused by one or more unfavourable exchanges with their external environments and/or failures in the structural and functional design of organisms . in the final analysis , human disease is attributable to the dependence of organisms on a fundamentally hostile external environment and to unfortunate evolutionary legacies . in all its manifestations , human disease is a reaction of organisms to , and/or a failure to cope with , one or more unbalancing changes in their internal environments . these are caused by one or more unfavourable exchanges with their external environments and/or failures in the structural and functional design of organisms . in the final analysis , human disease is attributable to the dependence of organisms on a fundamentally hostile external environment and to unfortunate evolutionary legacies . to be sure , there is more to health than mere absence of disease . an emphasis on the notion of the context or what naturalists call the statistically normal environment or the normativists call standard circumstancesis needed to understand how health is promoted in a positive sense , maintained or disrupted , and to give meaning to the theory of health as a continuum rather than as a binary concept of health versus disease . firstly , this context has to be seen in terms of internal external balance between the individual and her context or environment . secondly , the contextual balance must be causal in nature , at least in the counterfactual sense of being capable of leading to a different individual health if the balance were altered ( lewis 1973 ; greenland 2000 ; maldonado and greenland 2002 ; pearl 2000 ) . hume ( 1748 , p. 115 ) defines a cause to be an object , followed by another where , if the first object had not been , the second had never existed . an important aspect of this view of causation is its counterfactual concept : a certain outcome event ( the second object , or effect ) would not have occurred if , contrary to fact , an earlier event ( the first object , or cause ) had not occurred ( maldonado and greenland 2002 ; greenland 2005 ) . early life insults can and have been known to persist into adult life ( kuh and ben - shlomo 2004 ) , and to curb the ability to pursue life s vital goals ( nordenfelt 1995 ) or what one may have reason to value ( sen 1985 , 1992 ) . the foregoing properties re - define the context of health as being not merely observed but actually causative or determinant of the level , dynamics and distribution of health . this is in line with the popular use of the phrase determinants of health in the health literature ( arah and westert 2005).8 as we will see later on , the revitalization of the context part of the health concept allows us to evaluate the health relationship between individuals and across populations , in essence , linking individual and population health . when tolu broke her leg in a motor accident on a precariously narrow road in her home town in south - west nigeria , it seemed fair to say it was tolu s health , not that of her community or any such collective to which she belonged , that was primarily compromised . it turned out that tolu , who was a publicly employed physician , in her deprived town with few doctors , was on her way to the hospital , to respond to an emergency call from the local hospital to help out on a particularly busy day . typically , she would attend to a lot of patients , many of whom suffered from infectious diseases , were malnourished , and had been victims of road traffic accidents , and so on . being incapacitated by her injury , she was unable to attend to her patients who must now increase the workload of other already over - stretched doctors . the infants among the patients suffered disproportionately ; they were more vulnerable and had illnesses that rapidly consumed them without prompt care . unknown to most , the hospital was unable to save a number of such vulnerable patients who would have been seen by tolu had she not been reduced to a patient herself by a complex web of social and personal circumstances . not only did they suffer as a result of her inability to be a physician to them , but also they were subject to what ( dangerous roads , deprivation , and other standard circumstances ) shaped tolu s health and her pursuit of her vital goals ( which included being able to cycle , being an attending physician to the needy , and so on ) . actually , she chose to become a physician as a result of the telling experiences of growing up in the town s squalor . so , their lives , well - being and health , were co - dependent , at least on some level . in a sense , it was difficult for tolu to remain healthy in a town full of so many suffering people . indeed , it would be difficult to conclude that this town s population health was ideal , full or complete . the interacting individuals who made up the collective were often at risk of less - than - full health , largely due to the collective standard circumstances they lived in , a context they sculptured or was sculptured for them in some way , and which also sculptured who and what they became . admittedly , the foregoing illustration is a little dramatized . it serves its purpose nonetheless : health is not entirely individual ; it is relative to the individual s context , which in turn is fashioned out of the interactions that exist between members of any defined collective whose health ( read : population health ) is defined by the health and context of its members . the circularity of this concept and argument is not lost on us . many diseases such as allergic , cardiovascular , and even genetic9 disorders seem to have contextual antecedents ( mackenbach 2006 ) . and these contextual causes , determinants or facilitators tend to accumulate from , probably , before conception and birth through adult life ( kuh et al . we will return to this issue of life course and causal context of health in a population shortly . first , i want to broach two implicit views of population health : the simply - the - sum - of - the - parts and the greater - than - the - sum - of - the - parts views . the former hopefully with a dwindling proponents base sees population health as no more than a summary of health , aggregated across individuals within a population ( see for instance , the debate and work on designing summary measures of population : ( world bank 1993 ; murray and lopez 1994 ; murray et al . 1994 , 2001 , 2002 ; murray and evans 2003 ; murray 1994 ; anand and hanson 1997 , 1998 ; institute of medicine 1998 ; world health organization 2000 ; williams 2000 ; mathers et al . , summary measures of population health ( smph ) represent aggregated , singular indices of the quantity and sometimes distribution of health in a given population . these measures combine data on mortality and morbidity , including disability , obtained from the population in question or extrapolated from similar contemporary populations . the idea is that both the quantity and quality of life that an individual born into such a population could expect to enjoy can be captured by measures such as healthy life expectancy ( hale ) and disability - adjusted life years ( daly ) . critics have pointed that some of these metrics are not necessarily equitable or particularly suitable for the health policies they are purported to support:[disability - adjusted life years or ] dalys are an inequitable measure of aggregate ill - health and an inequitable criterion for resource allocation . through age - weighting and discounting , they place a different value on years lived at different ages and at different points in time . they value a year saved from illness more for the able - bodied than the disabled , more for those in middle age - groups than the young or the elderly , and more for individuals who are ill today compared with those who will be ill in the future . we regard such valuations to be inequitable both for the exercises of measuring the quantity of ill - health and for resource allocation . for resource allocation equity requires giving priority to the claims of the disadvantaged , which can not be achieved by using the restricted information set of the daly ( anand and hanson 1998 ) . [ disability - adjusted life years or ] dalys are an inequitable measure of aggregate ill - health and an inequitable criterion for resource allocation . through age - weighting and discounting , they place a different value on years lived at different ages and at different points in time . they value a year saved from illness more for the able - bodied than the disabled , more for those in middle age - groups than the young or the elderly , and more for individuals who are ill today compared with those who will be ill in the future . we regard such valuations to be inequitable both for the exercises of measuring the quantity of ill - health and for resource allocation . for resource allocation equity requires giving priority to the claims of the disadvantaged , which can not be achieved by using the restricted information set of the daly ( anand and hanson 1998 ) . the second implicit view of population health , the greater - than - the - sum - of - the - parts account as pursued in this article , would see population health as the indivisible health experience of a collective of individuals , where this collective is taken to be distinguishable from a mere collection or summation of individuals.10 the context would be seen as so defining and powerful that simple aggregations of health into singular measures would miss the richer information present in the context that shapes current and future health of the collective and of its individual members . at a minimum , population health should be measured in multidimensional terms , rich in information for different purposes and interpretations . greenland recently underscored this requirement as follows : my intention in raising these issues is not to offer a solution to a specific summarization problem . rather , it is to remind those facing a choice among measures that candidates need not ( and , for policy purposes , should not ) be limited to unidimensional summaries . while our ability to think in several dimensions is limited , it can be improved with practice . that practice has proven crucial in attacking problems in physics and engineering , and there is no reason to suppose it is less important in tackling more complex social policy issues . in instances in which many different people must make informed choices based on the same scientific data , but with different values , multidimensional measures are essential if we are to provide each person and each executive body with sufficient information for rational choice ( greenland 2005 ) . my intention in raising these issues is not to offer a solution to a specific summarization problem . rather , it is to remind those facing a choice among measures that candidates need not ( and , for policy purposes , should not ) be limited to unidimensional summaries . while our ability to think in several dimensions is limited , it can be improved with practice . that practice has proven crucial in attacking problems in physics and engineering , and there is no reason to suppose it is less important in tackling more complex social policy issues . in instances in which many different people must make informed choices based on the same scientific data , but with different values , multidimensional measures are essential if we are to provide each person and each executive body with sufficient information for rational choice ( greenland 2005 ) it is clear that how population health is measured is dependent on how it is conceptualized . if population health were seen only as aggregate health of a group , then unidimensional metrics such as hale and dalys might suffice . if , however , population health were conceived as a deeply contextual and causally charged notion , then metrics that went beyond the descriptive and dealt with the predictive , explanatory and evaluative would be needed ( mcdowell et al . 2004 ) . population health as a concept of health has been defined as the health outcomes of a group of individuals , including the distribution of such outcomes within the group ( kindig and stoddart 2003 ) . additionally , as a field , population health is said to address how and why some groups of people are healthy and others are not ( mcdowell et al . the late geoffrey rose once described the population [ health ] strategy as the attempt to control the determinants of incidence , to lower the mean level of risk factors , to shift the whole distribution of exposure in a favourable direction . in its traditional public health form it has involved mass environmental control methods ; in its modern form it is attempting ( less successfully ) to alter some of society s norms of behaviour ( rose 1985 ) . the attempt to control the determinants of incidence , to lower the mean level of risk factors , to shift the whole distribution of exposure in a favourable direction . in its traditional public health form it has involved mass environmental control methods ; in its modern form it is attempting ( less successfully ) to alter some of society s norms of behaviour ( rose 1985 ) . although the term population health could mean health outcome or health determinants in relation to public health outcomes or both , public health specialists mostly spend their time trying to influence the determinants or the so - called root causes of population health . this population health approach is quite old although there is no definitive history of this approach , with recent historic applications seen in the works of jerry morris and richard titmuss ( szreter 2003 ) and in the seminal lalonde model ( lalonde 1974 ; evans and stoddart 1990 ) . at its simplest level , the health determinants or lalonde model states that health has four classes of determinants : lifestyle , environment , human biology , and healthcare . this rather simple model was rather well - received , with no one seriously challenging the view that how we lived , where we lived , who we were ( born ) , and the care we used all shaped our health . as evans and stoddart ( 1990 ) noted , the policy response was not entirely clear given that one possible policy interpretation could have been that health was a personal choice . this is something that could be heard echoing in the corridors of many north american and european ministries , given the rise of consumerism , performance disclosure , market mechanisms and the information age in nearly all public policy areas . if anything , public policy on health missed the point about the health of populations being contextual , a reflection of the complex interplay of lifestyle , environment , human biology and even healthcare . recently , a global commission on social determinants of health was launched by the world health organization to focus health policies on the social context of health and inequalities ( lee 2005 ; marmot 2006 ; irwin et al . i can only hope the renewed interests will see the context of population health as both a means and an end , not just another series of inputs for attaining and subsequently aggregating health across members of a group . the context of population health comprises so much diversity , meaning and information which must be factored into any health evaluation exercise or intervention that seeing context as only given circumstances is to render the very concept of health of a person and of a group impotent . a crucial prerequisite for defining individual health and population health in terms of their context is that context must be dynamic and causal . even habitual lifestyles are rarely stationary ; they are subject to the enabling environment and resources that feed such habits . human biology is subject to numerous factors like micro - organisms , radiation , accidents , and so on . individuals are born ; they develop from childhood , adolescence through adulthood , learning the language and ways of life of their parents , imbibing their tastes , experiences , music , dance , art , and interacting with other people . they fall ill , survive , marry , have their own children , live with the marks of their experiential journey through life , and are continuously molded by their context as they search for and define who and what they become . social epidemiologists only recently discovered this life course interpretation of the health , well - being and over - all context of human beings , something that was already known for many years to psychologists , sociologists , anthropologists , biologists , and demographers ( kuh and ben - shlomo 2004 ) . life course epidemiology studies long term effects on later health or disease risk of physical or social exposures during gestation , childhood , adolescence , young adulthood and later adult life . it aims to elucidate biological , behavioural , and psychosocial processes that operate across an individual s life course , or across generations , to influence the development of disease risk ( kuh and ben - shlomo 2004 ) . epidemiology studies long term effects on later health or disease risk of physical or social exposures during gestation , childhood , adolescence , young adulthood and later adult life . it aims to elucidate biological , behavioural , and psychosocial processes that operate across an individual s life course , or across generations , to influence the development of disease risk ( kuh and ben - shlomo 2004 ) . parents social class , behaviors , wealth , education , and other childhood factors like cognitive and psychosocial developments have all been shown to determine who stays healthy , falls ill or dies prematurely in adult life ( kuh and ben - shlomo 2004 ; case et al . 2005 ) . if lifetime circumstances so evidently mold health and well - being and also subsequent social and other life circumstances in such cumulative ways , why must the health of persons and groups be seen as individual or concerted organ functioning given normal environment or circumstances ? the currently observed one ? or the one that has accumulated over the life course and may remain a harbinger of well - being in years to come ? neither individuals nor collectives can be understood in only cross - sectional , one - time views . all through their lifetimes , individuals become the collective just as the collective becomes them . and collectives age across generations of its members , evolving and defining and being defined through cumulative and adaptive experiences , events , and history . in all these this individualism within a collective should not be mistaken with the ordinary usage of individualism that seems to suggest a whiff of unsociability , but should be taken as the sort that forms the basis for an extensive concern for others ( appiah 2005 ) . this concern is the type needed throughout life to build a context worthy of individuality , freedom and collective well - being and health . so far , i have argued that neither individual nor population health is easily separable from the other . even when they are considered separable , as approaches to health , rather than health concepts , geoffrey rose would seem to choose the population approach because he was a strong believer in the context and distribution of health and its causes ( not that he would sacrifice individuals to achieve his objectives ) ( rose 1985 , 1992 ) . one might ask if the link between individual and population health could then be construed to imply that unhealthy individuals could not be found in healthy populations and vice versa . instances of incongruity between individual and population health may be best understood by considering a possible categorization of the individual - versus - population health relationship . therefore , borrowing terminology from epidemiologic methodology ( copas 1973 ; greenland and robins 1986 ) , i can classify the individual - versus - population health relationship into four categories : immune : individual health remains good irrespective of the population health or contextcausative : individual health is boosted in favorable population health or contextpreventive : individual health is compromised when population health or context is unfavorabledoomed : individual health is compromised irrespective of the population health or context . immune : individual health remains good irrespective of the population health or context causative : individual health is boosted in favorable population health or context preventive : individual health is compromised when population health or context is unfavorable doomed : individual health is compromised irrespective of the population health or context . they would include genetic diseases ( category 4 ) which progress irrespective what is done or experienced in the collective or medicine . a category 2 illustration : if along with the growing physician emigration ( arah et al . 2008 ) , tolu were to move from her impoverished circumstances in nigeria to a safer suburb somewhere in england , her health would no longer be what it was or would have been back in her hometown . she and her family might not only enjoy the healthful experiences of their new context , they might also acquire other non - health experiences and tastes which might subsequently redefine their immediate and long - term well - being . she , and in particular her children , would have escaped from a context where their life expectancies might have been the odd forty - something years to a place where they could live well into their seventh decade or longer . this would contrast with a category 3 scenario where jane , a brit , who might have lived to be an octogenarian in england , would end up cutting her life short in her thirties by moving to a mosquito - infested nigeria without proper anti - malarial prophylaxis or by being involved in a rather common road traffic accident there . similarly , it is very difficult to imagine populations that could be called healthy if the context for health is heavily compromised and individual members of the collective are at constant risk of dangerous exposures and events . it then seems to me that the relationship between individual and population health is a matter of ubi mel ibi apes where there is honey there are bees . to be sure , health is not entirely relative such a view would excuse the unfortunate morbidity and mortality suffered by millions of children in deprivation in africa . , this would imply denying a partial absolutist notion of health ( for instance , for these children not to be malnourished , to enjoy good health , and not be stricken dead before age five ) . in a purely relativist view , we could easily miss a widespread compromise of health in a context where health was already poor because we might erroneously infer that the relative distribution of health remained unchanged . the absolutist core of health implies that whenever health is compromised to the extent that functioning is obstructed that there is ill - health , no matter what the relative picture looks like . it is on this absolutist core of health that a relativist layer of the enabling context of health should be built . the relationship between individual and population health resides mostly in this relativist layer , although it requires the absolutist notion of health to exist in the first instance . without the informative contextual characterization of health at the individual or population level , there is little insight being gained by saying a person or a community is healthy . a possible criticism here is that this contextual re - interpretation of individual and population health includes almost every well - being oriented activity under the rubric of health . true , but this fear of all - inclusiveness that has already been leveled against the normativist school is not embarrassing . if anything , it is refreshingly bold to attempt to elevate the concept of health to the level of human well - being . if health is so integral to the notion of well - being and to the ability to conduct the life one may have reason to value ( including achieving one s vital goals ) ( nussbaum and sen 1993 ; nordenfelt 1995 , 2007 ) , then it is not surprising that the boundaries of health can easily encroach on the boundaries of well - being and life as a whole . after all , health represents both functioning ( the achieved ) and capability ( the achievable ) : a means to life s other vital goals or capabilities as well as an end in itself ( sen 1985 ; nussbaum and sen 1993 ) . i suspect that when some philosophers reject such ambitious notions of health , they are merely concerned with the overuse or abuse of possible responses or interventions to deal with not being in full health : a fear of medicalization . however , i think such criticisms miss the subtle but important distinctions between the boundaries of health ( and thus , health need ) and the boundaries of healthcare ( and thus , healthcare need).11 health need depicts the shortfall in ideal health ( in some sense , a gradual progression from the completely healthy end of the health spectrum to the disease end ) , whereby the shortfall and context combine to hinder the ability to flourish to a degree important to the individual . healthcare need , on the other hand , alludes to a shortfall in health which inhibits a person s ability to flourish and which is only amenable to healthcare or organized medicine . suffice it to say that while it is necessary to avoid medicalization , there is little reason for a concept of health to be bounded mainly by this medicalization avoidance or by any narrowly defined interpretation of what medicine is . medicine is largely a socially constructed response and therefore secondary , whereas health is more fundamental and therefore prior . nordenfelt has discussed some of the notions of medicine as health enhancement in both narrow and broad senses ( nordenfelt 1998 , 2001 ) . for now , i will submit that the prevailing dichotomization of medicine and its associated fields including ( bio)ethics into clinical medicine and public health aspects is erroneous , inefficient , and outdated , if not unethical . this criticism can also be leveled against the duality i have been discussing , namely , individual versus population health . such binary views which seem to pervade almost all of public policy on health fail to use the rich information and interpretations that stem from a more comprehensive approach to health over the life course ( i ) of the individual within the collective and ( ii ) of the collective of interacting individuals . i invite the reader to consider them : if the concepts of individual versus population health are so intimately interwoven , why do bioethicists see the need to separate public health ethics from main stream bioethics ? is it to give ethical considerations to , say , distributional issues that would otherwise be difficult at the individual or clinical level ? or are we young public health ethicists just busy building a parallel dichotomy similar to that seen between clinical medicine and public health , by way of argumentum ad verecundiam ? further , given the mounting evidence that health is compromised early in life and that the insults are borne forward into adult life and beyond and ultimately lead to expensive healthcare , why do health policies still concentrate overwhelmingly on healthcare in adulthood ? while we are at it , must healthcare represent the standard policy response to health problems , in effect being what norman daniels once called the ambulance at the bottom of the cliff after the free fall through life ? this article has argued that the relationship between individual and population health is one that is entrenched in the contextual definition of health and its life course causes . i have made an attempt to derive this relationship based on the concept of health ( if we were to continue pursuing such a concept anyway ) by including a population perspective on health . i emphasized the role of the context component of any notion of health , that is , the role of the standard circumstances or the so - called statistically normal environment . i then argued that this context is both individual and collective in nature , in largely inseparable ways , and that context must be causally seen across the life of an individual and the life of the collective . the meanings of both individual and population health lie in this revitalized life course and causally defined context , and have implications for how we measure and analyze health at all levels . armed with the reasoned scrutiny and the unresolved complexity of the concepts , i invite philosophers and other scientists to revisit the definitions of individual health and population health if the notions are to carry any more weight in ongoing discourses in public health , healthcare , and bioethics . i can only hope that this article will stimulate further debates on individual and population health concepts and on their associated policy - relevant fields . one conclusion of this article , for now , is that health , be it individual or population health , can be very context - dependent . after all , prior to the accident , tolu may have been absolutely healthy from her personal experiential point of view , but she was still contextually unhealthy , relatively speaking .

the relationship between individual and population health is partially built on the broad dichotomization of medicine into clinical medicine and public health . potential drawbacks of current views include seeing both individual and population health as absolute and independent concepts . i will argue that the relationship between individual and population health is largely relative and dynamic . their interrelated dynamism derives from a causally defined life course perspective on health determination starting from an individual s conception through growth , development and participation in the collective till death , all seen within the context of an adaptive society . indeed , it will become clear that neither individual nor population health is identifiable or even definable without informative contextualization within the other . for instance , a person s health can not be seen in isolation but must be placed in the rich contextual web such as the socioeconomic circumstances and other health determinants of where they were conceived , born , bred , and how they shaped and were shaped by their environment and communities , especially given the prevailing population health exposures over their lifetime . we can not discuss the what and how much of individual and population health until we know the cumulative trajectories of both , using appropriate causal language .

Introduction Lonely lives: from the concept and collective context of health to individual health Populations without individuals: from the concept of health and context of interacting individuals to population health The life course Healthy individuals, healthy populations Conclusions

population health is a relatively new , rather fashionable term in the medical field.1 from its probable origins in canada to its current use in the literature ( kindig and stoddart 2003 ; arah and westert 2005 ) , population health calls up images of non - individual health , at least in its literal meaning . both forms of health are , however , rarely analyzed together in the same papers,2 probably due to the prevailing dichotomy of medicine into clinical medicine ( with its personal or individual health purview ) and public health ( with its celebrated public or collective,3 and thus , population health purview ) ( jamrozik and hobbs 2002 ; arah 2005 ; arah et al . arguably , the birth of public health ethics as distinguishable from clinical ethics also rests on this dichotomization of medicine ( see ( beauchamp 1975 , 1983 ; dawson and verweij 2007 ) . this if - its - not - individual - its - collective approach begs the question if that is all there is to a possible relationship between individual health and population health . is it possible to study the relationship between individual and population health entirely in terms of the individual or the collective ? this article will argue that neither individual nor population health is identifiable or even definable without informativecontextualization within the other . for instance , a person s health can not be seen only in isolation but must be placed in the rich contextual web such as the socioeconomic circumstances and other health determinants of where they were conceived , born , bred , and how they shaped and were shaped by their environment and communities , especially given the prevailing population health exposures over their lifetime . what and how much of individual and population health until we know the cumulative trajectories of both , using appropriate causal language . indeed , the complementary relationship between individual and population health evokes important socially relevant causal inferences about both having the duality of being determinants and outcomes over time , and within and between places or societies . health as a concept is the focus of heated debates in the philosophy and medical literature.4 this literature is overwhelmingly concerned with the health of the individual and the medical or healthcare interpretations and interventions at the level of the diseased individual . in those instances , the term healthcare is often used to imply both personal medical care and public health.5 currently , there are at least two major schools of thought on the concept of health , namely , the naturalist and the normativist theories of health ( boorse 1975 , 1977 , 1997 ; schramme 2007 ; nordenfelt 1986 , 1995 , 2007 ) . within the normativist theory , the naturalist theory of health , which claims to be descriptive , value - free and consistent with evolutionary theory , states that an individual is completely healthy if and only if all her organs function normally , that is , given a statistically normal environment , her organs make at least their statistically normal contribution to her survival or to the human species survival ( boorse 1977 , 1997 ; schramme 2007 ) . on the other hand , the normativist account , which espouses a value - laden evaluative approach holds that an individual is completely healthy if and only if she has the ability , given standard circumstances , to reach all her vital or essential goals in life ( nordenfelt 2007 ) . this latter theory depicts a continuum where health accommodates disease , takes a holistic contextual approach , and instrumentalizes health in the larger scheme of vital life goals . for instance , according to the naturalist account , a person has a disease if and only if at least one of her organs functions subnormally , given a statistically normal environment , while the normativist theory asserts that a disease is a state or process in which the individual has at least one organ involved in any state that tends to reduce the individual s health . although engaging in a debate on the merits of each theory of health is beyond the scope of this article , i want to point out that both theories appear to take the context or circumstances or environment of any health - disease continuum as merely observed or passive , not active , interventional or causal.6 yet , we all know that many diseases7 arise from the complex interplay of the person and her context , be it social , psychological , physical , economic or not ( lalonde 1974 ; evans and stoddart 1990 ; van oers 2002 ; arah et al my argument is that any concept of individual health must emphasize the role of the person s circumstances in health maintenance or even in disease causation , fleshing out the imbalance between the internal and external functionings . in the final analysis , human disease is attributable to the dependence of organisms on a fundamentally hostile external environment and to unfortunate evolutionary legacies . in the final analysis , human disease is attributable to the dependence of organisms on a fundamentally hostile external environment and to unfortunate evolutionary legacies . an emphasis on the notion of the context or what naturalists call the statistically normal environment or the normativists call standard circumstancesis needed to understand how health is promoted in a positive sense , maintained or disrupted , and to give meaning to the theory of health as a continuum rather than as a binary concept of health versus disease . firstly , this context has to be seen in terms of internal external balance between the individual and her context or environment . secondly , the contextual balance must be causal in nature , at least in the counterfactual sense of being capable of leading to a different individual health if the balance were altered ( lewis 1973 ; greenland 2000 ; maldonado and greenland 2002 ; pearl 2000 ) . the foregoing properties re - define the context of health as being not merely observed but actually causative or determinant of the level , dynamics and distribution of health . this is in line with the popular use of the phrase determinants of health in the health literature ( arah and westert 2005).8 as we will see later on , the revitalization of the context part of the health concept allows us to evaluate the health relationship between individuals and across populations , in essence , linking individual and population health . when tolu broke her leg in a motor accident on a precariously narrow road in her home town in south - west nigeria , it seemed fair to say it was tolu s health , not that of her community or any such collective to which she belonged , that was primarily compromised . not only did they suffer as a result of her inability to be a physician to them , but also they were subject to what ( dangerous roads , deprivation , and other standard circumstances ) shaped tolu s health and her pursuit of her vital goals ( which included being able to cycle , being an attending physician to the needy , and so on ) . indeed , it would be difficult to conclude that this town s population health was ideal , full or complete . the interacting individuals who made up the collective were often at risk of less - than - full health , largely due to the collective standard circumstances they lived in , a context they sculptured or was sculptured for them in some way , and which also sculptured who and what they became . it serves its purpose nonetheless : health is not entirely individual ; it is relative to the individual s context , which in turn is fashioned out of the interactions that exist between members of any defined collective whose health ( read : population health ) is defined by the health and context of its members . many diseases such as allergic , cardiovascular , and even genetic9 disorders seem to have contextual antecedents ( mackenbach 2006 ) . we will return to this issue of life course and causal context of health in a population shortly . the former hopefully with a dwindling proponents base sees population health as no more than a summary of health , aggregated across individuals within a population ( see for instance , the debate and work on designing summary measures of population : ( world bank 1993 ; murray and lopez 1994 ; murray et al . the idea is that both the quantity and quality of life that an individual born into such a population could expect to enjoy can be captured by measures such as healthy life expectancy ( hale ) and disability - adjusted life years ( daly ) . they value a year saved from illness more for the able - bodied than the disabled , more for those in middle age - groups than the young or the elderly , and more for individuals who are ill today compared with those who will be ill in the future . for resource allocation equity requires giving priority to the claims of the disadvantaged , which can not be achieved by using the restricted information set of the daly ( anand and hanson 1998 ) . they value a year saved from illness more for the able - bodied than the disabled , more for those in middle age - groups than the young or the elderly , and more for individuals who are ill today compared with those who will be ill in the future . for resource allocation equity requires giving priority to the claims of the disadvantaged , which can not be achieved by using the restricted information set of the daly ( anand and hanson 1998 ) . the second implicit view of population health , the greater - than - the - sum - of - the - parts account as pursued in this article , would see population health as the indivisible health experience of a collective of individuals , where this collective is taken to be distinguishable from a mere collection or summation of individuals.10 the context would be seen as so defining and powerful that simple aggregations of health into singular measures would miss the richer information present in the context that shapes current and future health of the collective and of its individual members . in instances in which many different people must make informed choices based on the same scientific data , but with different values , multidimensional measures are essential if we are to provide each person and each executive body with sufficient information for rational choice ( greenland 2005 ) it is clear that how population health is measured is dependent on how it is conceptualized . if population health were seen only as aggregate health of a group , then unidimensional metrics such as hale and dalys might suffice . if , however , population health were conceived as a deeply contextual and causally charged notion , then metrics that went beyond the descriptive and dealt with the predictive , explanatory and evaluative would be needed ( mcdowell et al . population health as a concept of health has been defined as the health outcomes of a group of individuals , including the distribution of such outcomes within the group ( kindig and stoddart 2003 ) . additionally , as a field , population health is said to address how and why some groups of people are healthy and others are not ( mcdowell et al . the late geoffrey rose once described the population [ health ] strategy as the attempt to control the determinants of incidence , to lower the mean level of risk factors , to shift the whole distribution of exposure in a favourable direction . although the term population health could mean health outcome or health determinants in relation to public health outcomes or both , public health specialists mostly spend their time trying to influence the determinants or the so - called root causes of population health . this population health approach is quite old although there is no definitive history of this approach , with recent historic applications seen in the works of jerry morris and richard titmuss ( szreter 2003 ) and in the seminal lalonde model ( lalonde 1974 ; evans and stoddart 1990 ) . at its simplest level , the health determinants or lalonde model states that health has four classes of determinants : lifestyle , environment , human biology , and healthcare . this is something that could be heard echoing in the corridors of many north american and european ministries , given the rise of consumerism , performance disclosure , market mechanisms and the information age in nearly all public policy areas . if anything , public policy on health missed the point about the health of populations being contextual , a reflection of the complex interplay of lifestyle , environment , human biology and even healthcare . recently , a global commission on social determinants of health was launched by the world health organization to focus health policies on the social context of health and inequalities ( lee 2005 ; marmot 2006 ; irwin et al . i can only hope the renewed interests will see the context of population health as both a means and an end , not just another series of inputs for attaining and subsequently aggregating health across members of a group . the context of population health comprises so much diversity , meaning and information which must be factored into any health evaluation exercise or intervention that seeing context as only given circumstances is to render the very concept of health of a person and of a group impotent . a crucial prerequisite for defining individual health and population health in terms of their context is that context must be dynamic and causal . they fall ill , survive , marry , have their own children , live with the marks of their experiential journey through life , and are continuously molded by their context as they search for and define who and what they become . social epidemiologists only recently discovered this life course interpretation of the health , well - being and over - all context of human beings , something that was already known for many years to psychologists , sociologists , anthropologists , biologists , and demographers ( kuh and ben - shlomo 2004 ) . it aims to elucidate biological , behavioural , and psychosocial processes that operate across an individual s life course , or across generations , to influence the development of disease risk ( kuh and ben - shlomo 2004 ) . it aims to elucidate biological , behavioural , and psychosocial processes that operate across an individual s life course , or across generations , to influence the development of disease risk ( kuh and ben - shlomo 2004 ) . parents social class , behaviors , wealth , education , and other childhood factors like cognitive and psychosocial developments have all been shown to determine who stays healthy , falls ill or dies prematurely in adult life ( kuh and ben - shlomo 2004 ; case et al . if lifetime circumstances so evidently mold health and well - being and also subsequent social and other life circumstances in such cumulative ways , why must the health of persons and groups be seen as individual or concerted organ functioning given normal environment or circumstances ? all through their lifetimes , individuals become the collective just as the collective becomes them . in all these this individualism within a collective should not be mistaken with the ordinary usage of individualism that seems to suggest a whiff of unsociability , but should be taken as the sort that forms the basis for an extensive concern for others ( appiah 2005 ) . so far , i have argued that neither individual nor population health is easily separable from the other . even when they are considered separable , as approaches to health , rather than health concepts , geoffrey rose would seem to choose the population approach because he was a strong believer in the context and distribution of health and its causes ( not that he would sacrifice individuals to achieve his objectives ) ( rose 1985 , 1992 ) . one might ask if the link between individual and population health could then be construed to imply that unhealthy individuals could not be found in healthy populations and vice versa . instances of incongruity between individual and population health may be best understood by considering a possible categorization of the individual - versus - population health relationship . therefore , borrowing terminology from epidemiologic methodology ( copas 1973 ; greenland and robins 1986 ) , i can classify the individual - versus - population health relationship into four categories : immune : individual health remains good irrespective of the population health or contextcausative : individual health is boosted in favorable population health or contextpreventive : individual health is compromised when population health or context is unfavorabledoomed : individual health is compromised irrespective of the population health or context . immune : individual health remains good irrespective of the population health or context causative : individual health is boosted in favorable population health or context preventive : individual health is compromised when population health or context is unfavorable doomed : individual health is compromised irrespective of the population health or context . they would include genetic diseases ( category 4 ) which progress irrespective what is done or experienced in the collective or medicine . she , and in particular her children , would have escaped from a context where their life expectancies might have been the odd forty - something years to a place where they could live well into their seventh decade or longer . similarly , it is very difficult to imagine populations that could be called healthy if the context for health is heavily compromised and individual members of the collective are at constant risk of dangerous exposures and events . it then seems to me that the relationship between individual and population health is a matter of ubi mel ibi apes where there is honey there are bees . , this would imply denying a partial absolutist notion of health ( for instance , for these children not to be malnourished , to enjoy good health , and not be stricken dead before age five ) . the relationship between individual and population health resides mostly in this relativist layer , although it requires the absolutist notion of health to exist in the first instance . a possible criticism here is that this contextual re - interpretation of individual and population health includes almost every well - being oriented activity under the rubric of health . if health is so integral to the notion of well - being and to the ability to conduct the life one may have reason to value ( including achieving one s vital goals ) ( nussbaum and sen 1993 ; nordenfelt 1995 , 2007 ) , then it is not surprising that the boundaries of health can easily encroach on the boundaries of well - being and life as a whole . healthcare need , on the other hand , alludes to a shortfall in health which inhibits a person s ability to flourish and which is only amenable to healthcare or organized medicine . medicine is largely a socially constructed response and therefore secondary , whereas health is more fundamental and therefore prior . for now , i will submit that the prevailing dichotomization of medicine and its associated fields including ( bio)ethics into clinical medicine and public health aspects is erroneous , inefficient , and outdated , if not unethical . such binary views which seem to pervade almost all of public policy on health fail to use the rich information and interpretations that stem from a more comprehensive approach to health over the life course ( i ) of the individual within the collective and ( ii ) of the collective of interacting individuals . i invite the reader to consider them : if the concepts of individual versus population health are so intimately interwoven , why do bioethicists see the need to separate public health ethics from main stream bioethics ? or are we young public health ethicists just busy building a parallel dichotomy similar to that seen between clinical medicine and public health , by way of argumentum ad verecundiam ? further , given the mounting evidence that health is compromised early in life and that the insults are borne forward into adult life and beyond and ultimately lead to expensive healthcare , why do health policies still concentrate overwhelmingly on healthcare in adulthood ? this article has argued that the relationship between individual and population health is one that is entrenched in the contextual definition of health and its life course causes . i have made an attempt to derive this relationship based on the concept of health ( if we were to continue pursuing such a concept anyway ) by including a population perspective on health . i then argued that this context is both individual and collective in nature , in largely inseparable ways , and that context must be causally seen across the life of an individual and the life of the collective . the meanings of both individual and population health lie in this revitalized life course and causally defined context , and have implications for how we measure and analyze health at all levels . armed with the reasoned scrutiny and the unresolved complexity of the concepts , i invite philosophers and other scientists to revisit the definitions of individual health and population health if the notions are to carry any more weight in ongoing discourses in public health , healthcare , and bioethics . i can only hope that this article will stimulate further debates on individual and population health concepts and on their associated policy - relevant fields .

nanochemistry is predominating in major fields of science and technology , specifically in biotechnology and information technology . in the near future , nanochemistry will direct and guide towards nanomedicine and nanodiagnostics . however , obtaining suitable nanoparticles that can be used for diagnostic and medicinal purposes remains a significant challenge . moreover , the effect of these nanoparticles on biological entities such as proteins is considerably significant when it comes to ad . oligomeric aggregates a and tau protein or the protofibrils are considered as precursors for amyloid fibrillation in alzheimer 's disease . recently , the effect of fluorinated magnetic core - shell nanoparticles with the size range of 15.0 2.1 nm has been observed on amyloid model protein insulin ; these fluorinated nanoparticles show inhibition of insulin fibrils [ 3 , 4 ] . furthermore , the effect of various nanoparticles within the dimensions of 6200 nm on another model protein , 2 microglobulin , has been investigated . previously published studies have demonstrated that nanoparticles can act as catalysts for protein fibrillation [ 1 , 5 ] . very recently , li and coworkers have showed an inhibition effect of n - acetyl cysteine - capped cdte qds of size of 35 nm on a ( 140 ) fibrillation . in another case , dual effect of commercial polystyrene nanoparticles with amino modification having various sizes ( 57 , 120 , and 180 nm ) was observed on a ( 140 ) and recombinant a ( 140 ) and a ( 142 ) proteins . furthermore , there is only one recent publication on the effect of nanoparticles on a ( 142 ) fibrils . in this case , it was observed an increase in rate of amyloid fibrillation in presence of tio2 nanoparticles with size of approximately 20 nm . the interaction of nanoparticles with different proteins depends upon various factors such as surface coating of nanoparticles with ligands , surface properties , size , and composition of nanoparticles [ 1 , 5 ] . from the previous studies , [ 1 , 38 ] we can not generalize the concept that different nanoparticles can promote or inhibit the fibril formation for various amyloid model proteins . specifically , the only investigation that explains the effect of tio2 on a ( 142 ) shows that nanoparticles promote the fibrillation process by becoming nucleation centers . we report for the first time in our knowledge that cdse / zns qds of size of 2.5 1.3 nm can inhibit fibrillation of a ( 142 ) . in the present study , we have investigated the effect of the presence of dhla - capped cdse / zns qds either mixed with or conjugated to a ( 142 ) on fibrillation process of a ( 142 ) in aqueous phase . tem and afm studies show that the qds behave uniquely when they are conjugated to a ( 142 ) in comparison to a mixed sample of a ( 142 ) and qds . our study illustrates a considerable difference in morphology of the fibrils and the inhibition of fibrillation process when a ( 142 ) is conjugated to qds versus the mixed system a ( 142 ) and qds . these results are further supported by thioflavin t ( tht ) assay using fluorescence spectroscopy . all chemicals were commercially purchased and used without further purification . cadmium oxide ( cdo ) , selenium ( se ) , trioctylphosphine oxide ( topo ) , trioctylphosphine ( top ) , and hexamethyldisilathiane [ ( tms)2s ] were purchased from sigma - aldrich ( milwaukee , wi ) . the tetradecylphosphonic acid ( tdpa ) was obtained from alfa aesar ( ward hill , ma ) . the diethylzinc ( znet2 , 15 wt% solution in hexane ) was obtained from acros organics ( morrisplains , new jersey ) . dl--lipoic acid , a ( 142 ) , and tht were purchased from mp biomedicals ( solon , oh ) . briefly , cadmium oxide was reacted with a selenium reagent in the presence of a phosphine oxide surfactant at high temperature under argon flow . after the formation of the cdse core , the diethyl zinc and hexamethyldisilathiane in top was added dropwise at 130c . after the synthesis of topo - capped hydrophobic qds , modification to hydrophilic dhla - capped qds was carried out . briefly , first dl--lipoic acid ( 1 g ) was reduced using sodium borohydride ( 2 g ) in methanol / water ( v / v , 1:1 ) solution . after workup product was isolated in chloroform and characterized using h nmr ( 400 mhz , cdcl3 ) : ( ppm ) 1.3 ( d , 1h ) , 1.35 ( t , 1h ) , 1.41.8 ( m , 6h ) , 1.9 ( m , 2h ) , 2.4 ( t , 2h ) , 2.62.8 ( m , 2h ) , 2.9 ( m , 1h ) , and 11 ( s , 1h ) . dhla was used for ligand exchange with topo ; excess of dhla ( 0.5 g ) was added in 5 ml of topo - capped qds in methanol and heated at 60c70c for 4 h. once a homogeneous qds solution was obtained , solution was basified using potassium tert - butoxide and centrifuged to get the precipitates . the water - soluble qds were filtered through 0.2 m filter to get a clear solution . a(142 ) was chemically conjugated to qds by the formation of an amide bond between asp - nh2 end of the polypeptide chain and the cooh end of the dhla ligand using the protocol to conjugate proteins . freshly prepared dhla - capped qds ( 1.51 10 m , 100 l ) were taken in a clean borosilicate glass vial , and 500 l pbs buffer ( ph 7.4 ) was added to the qds . to lyophilize the peptide , 0.5 mg a ( 142 ) was dissolved in hexafluoroisopropanol ( hfip ) to bring the peptide in monomer form and evaporated under gentle flow of n2 . the dried protein was then dissolved in 500 l pbs buffer ( ph 7.4 ) to get the final concentration of 1 mg / ml . freshly prepared a(142 ) solution was then mixed in qds solution . 57 l of freshly prepared 10 mg / ml edc ( 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride ) solution in deionized water was then added to the mixture of peptide and qds , total volume of solution prepared was 1157 l . the solution was stirred for 4 h at the speed of 200 rpm . a ( 142 ) mixed with dhla - capped qds was prepared according to the above - mentioned protocol except for the addition of edc . total volume of the solution was kept at 1157 l . for the induction of fibrillation pure a ( 142 ) solution was prepared similarly , first by lyophilizing the peptide in hfip and evaporating the solvent under gentle nitrogen flow and then redissolving the dried peptide in 1157 l of pbs buffer ( ph 7.4 ) . electrophoresis of qds was performed using a minicuve 8.10 electrophoresis unit ( mp biomedicals , solon , oh ) . hand cast gels were composed of 1% agarose in 1 tbe ( 0.089 m tris base , 0.089 m boric acid , and 0.002 m ethylenediaminetetraaceticacid , ph 8.3 ) . 10 l of each sample was loaded into wells on the agarose gel using a micropipet . the samples were run in 1 tbe buffer on the 1% agarose gel at 84 v for 75 min . for visualization , the gel was placed on a uv transilluminator , and an image was captured with a gel doc xr system ( bio - rad , hercules , ca ) . tem measurements were performed at the center of advanced microscopy , scripps research institute ( la jolla , ca ) and at the center of advanced microscopy , ( cma ) , trinity college dublin ( ireland ) . for the images containing amyloid , briefly , copper grids ( carbon and formvar coated 400 mesh : electron microscopy sciences , hatfield , pa ) were glow discharged and inverted on an 5 l aliquot of sample for 3 min . excess sample was removed and the grids immediately placed briefly on a droplet of double - distilled water . grids requiring the negative stain were then placed on droplets of 2% uranyl acetate solution for 2 min . excess stain was removed and the grid was allowed to dry thoroughly . for unstained grids , the excess water was removed , and the dried grids were examined on a philips cm100 electron microscope ( fei , hillsbrough , or ) at 80 kv and images collected using a megaview iii ccd camera ( olympus soft imaging solutions , lakewood , co ) . grids at ireland were examined on a jeol 2100 electron microscope ( zeiss ) operating at 200 kv and images collected using a ccd camera . analysis of tem images was performed using image j software from nih ( http://rsbweb.nih.gov/ij/ ) . briefly , 4 l aliquots of a solutions were deposited on freshly cleaned and dried silicon wafers ( approximately 1 mm thick ) . after waiting for 10 min , nonadsorbed portions of the samples were washed with deionized water ( 400 l ) . the wet surface of the silicon wafer was then dried using gentle flow of air . the samples were analyzed by atomic force microscopy ( afm , a multimode spm , model no . tapping mode approach was used to acquire the images , which allows intermittent contact of the tip with the sample and minimizes the chances of deformation of the peptide samples . the cantilever force constant was approximately 20100 n / m with the resonance frequency between 200 and 400 khz . the software used for the analysis of fibrils was the nanoscope control , version 5.30 and the histogram analysis was performed using the postanalysis pico image software ( pico view version 1.6.2 ) . uv - vis spectra of solutions were recorded on a perkin - elmer lambda 900 uv / vis / nir spectrometer ( norwalk , ct ) . fluorescence spectra were obtained using spex fluorolog fluorospectrometer ( horiba jobin yvon , edison , nj ) . both uv - vis and fluorescence measurements were obtained using quartz cuvette with1 cm optical path length . 30 l of a ( 142 ) aliquots were extracted at different time periods , and 300 l of tht ( 10 m ) was added to the samples . the tht fluorescence was measured at 482 nm at an excitation wavelength of 440 nm in a semimicro quartz cuvette of an optical pathlength of 1 cm . characterization of dhla - capped qds was carried out using uv - vis and fluorescence spectroscopy ( figure 1(a ) ) . the fluorescein ( qy is 0.94 in 0.1 m naoh ) was employed as reference . the qy was calculated by using the following equation : ( 1)qyq = qyf[afnq2iq()d][aqnf2if()d ] . a is the absorbance at the excitation wavelength , n is the refractive index of the solvent used , i is the emission wavelength - dependent emission intensity , and is the emission wavelength . qy was calculated from the intensity of luminescence and the absorbance in figures 1(b ) and 1(c ) for fluorescein ( 0.1 m naoh as solvent ) and qds ( water as solvent ) , respectively . the qy of the dhla - capped cdse / zns qds was around 25% . to characterize the qds conjugated to a ( 142 ) , it is important to estimate the number of a monomers bound to qds after conjugation . firstly , characterization of samples of pure a ( 142 ) , a ( 142 ) mixed and conjugated to qds was performed using uv - vis and fluorescence spectroscopy ( figure 2 ) . the emission band for the tyrosine moiety in a ( 142 ) was observed at 309 nm at the excitation wavelength of 280 nm , slit width at emission and excitation was set at 5 nm . the absorption band for a ( 142 ) was observed at 275 nm ( figure 2(a ) ) , whereas the emission band for the a ( 142 ) mixed and conjugated to qds was observed at 560 nm at the excitation wavelength of 467 nm . the absorption band for the qds was observed at 547 nm and for the a ( 142 ) a little hump was observed at 275 nm as shown in figure 2(b ) . molar concentration of the qds and a ( 142 ) was calculated from uv - vis spectrum of the solution [ 12 , 13 ] , the optical path length used was 1 cm . the ratio of these concentration values gave the average number of a per quantum dot nanoparticles . the molar extinction coefficient of qds at 547 nm is 105.8 10 m cm and at 275 nm is 2.6 10 m cm . extinction coefficient for a ( 142 ) at 275 nm is 1.4 10 m cm . the calculations to determine the ratio of a ( 142 ) and qds are shown below . qds concentration ( conjugated sample ) is ( 2)abs547547l=0.1561.05 105 m1 cm11 cm=1.5106 m. absorption at 275 nm from qds is ( 3)abs547275547=0.1562637.21.05105=3.9103 . absorption at 275 nm from a ( 142 ) is ( 4)abs275[abs547275547]=2.23.9 103=2.2 . a ( 142 ) concentration in the sample conjugated to qds is ( 5)abs for a at 275 for a at 275l=2.21390 m1cm11 cm=1.6103 m , loading = a concentrationqds concentration=1.6103 m1.5106 m=1.1103 . theoretically , the total number of molecules / particle of a ( 142 ) ( molecular weight 4514.1 g ) present in 0.5 mg of the 1157 l of sample can be calculated by multiplying the number of moles with na ( avogadro 's number ) number of molecules . hence , the total number of molecules of a ( 142 ) present in the sample is 5.8 10 . similarly , the total number of qds particles present in the solution is 9.0 10 . therefore , the ratio of a ( 142 ) molecules per qd particle is 6400 . subtracting the experimental value ( 1100 ) from the theoretical value ( 6400 ) , it can be inferred that there are 5300 molecules of a ( 142 ) that are free in the solution per qd particle that is conjugated to a ( 142 ) . to confirm that the qds were indeed conjugated to a ( 142 ) , we have used the agarose gel electrophoresis for the control dhla - capped cdse / zns qds along with a ( 142 ) mixed and conjugated to the qds ( figure 3 ) . the gel was run in tae buffer ( ph 7.4 ) at 84 v for 75 min , and the volume of the samples in each well was 10 l . figure 3 shows the distance moved by three different samples : dhla - capped qds ( 1 ) , a ( 142 ) mixed with dhla - capped qds ( 3 ) , and a ( 142 ) conjugated with dhla - capped qds ( 5 ) . from the gel electrophoresis , we can clearly distinguish that the distance moved by the qds mixed with a ( 142 ) is the same as for the pure dhla - capped qds . whereas when the qds are conjugated to a ( 142 ) , the distance moved is lower . this shows that when the qds are chemically conjugated to a ( 142 ) , the distance moved is reduced due to the higher molecular weight . moreover , comparison of polyethylene glycol ( peg)-capped qds ( 2 ) , a ( 142 ) mixed peg - capped qds ( 4 ) , and a ( 142 ) conjugated peg - capped qds ( 6 ) shows that peg - capped qds when conjugated travel the least distance . purified fractions of dhla- ( 7 ) and peg - capped ( 8) qds conjugated to peptide show that free a ( 142 ) can be separated from the a ( 142 ) conjugated qds . different fractions of 1 ml each obtained from gel chromatography were checked for the presence of free a ( 142 ) using uv - vis absorption and fluorescence spectroscopy . first evidence on the inhibition in fibrillation comes from tem images ( figure 4 ) taken on the 7th day of incubation at 37c . tem images containing a ( 142 ) are negatively stained using 2% uranyl acetate solution . three different samples , namely , pure a ( 142 ) , a ( 142 ) mixed with dhla - capped cdse / zns qds , and a ( 142 ) conjugated to dhla - capped cdse / zns qds , are shown in figures 4(a ) , 4(c ) , and 4(d ) , respectively . in all the three samples , the concentration of a ( 142 ) is 0.96 10 m and the concentration of qds is 1.4 m . analysis of this image indicates that the length of the fibrils varies from 30 to 1730 nm . the width of the shorter fibrils is 4.0 0.7 nm whereas for the longer fibrils it is 7.5 0.5 nm . figure 4(b ) presents the pure dhla - capped cdse / zns qds , the average particle size is 2.5 1.3 nm ( figure 5(a ) ) . the size of qds is an important parameter for the biodiagnostic studies , since smaller size qds are capable of passing through the blood - brain barrier [ 15 , 16 ] . figure 4(c ) shows the incubated sample of a ( 142 ) in the presence of qds whereas figure 4(d ) shows a ( 142 ) conjugated qds . comparison of the results in the three images containing a ( 142 ) illustrates very interesting pattern . sample containing pure a ( 142 ) , figure 4(a ) , has a large number of fibrils ranging from short fibrils around 30 nm to long fibrils around 2 micron whereas images of samples in presence of qds are very different . figure 4(c ) is the image of a ( 142 ) mixed with qds ; it shows long - length fibrils of around 1 micron . the width of the fibrils for the sample having mixed qds is 7.7 0.7 nm . figure 4(d ) for a ( 142 ) conjugated to qds shows short - length fibrils ranging from 30 to 80 nm . for this sample , the variation in the thickness of fibrils is significant in this case as compared to the other two . one can see a distinguishable inhibition of the fibrillation when a ( 142 ) is conjugated to qds . histogram showing the size of dhla - capped qds is shown in figure 5(a ) . the average size analyzed from the histogram obtained from tem images is 2.5 1.3 nm . furthermore , to consolidate the results , we have performed the statistical analysis on the tem images ( 1318 images per sample ) . the number of short - length fibrils ( 80150 nm ) observed in the sample containing pure a ( 142 ) was extremely high as compared to samples containing qds . statistical analysis showed ( figure 5(b ) ) that the number of fibrils having length 50100 nm dropped to 90% in the case of a ( 142 ) mixed to qds . there were only 26% of short - length fibrils observed for a ( 142 ) conjugated to qds . the total number of fibrils in the samples containing qds mixed or conjugated to a ( 142 ) was similar ( 35 fibrils approximately ) . . figures 6(a ) and 6(b ) show the tem images of unstained samples of a ( 142 ) mixed with qds . contrary to the tem images of stained samples where we can observe only fibrils and no qds , in this case , we were able to observe the qds , as well as fibrils . it could be discerned from the results that qds are enveloping the fibrils and more number of qds are observed at the ends of the fibrils . for the samples containing a ( 142 ) conjugated to qds ( figures 6(c ) and 6(d ) ) , we could observe less number of qds and short - length fibrils . the qds observed in this case are segregated at one end of the fibrils as seen in figure 6(d ) . it could be infered from the results obtained using unstained samples ( figure 6 ) that qds envelop the fibrils and could block the ends to elongate . importance of the c terminus of the a ( 142 ) in controlling the self - assembly of fibrillation was revealed before . the reason for the inhibition of fibrillation in presence of qds could be that the small size of particles could block the c - terminal end of the fibrils ( ~10 nm ) or the protofibrils ( ~5 nm in diameter ) , which is considered as the terminus with lower degree of freedom and accessibility for elongation mechanism . furthermore , binding between the qds and the a ( 142 ) could block the active sites leading to low local protein concentration , hence increasing the lag time for nucleation or disrupting the nucleation process leading to inhibition of the fibrillation process . besides , presence of a ( 142 ) conjugated to qds in the sample containing free a ( 142 ) may perturb the nucleation mechanism with decrease in localised concentration of the a ( 142 ) as thereby inhibiting fibrillation process [ 19 , 20 ] . however , mixed sample of a ( 142 ) and the qds might increase the localised concentration of the polypeptide , thereby increasing the length of fibrils but the number of fibrils still remains low suggesting that there is a perturbation in the mechanism of the fibrillation process . the tem results are supported by the afm images as shown in figure 7 for the 7th day of incubation at 37c . analysis of afm image for a ( 142 ) in absence of qds ( figure 7(a ) ) shows that the length of the longest fibril is 523 nm and the shortest fibril is 30 nm . a bigger number of short length fibrils ( 3080 nm ) are observed as witnessed by the tem images whereas in the sample containing a ( 142 ) mixed to qds ( figure 7(c ) ) the length of the longest fibrils is 849 nm , comparable to the length of fibrils ( 1 m ) found in the tem images . however , when we compare the length of fibrils ( figure 7(d ) ) in the sample containing a ( 142 ) conjugated to qds , very few long fibrils were observed , which correspond to the tem images of the same sample . the length of the longest fibrils found is 468 nm and the shortest fibril is 58 nm , while in the case of the tem images the length of fibrils is between 30 and 80 nm . the height analysis of qds ( figure 7(b ) ) shows that the root mean square height of qds is 2.3 nm , which is comparable to the average height of qds found in tem images ( 2.5 1.3 nm ) . the measurement of z - height of the a aggregates shows that when a ( 142 ) is conjugated to qds the height distribution histogram changes significantly . it is known for the a oligomers that the average height is between 3 and 5 nm , and for the fibrils it varies from 3 to 9 nm [ 21 , 22 ] . from the height distribution curves ( figure 8) one can see that in the case of amyloid fibrils in absence of qds 40% of aggregates have height greater than 8 nm whereas 60% of aggregates have height between 6 and 8 nm . for the sample of a ( 142 ) mixed with qds , 30% of the aggregates have height greater than 7 nm , and 70% of the aggregates have height between 1 and 3 nm . interestingly , when we analyze the height distribution for a ( 142 ) conjugated to qds only 14% of the aggregates have height greater than 7 nm . almost 90% of the aggregates have height between 2 and 4 nm . to further examine the fibrillation process and to support the image analysis , it is known that the fluorescence intensity of the tht dye grows with increasing concentration of fibrils . it has to be pointed out that previous studies confirmed that the fluorescence enhancement of tht depends upon the structure of the aggregated state of the amyloid peptides [ 23 , 24 ] . figure 9(a ) shows the tht assay on the 7th day for the samples incubated at 37c . when the pure solution of 10 m tht in pbs buffer ( ph 7.4 ) is excited at 440 nm , the emission band at 482 nm is observed with a very low intensity . in the presence of amyloid fibrils , the tht emission band at 482 nm is enhanced significantly . for the sample containing qds mixed to the fibrils , the intensity of the emission band at 482 nm is decreased by 66% as compared to the band for pure amyloid fibrils . whereas the amyloid fibrils , conjugated to qds show a decrease in intensity for the emission band by 40% as compared to the qds mixed to the fibrils . the variation of tht intensity yields information regarding the extent of fibrillation . for the sample containing pure a ( 142 ) , a sigmoidal curve is observed , lag phase is between 0 and 24 h , and rapid progress in fibrillation is observed after 50 h of incubation . however , for the sample containing dhla - capped qds mixed to a ( 142 ) , lag time is increased to 48 h and it can be observed with decrease in intensity of fluorescence that the qds are inhibiting the fibril formation . similarly , in the case of sample containing a ( 142 ) conjugated to the dhla - capped qds , a decrease in intensity of fluorescence and completion of saturation in fibrillation are observed at 72 h. these results show , in the presence of qds , that the self - assembly of a ( 142 ) is perturbed . a remarkable diminution in fibrillation process in the presence of qds and a significant change in morphology contrary to the results that have been published previously on the nanoparticles such as tio2 , copolymer particles , cerium oxide , qds , and carbon nanotubes behaving as catalyst for fibrillation [ 5 , 8 ] , we did not observe the same behavior for the dhla - capped cdse / zns qds . moreover , our results are in line with a very recent publication showing the inhibition of a ( 140 ) by cdte nanoparticles which have similar diameter size ( 35 nm ) . major difference from other set of nanoparticles being used could be the composition and the size of the particles . the size range for the particles that have been used for the previous studies varies from 16 to 200 nm whereas the qds used in our work have an average size of 2.5 1.3 nm . to investigate the effect on the tyrosine residue , which is an intrinsic probe of a ( 142 ) , we have examined the tyrosine fluorescence spectra for the three samples on the 7th day of incubation at 37c . there is a notable quenching of the tyrosine fluorescence intensity at 309 nm ( figure 9(c ) ) in the presence of mixed or conjugated qds . this effect could be due to the fact that the tyrosine moiety ( tyr ) interacts with the qds . for example , the three histidine residues ( his , his , and his ) in the vicinity of the tyrosine may interact or form coordination bond with the surface of qds . this phenomenon happens due to the presence of overcoated zns shell offering zn ions , hence rendering tyrosine to interact with qds and consequently decreasing significantly the fluorescence intensity of tyrosine band . another explanation could be the fret mechanism between the donor ( tyrosine moiety ) and the acceptor ( qds ) , since there is an overlap between the absorption spectrum of the acceptor ( qds ) and the emission spectrum of the donor ( tyrosine ) . fret efficiency in case of a ( 142 ) conjugated to qds was 0.84 whereas for a ( 142 ) mixed with qds was 0.94 . it could be interpreted that the forster distance ( ro ) between the a ( 142 ) and qds in aqueous solution was less than 60 which is the critical distance for energy transfer . it means that in both the samples , a ( 142 ) mixed with or conjugated to qds , a ( 142 ) is present very near to the qds . this is an indirect evidence that qds are present very near to fibrils ; that is also observed in the tem images of the unstained samples ( figure 6 ) . furthermore , to examine the inhibition effect of cdse / zns qds , we performed the same set of experiments using polyethylene glycol ( peg ) ( mw 400)-capped cdse / zns qds . no inhibition on fibrillation process is observed when peg - capped qds are mixed or conjugated to a ( 142 ) . figures 10(a ) and 10(b ) show the afm images of peg - capped qds conjugated and mixed to a ( 142 ) , respectively , after 2 days . the length of the fibrils is between 700 nm and 3 m and the height of oligomers is observed between 2.5 and 5.9 nm . increase in intensity of fluorescence at 482 nm for the peg - capped qds mixed or conjugated with a ( 142 ) shows that there is increase in fibrillation in presence of peg - capped qds . experiments were also designed where dhla- or peg - capped qds were purified using gel chromatography to check the inhibition effect . similar results are obtained over a period of 72 h with the purified fractions of dhla - or peg - capped qds , that is , inhibition and absence of inhibition on fibrillation process , respectively . it shows that if we change the ligand of the qds , it changes its behavior towards the fibrillation process . the emission band for the peg - capped qds was observed at 472 nm , when the excitation wavelength was set at 467 nm . the average size for the peg - capped qds obtained using tem analysis was 19.4 4.7 nm ( figure 10 ) . this change in behavior might be due to the fact that peg - capped qds tend to aggregate in buffer solutions , and peg polymer increases the size of the qds . these two factors make the qds nanoparticles less dynamic in solution and less accessible for the a ( 142 ) monomers , where the nanoparticles can block the active sites for extended fibrillation . figures 11(a ) and 11(b ) show the tem images of peg - capped qds . the average size for the peg - capped qds obtained using tem analysis was 19.4 4.7 nm as illustrated in the histogram ( figure 11(c ) ) . qds mixed or conjugated to a ( 142 ) show a decrease in the fibrillation as compared to pure a ( 142 ) , when incubated at 37c for 7 days . longer fibrils ( 2 micron ) are observed in the sample containing a ( 142 ) mixed with qds . pure a ( 142 ) sample contained large number of short- and long - length fibrils ( 301730 nm ) . thicker and shortest length fibrils ( 3080 nm ) are observed in the case of a ( 142 ) conjugated to qds . the height analysis of afm images shows significant decrease in height of aggregates greater than 7 nm ( only 14% ) when qds are conjugated to a ( 142 ) and 30% when qds are mixed to a ( 142 ) as compared to pure a ( 142 ) solution . tht assay for the samples confirmed the inhibition of the fibrillation process when a ( 142 ) is mixed or conjugated to qds . moreover , quenching of tyrosine signal is observed in the presence of qds , which indicates an interaction of the qds with the tyr residue in a ( 142 ) . however , in presence of peg - capped qds mixed or conjugated to a ( 142 ) , an absence of inhibition on fibrillation is observed as revealed by afm images and tht fluorescence . the conclusion of this work is presented in figure 12 that shows diminution in fibrillation in presence of dhla - capped qds , either mixed with or conjugated to a ( 142 ) . to investigate the use of qds in vivo studies is very important part of biomedical applications , there is a recent investigation showing the use of qds for imaging and delivery purposes , where qds carrying snare - tagged rbd were delivered at the synaptic contacts in the cultures from hippocampal neurons obtained from mice . moreover , qds doped with sio2 nanoparticles showed imaging and gene carrier capabilities , it was demonstrated that these qds were internalized by primary cortical neural cells without inducing cell death in vitro and in vivo . point to be noted is cdse quantum dots are toxic and might not be used for medicinal purposes . however , some toxicology studies have shown that the toxicity of qds is size and concentration dependent . for example , cytotoxicity studies of cdse qds on b16 f10 melanoma cells , and c57/bl6 mice showed no detectable toxicity . early studies have shown high toxicity of cdse qds due to the release of toxic cd ions ; however , coating of zns has shown to reduce the toxicity in cell culture to a great extent . nevertheless , extensive studies are required in the field of toxicology . in the light of these studies , it would be important to test a ( 142 ) mixed with or conjugated to qds in the cultures from neurons of mice to investigate the effect of qds in in vivo systems .

nanoparticles have enormous potential in diagnostic and therapeutic studies . we have demonstrated that the amyloid beta mixed with and conjugated to dihydrolipoic acid- ( dhla ) capped cdse / zns quantum dots ( qds ) of size approximately 2.5 nm can be used to reduce the fibrillation process . transmission electron microscopy ( tem ) and atomic force microscopy ( afm ) were used as tools for analysis of fibrillation . there is a significant change in morphology of fibrils when amyloid ( 142 ) ( a ( 142 ) ) is mixed or conjugated to the qds . the length and the width of the fibrils vary under modified conditions . thioflavin t ( tht ) fluorescence supports the decrease in fibril formation in presence of dhla - capped qds .

1. Introduction 2. Experimental Section 3. Results and Discussion 4. Imaging and Analysis 5. Conclusion

however , obtaining suitable nanoparticles that can be used for diagnostic and medicinal purposes remains a significant challenge . in another case , dual effect of commercial polystyrene nanoparticles with amino modification having various sizes ( 57 , 120 , and 180 nm ) was observed on a ( 140 ) and recombinant a ( 140 ) and a ( 142 ) proteins . furthermore , there is only one recent publication on the effect of nanoparticles on a ( 142 ) fibrils . specifically , the only investigation that explains the effect of tio2 on a ( 142 ) shows that nanoparticles promote the fibrillation process by becoming nucleation centers . we report for the first time in our knowledge that cdse / zns qds of size of 2.5 1.3 nm can inhibit fibrillation of a ( 142 ) . in the present study , we have investigated the effect of the presence of dhla - capped cdse / zns qds either mixed with or conjugated to a ( 142 ) on fibrillation process of a ( 142 ) in aqueous phase . tem and afm studies show that the qds behave uniquely when they are conjugated to a ( 142 ) in comparison to a mixed sample of a ( 142 ) and qds . our study illustrates a considerable difference in morphology of the fibrils and the inhibition of fibrillation process when a ( 142 ) is conjugated to qds versus the mixed system a ( 142 ) and qds . these results are further supported by thioflavin t ( tht ) assay using fluorescence spectroscopy . dl--lipoic acid , a ( 142 ) , and tht were purchased from mp biomedicals ( solon , oh ) . after the synthesis of topo - capped hydrophobic qds , modification to hydrophilic dhla - capped qds was carried out . freshly prepared dhla - capped qds ( 1.51 10 m , 100 l ) were taken in a clean borosilicate glass vial , and 500 l pbs buffer ( ph 7.4 ) was added to the qds . to lyophilize the peptide , 0.5 mg a ( 142 ) was dissolved in hexafluoroisopropanol ( hfip ) to bring the peptide in monomer form and evaporated under gentle flow of n2 . a ( 142 ) mixed with dhla - capped qds was prepared according to the above - mentioned protocol except for the addition of edc . for the induction of fibrillation pure a ( 142 ) solution was prepared similarly , first by lyophilizing the peptide in hfip and evaporating the solvent under gentle nitrogen flow and then redissolving the dried peptide in 1157 l of pbs buffer ( ph 7.4 ) . the samples were analyzed by atomic force microscopy ( afm , a multimode spm , model no . 30 l of a ( 142 ) aliquots were extracted at different time periods , and 300 l of tht ( 10 m ) was added to the samples . characterization of dhla - capped qds was carried out using uv - vis and fluorescence spectroscopy ( figure 1(a ) ) . the qy of the dhla - capped cdse / zns qds was around 25% . to characterize the qds conjugated to a ( 142 ) , it is important to estimate the number of a monomers bound to qds after conjugation . firstly , characterization of samples of pure a ( 142 ) , a ( 142 ) mixed and conjugated to qds was performed using uv - vis and fluorescence spectroscopy ( figure 2 ) . the emission band for the tyrosine moiety in a ( 142 ) was observed at 309 nm at the excitation wavelength of 280 nm , slit width at emission and excitation was set at 5 nm . the absorption band for a ( 142 ) was observed at 275 nm ( figure 2(a ) ) , whereas the emission band for the a ( 142 ) mixed and conjugated to qds was observed at 560 nm at the excitation wavelength of 467 nm . the absorption band for the qds was observed at 547 nm and for the a ( 142 ) a little hump was observed at 275 nm as shown in figure 2(b ) . molar concentration of the qds and a ( 142 ) was calculated from uv - vis spectrum of the solution [ 12 , 13 ] , the optical path length used was 1 cm . the calculations to determine the ratio of a ( 142 ) and qds are shown below . absorption at 275 nm from a ( 142 ) is ( 4)abs275[abs547275547]=2.23.9 103=2.2 . a ( 142 ) concentration in the sample conjugated to qds is ( 5)abs for a at 275 for a at 275l=2.21390 m1cm11 cm=1.6103 m , loading = a concentrationqds concentration=1.6103 m1.5106 m=1.1103 . theoretically , the total number of molecules / particle of a ( 142 ) ( molecular weight 4514.1 g ) present in 0.5 mg of the 1157 l of sample can be calculated by multiplying the number of moles with na ( avogadro 's number ) number of molecules . therefore , the ratio of a ( 142 ) molecules per qd particle is 6400 . subtracting the experimental value ( 1100 ) from the theoretical value ( 6400 ) , it can be inferred that there are 5300 molecules of a ( 142 ) that are free in the solution per qd particle that is conjugated to a ( 142 ) . to confirm that the qds were indeed conjugated to a ( 142 ) , we have used the agarose gel electrophoresis for the control dhla - capped cdse / zns qds along with a ( 142 ) mixed and conjugated to the qds ( figure 3 ) . figure 3 shows the distance moved by three different samples : dhla - capped qds ( 1 ) , a ( 142 ) mixed with dhla - capped qds ( 3 ) , and a ( 142 ) conjugated with dhla - capped qds ( 5 ) . from the gel electrophoresis , we can clearly distinguish that the distance moved by the qds mixed with a ( 142 ) is the same as for the pure dhla - capped qds . whereas when the qds are conjugated to a ( 142 ) , the distance moved is lower . this shows that when the qds are chemically conjugated to a ( 142 ) , the distance moved is reduced due to the higher molecular weight . moreover , comparison of polyethylene glycol ( peg)-capped qds ( 2 ) , a ( 142 ) mixed peg - capped qds ( 4 ) , and a ( 142 ) conjugated peg - capped qds ( 6 ) shows that peg - capped qds when conjugated travel the least distance . purified fractions of dhla- ( 7 ) and peg - capped ( 8) qds conjugated to peptide show that free a ( 142 ) can be separated from the a ( 142 ) conjugated qds . different fractions of 1 ml each obtained from gel chromatography were checked for the presence of free a ( 142 ) using uv - vis absorption and fluorescence spectroscopy . three different samples , namely , pure a ( 142 ) , a ( 142 ) mixed with dhla - capped cdse / zns qds , and a ( 142 ) conjugated to dhla - capped cdse / zns qds , are shown in figures 4(a ) , 4(c ) , and 4(d ) , respectively . in all the three samples , the concentration of a ( 142 ) is 0.96 10 m and the concentration of qds is 1.4 m . analysis of this image indicates that the length of the fibrils varies from 30 to 1730 nm . the width of the shorter fibrils is 4.0 0.7 nm whereas for the longer fibrils it is 7.5 0.5 nm . figure 4(b ) presents the pure dhla - capped cdse / zns qds , the average particle size is 2.5 1.3 nm ( figure 5(a ) ) . figure 4(c ) shows the incubated sample of a ( 142 ) in the presence of qds whereas figure 4(d ) shows a ( 142 ) conjugated qds . comparison of the results in the three images containing a ( 142 ) illustrates very interesting pattern . sample containing pure a ( 142 ) , figure 4(a ) , has a large number of fibrils ranging from short fibrils around 30 nm to long fibrils around 2 micron whereas images of samples in presence of qds are very different . figure 4(c ) is the image of a ( 142 ) mixed with qds ; it shows long - length fibrils of around 1 micron . the width of the fibrils for the sample having mixed qds is 7.7 0.7 nm . figure 4(d ) for a ( 142 ) conjugated to qds shows short - length fibrils ranging from 30 to 80 nm . one can see a distinguishable inhibition of the fibrillation when a ( 142 ) is conjugated to qds . histogram showing the size of dhla - capped qds is shown in figure 5(a ) . statistical analysis showed ( figure 5(b ) ) that the number of fibrils having length 50100 nm dropped to 90% in the case of a ( 142 ) mixed to qds . there were only 26% of short - length fibrils observed for a ( 142 ) conjugated to qds . the total number of fibrils in the samples containing qds mixed or conjugated to a ( 142 ) was similar ( 35 fibrils approximately ) . figures 6(a ) and 6(b ) show the tem images of unstained samples of a ( 142 ) mixed with qds . for the samples containing a ( 142 ) conjugated to qds ( figures 6(c ) and 6(d ) ) , we could observe less number of qds and short - length fibrils . the qds observed in this case are segregated at one end of the fibrils as seen in figure 6(d ) . importance of the c terminus of the a ( 142 ) in controlling the self - assembly of fibrillation was revealed before . the reason for the inhibition of fibrillation in presence of qds could be that the small size of particles could block the c - terminal end of the fibrils ( ~10 nm ) or the protofibrils ( ~5 nm in diameter ) , which is considered as the terminus with lower degree of freedom and accessibility for elongation mechanism . furthermore , binding between the qds and the a ( 142 ) could block the active sites leading to low local protein concentration , hence increasing the lag time for nucleation or disrupting the nucleation process leading to inhibition of the fibrillation process . besides , presence of a ( 142 ) conjugated to qds in the sample containing free a ( 142 ) may perturb the nucleation mechanism with decrease in localised concentration of the a ( 142 ) as thereby inhibiting fibrillation process [ 19 , 20 ] . however , mixed sample of a ( 142 ) and the qds might increase the localised concentration of the polypeptide , thereby increasing the length of fibrils but the number of fibrils still remains low suggesting that there is a perturbation in the mechanism of the fibrillation process . analysis of afm image for a ( 142 ) in absence of qds ( figure 7(a ) ) shows that the length of the longest fibril is 523 nm and the shortest fibril is 30 nm . a bigger number of short length fibrils ( 3080 nm ) are observed as witnessed by the tem images whereas in the sample containing a ( 142 ) mixed to qds ( figure 7(c ) ) the length of the longest fibrils is 849 nm , comparable to the length of fibrils ( 1 m ) found in the tem images . however , when we compare the length of fibrils ( figure 7(d ) ) in the sample containing a ( 142 ) conjugated to qds , very few long fibrils were observed , which correspond to the tem images of the same sample . the length of the longest fibrils found is 468 nm and the shortest fibril is 58 nm , while in the case of the tem images the length of fibrils is between 30 and 80 nm . the height analysis of qds ( figure 7(b ) ) shows that the root mean square height of qds is 2.3 nm , which is comparable to the average height of qds found in tem images ( 2.5 1.3 nm ) . the measurement of z - height of the a aggregates shows that when a ( 142 ) is conjugated to qds the height distribution histogram changes significantly . for the sample of a ( 142 ) mixed with qds , 30% of the aggregates have height greater than 7 nm , and 70% of the aggregates have height between 1 and 3 nm . interestingly , when we analyze the height distribution for a ( 142 ) conjugated to qds only 14% of the aggregates have height greater than 7 nm . to further examine the fibrillation process and to support the image analysis , it is known that the fluorescence intensity of the tht dye grows with increasing concentration of fibrils . whereas the amyloid fibrils , conjugated to qds show a decrease in intensity for the emission band by 40% as compared to the qds mixed to the fibrils . however , for the sample containing dhla - capped qds mixed to a ( 142 ) , lag time is increased to 48 h and it can be observed with decrease in intensity of fluorescence that the qds are inhibiting the fibril formation . similarly , in the case of sample containing a ( 142 ) conjugated to the dhla - capped qds , a decrease in intensity of fluorescence and completion of saturation in fibrillation are observed at 72 h. these results show , in the presence of qds , that the self - assembly of a ( 142 ) is perturbed . a remarkable diminution in fibrillation process in the presence of qds and a significant change in morphology contrary to the results that have been published previously on the nanoparticles such as tio2 , copolymer particles , cerium oxide , qds , and carbon nanotubes behaving as catalyst for fibrillation [ 5 , 8 ] , we did not observe the same behavior for the dhla - capped cdse / zns qds . to investigate the effect on the tyrosine residue , which is an intrinsic probe of a ( 142 ) , we have examined the tyrosine fluorescence spectra for the three samples on the 7th day of incubation at 37c . there is a notable quenching of the tyrosine fluorescence intensity at 309 nm ( figure 9(c ) ) in the presence of mixed or conjugated qds . another explanation could be the fret mechanism between the donor ( tyrosine moiety ) and the acceptor ( qds ) , since there is an overlap between the absorption spectrum of the acceptor ( qds ) and the emission spectrum of the donor ( tyrosine ) . fret efficiency in case of a ( 142 ) conjugated to qds was 0.84 whereas for a ( 142 ) mixed with qds was 0.94 . it could be interpreted that the forster distance ( ro ) between the a ( 142 ) and qds in aqueous solution was less than 60 which is the critical distance for energy transfer . it means that in both the samples , a ( 142 ) mixed with or conjugated to qds , a ( 142 ) is present very near to the qds . no inhibition on fibrillation process is observed when peg - capped qds are mixed or conjugated to a ( 142 ) . figures 10(a ) and 10(b ) show the afm images of peg - capped qds conjugated and mixed to a ( 142 ) , respectively , after 2 days . the length of the fibrils is between 700 nm and 3 m and the height of oligomers is observed between 2.5 and 5.9 nm . increase in intensity of fluorescence at 482 nm for the peg - capped qds mixed or conjugated with a ( 142 ) shows that there is increase in fibrillation in presence of peg - capped qds . similar results are obtained over a period of 72 h with the purified fractions of dhla - or peg - capped qds , that is , inhibition and absence of inhibition on fibrillation process , respectively . it shows that if we change the ligand of the qds , it changes its behavior towards the fibrillation process . this change in behavior might be due to the fact that peg - capped qds tend to aggregate in buffer solutions , and peg polymer increases the size of the qds . these two factors make the qds nanoparticles less dynamic in solution and less accessible for the a ( 142 ) monomers , where the nanoparticles can block the active sites for extended fibrillation . qds mixed or conjugated to a ( 142 ) show a decrease in the fibrillation as compared to pure a ( 142 ) , when incubated at 37c for 7 days . longer fibrils ( 2 micron ) are observed in the sample containing a ( 142 ) mixed with qds . thicker and shortest length fibrils ( 3080 nm ) are observed in the case of a ( 142 ) conjugated to qds . the height analysis of afm images shows significant decrease in height of aggregates greater than 7 nm ( only 14% ) when qds are conjugated to a ( 142 ) and 30% when qds are mixed to a ( 142 ) as compared to pure a ( 142 ) solution . tht assay for the samples confirmed the inhibition of the fibrillation process when a ( 142 ) is mixed or conjugated to qds . moreover , quenching of tyrosine signal is observed in the presence of qds , which indicates an interaction of the qds with the tyr residue in a ( 142 ) . however , in presence of peg - capped qds mixed or conjugated to a ( 142 ) , an absence of inhibition on fibrillation is observed as revealed by afm images and tht fluorescence . the conclusion of this work is presented in figure 12 that shows diminution in fibrillation in presence of dhla - capped qds , either mixed with or conjugated to a ( 142 ) . in the light of these studies , it would be important to test a ( 142 ) mixed with or conjugated to qds in the cultures from neurons of mice to investigate the effect of qds in in vivo systems .

the essential and highly conserved snare ( soluble n - ethylmaleimide - sensitive factor attachment protein receptor ) proteins are the molecular machines that drive membrane fusion , which represents the final step in every trafficking pathway . the neuronal snare complex , consisting of vamp2 bound to the synaptic vesicle ( v - snare ) and syntaxin1a and snap25 on the target membrane ( t - snare ) , has been extensively studied and serves as a model system for understanding complex assembly and snare - mediated membrane fusion . snare proteins are characterized by the presence of the heptad repeat motif that consists of mostly hydrophobic layer residues at every third or fourth position ( layers numbered 7 to + 8 , figure 1a ) . isolated snares are largely unstructured but adopt an -helical conformation upon interaction with their cognate binding partners . a progressive zippering model was proposed for snare assembly , which suggests that the snare motifs from vamp2 , syntaxin1a and snap25 , zipper directionally from their membrane - distal n - terminal domains ( ntds ) to their c - terminal domains ( ctds ) into a four - helical coiled - coil bundle . interactions extend through the membrane - proximal linker domains ( lds ) and the transmembrane domains ( tmds ) helices of snares . the energy from complex formation is thought to be used to overcome the thermodynamic barrier of membrane fusion . the zippering reaction has been historically considered to take place continuously as a single event , but recent biophysical studies are more consistent with the idea that it occurs in discrete steps . however , the functional significance of these biophysical observations has not been tested , which is our goal here . a switch that activates the t - snare exists in ntd assembly of snare complex . ( a ) illustration of 4 domains and 16 layers in postfusion snare complex . the snare motifs form a four - helix bundle ( syntaxin 1a , red ; snap25 , green ; vamp2 , blue ) . ( b ) vn peptide activates fusion between flt - liposomes and vc - liposomes . standard liposome fusion assays were performed in the absence and presence of prebound vn ( vamp2 residues from layers 7 to 1 ) peptide . to prebind the vn peptide , flt - liposomes and 20 m vn peptide were incubated together at 37 c for 60 min prior to mixing with vc - liposomes . positive control represents fusion of flt - liposomes ( full length t - snare ) and flv - liposomes ( full length vamp2 ) , and negative control shows fusion of flt - liposomes preincubated with cdv ( the cytosolic domain of vamp2 , residues 194 ) and flv - liposomes . ( c ) activation of the t - snare requires snares to assemble at least to layer 1 . fusion reactions were performed between vc - liposomes and flt - liposomes prebound with vn , vn , vn , vn , or vn , respectively . ( d ) layer 1 is required for vn to bind tightly with the t - snare . each individually labeled vn ( 200 nm ) was incubated with cytosolic t - snare at various concentrations at 37 c for 60 min followed by 24 h on ice . for each vn , the increase in anisotropy was plotted versus t - snare concentration and fitted using eq 9 in experimental section to obtain the affinity constants . neuronal snare assembly is positively regulated by the sm protein munc18 and controlled by a clamping system consisting of complexin and synaptotagmin for ca - dependent rapid and synchronized fusion . it is not known how all these regulatory proteins interact with snares on a molecular level . the hypothesis we will explore here is that a partially assembled snare complex represents a folding intermediate on which regulators might act to accelerate or decelerate snare assembly . here we demonstrate for the first time that even in the absence of any regulatory protein , a half - zippered snare complex represents a functional intermediate in a two - step folding process , and this intrinsic property of snares provides a molecular basis that supports the models put forward for the function of complexin . we show distinct functions for n- and c - terminal snare zippering , namely , prestructuring the t - snare and driving fusion , respectively . the abbreviations of constructs used in this study are summarized in supporting table s1 . the full length t - snare complex , which includes full length , wild - type mouse his6-snap25 and rat syn1a , was produced by expression of the polycistronic plasmid ptw34 in the bl-21 gold ( de3 ) escherichia coli bacterial strain and purified as described before . the full length vamp2 , which includes full length , wild - type mouse vamp2 , was produced by expression of the plasmid ptw2 in the bl-21 gold ( de3 ) escherichia coli bacterial strain and purified as described before . the soluble t - snare complex , made of the cytoplasmic domain of rat syntaxin 1a ( residues 1265 ) and mouse his6-snap25 ( residues 1206 ) , was produced by coexpression of pjm57 and pjm72 plasmids in the bl-21 gold ( de3 ) escherichia coli bacterial strain and purified as described before . the protein concentration was typically 510 mgml as determined by bradford protein assay with bsa as the standard . the cytoplasmic domain of mouse his6-vamp2 ( residues 194 , for fusion assay ) and his6-sumo - vamp2 ( residues 2894 , for circular dichroism assay ) were produced by expression in the bl-21 gold ( de3 ) escherichia coli bacterial strain and purified as previously described . the protein concentration was typically 1.53 mgml as determined by bradford protein assay with bsa as the standard . the plasmids for the vn variants were produced by cloning the n - terminus of vamp2 of various lengths into a pcdfduet-1 vector containing gst - prescission - vn ( containing mouse vamp2 n - terminal residues ) . the vn constructs generated were vn ( vamp2 residues 2860 ) , vn ( vamp2 residues 2857 ) , vn ( vamp2 residues 2855 ) , vn ( vamp2 residues 2850 ) , vn ( vamp2 residues 2847 ) , and vn ( vamp2 residues 2844 ) . these constructs were used in liposome liposome fusion assay and circular dichroism experiments . the plasmids for the tm - vc variants were produced by cloning the c - terminus of vamp2 of various lengths into a pet sumo vector containing n - terminal his6 tag . the tm - vc constructs generated were vc ( vamp2 residues 49116 ) , vc ( vamp2 residues 55116 ) , vc ( vamp2 residues 60116 ) , vc ( vamp2 residues 62116 ) , vc ( vamp2 residues 65116 ) , vc ( vamp2 residues 69116 ) , vc ( vamp2 residues 79116 ) , and vc ( vamp2 residues 85116 ) . all tm - vc variants were expressed in bl21 gold ( de3 ) escherichia coli bacterial strain . the lysate was centrifuged , and the supernatant was incubated with nickel - nta beads . the his6-sumo tag was cleaved by incubating the protein ( attached to nickel - nta beads ) with sumo protease . the protein was eluted with a buffer containing 25 mm hepes ( ph 7.4 ) , 400 mm kcl , 10% glycerol , 1 mm dtt , and 1% ( w / v ) n - octyl--d - glucopyranoside ( og ) . the full length t - snare complex was reconstituted with the acceptor lipid mix made of 85 mol % popc and 15 mol % dops . full length vamp2 and tm - vc were reconstituted with the donor lipid mix comprising 82 mol % popc , 15 mol % dops , 1.5 mol % dppe - rho , and 1.5 mol % dppe - nbd . the snare - liposome was prepared using the standard detergent removal method , which was previously reported . typically , flt - liposome had 400:1 lipid / protein ratio and the flv - liposome and vc - liposome had 200:1 lipid / protein ratio . for a typical liposome liposome fusion assay , 45 l of flt - liposome was mixed with 15 l of buffer or vn peptide ( final concentrations of lipids and vn peptide were 2 mm and 20 m , respectively ) and incubated at 37 c for 60 min , then transferred to a 96-well fluoronunc plates ( nalge nunc , rochester , ny ) and kept at 37 c for 5 min . the fusion reaction was initiated by adding 5 l of vc - liposome or flv - liposome . fusion between flt - liposome and vc - liposome was measured by monitoring the dequenching of the dppe - nbd fluorescence resulting from its dilution into the fused liposomes , at 1 min intervals for 120 min , with excitation wavelength at 460 nm and emission wavelength at 538 nm , by a plate reader ( synergy h1 hybrid microplate reader , bio - tek ) . after 120 min , 10 l of 2.5% ( w / v ) n - dodecylmaltoside ( boehringer , ingelheim , germany ) was added to completely dissolve the liposomes . measurement of the dppe - nbd fluorescence was continued for another 40 min to obtain the dppe - nbd fluorescence at infinite dilution . as reported previously , the normalized fluorescence was obtained by using the fluorescence intensity of dppe - nbd during fusion divided by the average intensity the dppe - nbd fluorescence at infinite dilution . the vn - s28c variants and cytosolic vc - cys ( contains vamp2 residues 5894 and a single cysteine at the end of sequence ) were labeled with texas red c2 maleimide ( invitrogen ) according the protocol recommended by the manufacturer . t - format polarization was used with a 625 nm long - path filter on the left - emission channel and a monochromator on the right - emission channel . the temperature of sample chamber was controlled with 0.1 c accuracy . for texas red labeled protein , the excitation wavelength was 580 nm and the emission wavelength at the right - emission channel was 612 nm . for steady - state anisotropy measurements , the anisotropy of various texas red labeled vn - s28c peptides was first measured in the absence of t - snare . cytosolic t - snare ( cdt ) of various concentrations was then added to each labeled vn peptide ( 200 nm ) . the mixtures were incubated at 37 c for 60 min , followed by 24 h on ice . the texas red labeled cytosolic vc peptide ( 72 nm ) solution was introduced to a quartz cuvette ( hellma ) with continuous and rapid magnetic stirring . cytosolic t - snare prebound with vn peptide or t - snare alone , at various concentrations , was injected into the cuvette and mixed rapidly . the data were plotted as anisotropy versus time , and the beginning of mixing was set as time zero . to obtain the kinetics and thermodynamics parameters , we consider the following binding reaction : the kinetics equation is1where kon is the on - rate , koff is the off - rate , and [ v ] , [ t ] , and [ vt ] are the concentrations of vamp2 peptide , t - snare , and snare complex at time t , respectively . let v0 and t0 be the initial concentration ( or total concentration ) of vamp2 peptide and t - snare , respectively . thenthe measured anisotropy a at time t is an average of anisotropy of the fluorophores associated with vamp2 peptide and the fluorophores associated with the snare complex . let av be the anisotropy of vamp2 peptide ( all of the fluorophores are associated with vamp2 ) and avt be the anisotropy of complex ( all of the fluorophores are associated with the complex ) , thenthus,2equation 1 can be written as3at the initial stage of the binding reaction , [ vt ] is close to zero . hence , eq 3 can be simplified as4combining eqs 2 and 4 , we obtain5to obtain kon , we performed a series of reactions that labeled vamp2 peptide binds to t - snare at various initial concentrations , t0 , and monitored the variation of a with t. for each t0 , we obtained the initial rate da / dt from the a versus t curve , then plotted ( da / dt)/(avt av ) versus t0 . the resulting data points were fitted with a simple linear regression , and the slope gave kon . let kd be the affinity constant , ap be the measured anisotropy at equilibrium , and [ v]p , [ t]p , and [ vt]p be the concentrations of vamp2 peptide , t - snare , and snare complex at equilibrium , respectively . then67solving eq 6 for [ vt]p , and then entering into eq 7 , we have8or9by changing the initial concentration of t - snare , t0 , while keeping v0 constant , one can obtain a curve of ap as a function of t0 . kd is obtained using eq 8 or eq 9 and applying a nonlinear regression fit to the ap versus t0 curve . the model for complexin clamping is that complexin binds the half - assembled , intermediate snare complex and arrests further zippering and that upon clamp release , the zippering of the c - terminal portions provides sufficient energy to drive fusion ( figure 1a ) . to test this directly , we topologically separated ntd from later zippering reactions , thereby isolating c - terminal assembly as the only source of energy for bilayer fusion . we truncated vamp2 at layer + 1 , right after the zeroth layer , generating a protein , vc , containing only ctd , ld , and tmd ( vamp2 residues from 60 to 116 ) . vc was reconstituted into liposomes ( vc - liposomes , supporting information figure s1 ) and nanodiscs ( vc - nanodiscs ) . lipid mixing of vc - liposomes or vc - nanodiscs with full length t - snare liposomes ( flt - liposomes ) was monitored by dequenching of membrane dye . in content release assay , cacl2 that was encapsulated within flt - liposomes was released through the fusion pores formed between flt - liposomes and vc - nanodiscs and monitored by measuring the fluorescence of a ca sensor , mag - fluo-4 . however , fusion was restored when covalently separated n - terminal portion of the v - snare ( vn ) was added . specifically , we preincubated flt - liposomes with vn peptide ( the ntd region of vamp2 from layer 7 to 1 , right before the zeroth layer ) and then added vc - liposomes or vc - nanodiscs to start the fusion assays ( figure 1b and supporting information figure s2 ) . the initial fusion rates of lipid mixing were 12-fold that of the positive control , where both flt - liposomes and flv - liposomes ( or flv - nanodiscs ) contain wild type , full length snares . interestingly , the magnitude of activation by prebound vn is very similar to the level of activation by munc18 ( 10-fold ) . these results show that ( i ) the c - terminal ( ctd - ld - tmd ) assembly of snares provides sufficient energy needed to drive fusion whereas the energy from the n - terminal assembly has no direct contribution to fusion and ( ii ) n - terminal assembly with the t - snare is a prerequisite to enable fusion driven by ctd - ld - tmd assembly . ntd binding switches the t - snare to an activated state that must be reached before fusion can occur . therefore , the process of snare - mediated fusion can be functionally divided into two distinct and sequential steps , and the fusion in the positive control with full length snares follows the same two - step pattern : the ntd of snares assembles first and activates the t - snare , after which the c - termini assemble to drive fusion . the positive control is 12 times slower than the fusion between vc - liposomes and preactivated flt - liposomes . this shows that ( i ) c - terminal assembly of snares is rapid and not rate - limiting and that ( ii ) the n - terminal assembly with the t - snare is the rate - limiting factor . we further examined the ability of ntd to activate fusion for a potential length requirement for the sequence of ntd . we generated a series of ntd peptides that are truncated after the respective hydrophobic layer , ranging from vn ( which contains vamp2 residues from layer 7 to layer 4 ) to vn ( layer 7 to + 1 ) , and tested whether they are able to activate fusion between vc - liposomes or vc - liposomes and flt - liposomes ( figure 1c and supporting information figure s3 ) . we observed a distinct transition : vn peptides containing layer 1 ( vn and vn ) both enabled fusion at similar rates , while peptides lacking layer 1 ( vn , vn and vn ) did not activate fusion . next we used fluorescence anisotropy to measure the binding between these vn peptides and cytosolic t - snare . the binding curves in figure 1d show a similar transition regarding the sequence of vn : vn , vn , and vn bound tightly to the t - snare , and the binding constants for these peptides are virtually identical ( 70 to 100 nm ) ; however , vn and vn exhibited a dramatic reduction in affinity , with affinity constants of 10 m , showing that truncations of layer 1 of vamp2 lead to a large loss of binding to the t - snare . accordingly , we were able to reconstitute partially assembled snare complexes from syntaxin 1a , snap25 , and vn or longer but not with vn , as assessed by gel filtration analysis ( supporting information figure s4 ) . we observe an all - or - nothing transition , revealing a binary switch on the n - terminus of snares , with the minimum sequence being layer 7 to layer 1 ( figure 1c and figure 1d ) . only upon the binding of vn or longer versions is the t - snare switched on for fusion . to investigate the molecular basis for the n - terminal activation , we compared the crystal structures of a postfusion , fully zippered snare complex and a partially assembled , half - zippered complex . the fully assembled complex displays a four - helix bundle structure from n- to c - termini , as there are four helices present on both n- and c - termini . the partially assembled snare complex mimicked an intermediate folding state with a vn peptide , vn , which was reconstituted into a complex with the complete t - snare motifs . the crystal structure of the resulting half - zippered snare complex ( snare ) showed that surprisingly the snare motifs of syntaxin and snap25 almost entirely adopt an -helical conformation ( figure 2a ) : even though the c - terminal half of the complex only consists of three helices , they still display exactly the same four - helix bundle configuration as in the fully zippered complex . previous studies have shown that the binary syntaxin / snap25 t - snare complex is unstructured in its c - terminal portion , implying together with our data that binding of vamp2 ntd to the t - snare triggers a binary switch that propagates the four - helix bundle geometry to the t - snare c - terminal domain ( a ) binding of vn prestructures the c - terminus of the t - snare , as seen in a comparison of the crystal structures of the postfusion snare complex ( left ) and a partially assembled complex ( right ) . the postfusion complex exhibits four - helix structure , as four helices are present on both its n- and c - termini ( synxin 1a , red ; snap25 , green ; vamp2 , blue ) . the partially assembled snare complex contains a peptide vn binding to the t - snare motif , and similar to the postfusion complex , it also shows four - helix structure on both n- and c- termini , even though only three helices are present on its c - terminus . t - snare was incubated with various vn peptides at equimolar ratio , and their structures were monitored by circular dichroism . after incubating with vn , vn or vn , respectively , the cd spectra of snares became similar to postfusion snare complex , whereas the cd spectra of t - snare were not altered after incubating with vn and vn , respectively . to confirm this by an independent method , we next tested if the vn peptides that enable fusion mediated by membrane attached vc are capable of structuring the t - snare in solution . the transition from the partially unstructured t - snare to the folded snare core complex can be monitored using circular dichroism ( cd ) where the higher helical content of the ternary complex leads to a strong increase in ellipticity ( figure 2b ) . importantly , the truncated snare complex adopts a state of intermediate folding / helicity more similar to the snare core than the t - snare . this shows that the structure observed in the crystal is a true representation of the solution structure . the increase in ellipticity for vn and vn is similar to that of vn , suggesting the formation of a similar structure . in contrast , vn and vn , which do not activate fusion , have no effect on t - snare conformation when they were added at equimolar ratio or 5-fold molar excess ( figure 2b ) . as a concentration - independent measure of helical content , the ratio of ellipticity at 222 and 208 nm can be compared ( supporting information figure s5 ) . this result follows the same length requirement as the fusion assay and binding assay for different lengths of vn peptides . taken together , our observations show that activation of fusion , peptide binding , and induction of helicity are all coupled in an all - or - nothing transition , revealing that the molecular basis of this binary switch is the structuring of the c - terminal domain of the t - snare . specifically , binding of vn induces the three helices of the t - snare ctd to adopt the same configuration as in the fully assembled , postfusion complex , resulting in a preformed binding site for the fourth helix , the c - terminal domain of vamp2 . the n - terminal activation of the snares profoundly impacts the assembly and fusion on their c - termini . to accurately quantify this effect , we used fluorescence anisotropy measurements and monitored the rate of assembly of a soluble vc peptide , vc ( which includes vamp2 residues 5894 , layers + 1 to + 8 plus linker domain ) , with soluble , cytosolic portion of the t - snare in the presence and absence of vn in real time ( figure 3a and figure 3b , supporting information figures s6 and s7 ) . in the absence of vn , rate of binding of vc to the t - snare is slow ( kon = ( 9 1 ) 10 m s ) and thermodynamically less favorable ( kd = 352 60 nm ) , which corresponds to a free energy of 14.9 0.2 kbt , where kb is the boltzmann constant . when the t - snare was switched on upon binding vn , the kinetics of c - terminal domain assembly was increased 70 times ( now kon = ( 6 1 ) 10 m s ) and affinity of vc was increased by 30-fold to kd = 12 2 nm , corresponding to a free energy 18.3 0.2 kbt . as shown above , the energy that overcomes the fusion barrier comes from the assembly of c - terminus ( layer + 1 to layer + 8 plus linker domain ) . this suggests that an extra free energy of 3.4 kbt is generated when t - snare is in the on state and that this additional energy is required for rapid fusion to occur . structuring of the t - snare facilitates c - terminal assembly both energetically and kinetically . fluorescence anisotropy experiments were performed to monitor the binding process of vc ( vamp2 residues 5894 ) to the cytosolic t - snare at various concentrations with and without prebound vn peptide in real time . ( a ) vn binding to the t - snare improves binding affinity of vc . plateau anisotropy values were plotted versus the concentration of t - snare ( squares ) . the solid lines were fits using eq 8 in experimental section to obtain the affinity constants . ( b ) vn binding to the t - snare increases the on - rate of c - terminal assembly . the initial binding rate was plotted versus the concentration of t - snare according to eq 5 in experimental section to obtain the on - rate . the on - rate of vc assembling with preactivated t - snare is also rapid , with kon = ( 6 1 ) 10 m s. considering the concentration of snares between two docked membranes is 1 mm , the on - rate becomes 10 s ( time constant of 1 ms ) , which is very close to the rate measured by optical tweezers . this kinetics is of the same order of magnitude as the submillisecond scale kinetics of synaptic vesicle measured by electrophysiology studies . what we measured was an intermolecular binding , and this underestimated the real kinetics . under physiological conditions , vc and vn are located within a single molecule , and after vn prebinds the t - snare , c - terminal assembly will be an intramolecular binding , making the local concentration of vc even higher ( probably 0.1 m ) , and thus , the reaction will be even faster than the kinetics we measured here . hence the rapid c - terminal assembly of the snares should be capable of driving fusion at the time scale required in synaptic vesicle fusion . therefore , structuring of the t - snare c - terminal domain accelerates assembly of vc both energetically and kinetically by lowering the entropy of the t - snare as well the activation barrier of assembly , which is the mechanism underlying activation of fusion . we also examined the length requirements of the liposome - attached vc by systematically testing a series of vc - liposomes ( supporting information figure s1 ) in the fusion assay using flt - liposomes . when the flt - liposomes were not preincubated with vn , no specific fusion occurred for all these vc constructs ( supporting information figure s8 ) . when the t - snares were preassembled with vn ( figure 4a , supporting information figures s9 ) or with vn ( supporting information figures s10 and s11 ) to make sure that the t - snare was in the on state , the fusion capability of these vc - liposomes also exhibited an all - or - nothing behavior . all vc - liposomes containing layer + 1 ( vc - liposomes or longer ) fused with prestructured flt - liposomes with an elevated rate , while all vc - liposomes lacking layer + 1 ( vc - liposomes or shorter ) did not fuse with prestructured flt - liposomes . fusion results between flt - liposomes and vc - nanodiscs showed similar transition between layers + 1 and + 2 ( supporting information figure s12 ) . this shows that ( i ) the ctd - ld - tmd assembly that triggers fusion also behaves as a binary switch and ( ii ) layer + 1 has a critical role in fusion , without which fusion is completely abolished . the c - termini of snares are required to assemble from layer + 1 to their end to drive membrane merging . ( a ) fusion assay between flt - liposomes with prebound vn and various vc - liposomes : vc - liposome , vc - liposome , vc - liposome , vc - liposome , vc - liposome , vc - liposome , respectively . a sharp transition was observed between layers + 1 and + 2 . in the absence of layer ( b ) vc - liposomes and vc - liposomes were able to dock to flt - liposomes . in a his - tag pull - down , flt - liposomes were first incubated with vn or cdv at 37 c for 1 h , followed by incubation with various vc - liposomes at 37 c for 2 h , then pulled down by nickel - nta through the his - tag on snap25 . ( c ) a map of fusion activation that illustrates the sequence requirements for both n- and c - termini . two factors may be responsible for fusion incompetency of vc - liposomes or shorter : ( i ) a docking defect which means that vc - liposomes or shorter do not bind prestructured flt - liposomes ; ( ii ) fusion defect , where the energy obtained from zippering layer + 1 to tmd is just enough to overcome the energy barriers of c - terminal assembly and fusion , while zippering from layer + 2 to tmd does not provide enough energy . to determine which the dominant factor is , we performed a his - tag pull - down assay ( figure 4b ) . vc - liposomes contained no his - tag , while flt - liposomes were his - tagged . vc - liposomes were only pulled down when they docked on flt - liposomes . page analysis showed that the fusion - potent construct , vc - liposomes , and the fusion - incompetent construct , vc - liposomes , could both dock to flt - liposomes . even though the pull - down experiment can not prove that docking is quantitatively the same , this result suggests that fusion incompetency of vc - liposomes or shorter versions was most likely due to their inability to generate sufficient energy required for c - terminal assembly and fusion . by systematically testing the sequence requirements for n - terminal activation and c - terminal fusion , we are able to generate a comprehensive map of fusion activation ( figure 4c ) . the optimal combination is that flt - liposome is activated by vn and then fuses with vc - liposome . compared with the standard flv - liposome / flt - liposome fusion reaction , this pair results in 12-fold activation . membrane fusion ultimately requires the assembly of t- and v - snares into a four - helix bundle which brings the membranes into close proximity and triggers bilayer merging . formation of cis - snare complex was proposed to occur through continuous and progressive zippering from n - termini to c - termini and to culminate in a release of energy to drive membrane fusion . our data show that functionally , a two - step sequential zippering pathway is required in membrane fusion , and each step has its specific and distinct function ( figure 5 ) . in both steps , zippering exhibits all - or - nothing , binary - switch - like behavior . the first step is characterized by docking and t - snare structuring and requires t- and v - snares to zipper to at least layer 1 ( assembly of layer 7 to layer 2 or shorter can barely dock the v - snare to the t - snare because binding of vn or shorter versions to the t - snare is extremely weak , with affinities 10 m ) . the long - range effect of vn binding on the structure of the t - snare c - terminus can best be explained by an induced - fit conformational transition of the t - snare from a triple - helix to a four - helix bundle configuration . the second step defines actual fusion , where t- and v - snares assemble from layer + 1 to the transmembrane domain and energy generated from this step of zippering is used to overcome the fusion barrier . these findings are intrinsic properties of the snares ; in vivo , regulatory proteins such as synaptotagmin may help stalk formation and pore opening . whereas the fusion step occurs very rapidly our data suggest that the ionic layer , layer 0 , does not have a functional role in either of the two assembly steps . however , it is possible that it separates ntd and ctd - ld - tmd from each other . first the n - termini of snares assemble to at least layer 1 and switch the t - snare into fusion - ready conformation ( docking and structuring ) . then the c - termini of snares assemble ( starting at least from layer + 1 ) and provide energy to overcome the fusion barrier . the docking and structuring step is the rate - limiting step . in the conventional n - to - c zippering model , the very n - termini of the snares we show that n - terminal assembly has to reach the middles layers ( around layer 1 ) to induce a dramatic transition that ( i ) achieves a much higher binding affinity than that in the n - terminal layers , ( ii ) introduces a significant structural change in the t - snare , and ( iii ) facilitates c - terminal zippering of the snare complex . these data suggest that the middle layers are the critical part for assembly of the entire snarepin . previously , a soluble vc peptide ( vamp2 residues 5792 ) was found to accelerate fusion between flt - liposomes and flv - liposomes . proposed a molecular mechanism for this finding , suggesting that binding of vc to the t - snare displaced the n - terminal regulatory domain of syntaxin and opened up the t - snare . however , in the current view of the folding pathway of the snares , because of the topological constraints , the n - termini zipper first , then followed by c - terminal assembly . here we use vn peptides to prebind the t - snare and liposome - reconstituted vc to initiate fusion . this design perfectly matches the folding pathway of the snares because it is completely viable that physiologically full length vamp2 uses its n - terminal portion to bind and prestructure the t - snare and then further zippers up its c - terminal portion with the t - snare to drive fusion . hence , activation of fusion by vn , but probably not vc , is likely to be relevant under physiological conditions . other groups reported that vn peptides that contained layers 7 to 0 ( or longer ) inhibited assembly and fusion of full length snares . in our experiments , when flt - liposomes were prebound with vn ( supporting information figure s10 ) , the rate of fusion with flv - liposomes was about half the rate of the positive control ( fusion between flt - liposomes and flv - liposomes in the absence of vn ) , the result was inhibition , which is consistent with these reports . however , when flt - liposomes were prebound with vn ( supporting information figure s9 ) , the rate of fusion with flv - liposomes was about twice the rate of the positive control , the result was activation . there are two factors affecting the fusion rate : ( i ) prestructuring of the t - snare and ( ii ) overlap of residues between vn peptide and flv - liposomes . such overlap decreases the efficiency of collision and thus decreases fusion rate in a systematic manner . the overall effect is a combination of these factors . if flt - liposomes were prebound with vn , prestructuring was the dominant factor and the result was activation ; however , if flt - liposomes were prebound with vn or longer , more residues overlapped and this factor overcame the prestructuring , and the overall result was inhibition . supporting information table the significance of the two - step assembly pathway becomes apparent in the context of regulatory proteins that influence fusion rates . complexin has been suggested to promote t- and v - snare interaction by binding with its central domain to a groove formed by vamp2 and syntaxin , while its accessory domain binds the t - snare to block progression of fusion . the n - terminal switch allows recruiting complexin and creation of a clamped state , as both interactions occur with the half - zippered snare complex . the three aspartic acid residues , which are required by ca - dependent removal of the clamp , are located on the ctd of vamp2 ( between layers + 2 and + 4 ) . as soon as the snares zipper to around layer + 1 or + 2 , complexin switches to the closed conformation simultaneously . a further physiologically meaningful intermediate pause in the snare folding pathway can not exist after the action of complexin switch occurs , which indicates that the c - terminal zippering of the snares may happen as a single event . this is consistent with our finding that assembly of vc or shorter versions with the prestructured t - snare is not capable of driving fusion . these results suggest that it is unlikely that there is another relevant intermediate state in the c - terminal assembly step of the snares . the two - step assembly becomes both kinetically and thermodynamically observable in the presence of complexin . therefore , this switchlike , two - step folding pathway plays a critical role under physiological conditions and the half - zippered snare complex represents a previously unrecognized important intermediate stage of the snare assembly .

snare ( soluble n - ethylmaleimide - sensitive factor attachment protein receptor ) proteins mediate fusion by pulling biological membranes together via a zippering mechanism . recent biophysical studies have shown that t- and v - snares can assemble in multiple stages from the n - termini toward the c - termini . here we show that functionally , membrane fusion requires a sequential , two - step folding pathway and assign specific and distinct functions for each step . first , the n - terminal domain ( ntd ) of the v - snare docks to the t - snare , which leads to a conformational rearrangement into an activated half - zippered snare complex . this partially assembled snare complex locks the c - terminal ( ctd ) portion of the t - snare into the same structure as in the postfusion 4-helix bundle , thereby creating the binding site for the ctd of the v - snare and enabling fusion . then zippering of the remaining ctd , the membrane - proximal linker ( ld ) , and transmembrane ( tmd ) domains is required and sufficient to trigger fusion . this intrinsic property of the snares fits well with the action of physiologically vital regulators such as complexin . we also report that ntd assembly is the rate - limiting step . our findings provide a refined framework for delineating the molecular mechanism of snare - mediated membrane fusion and action of regulatory proteins .

Introduction Experimental Section Results Discussion

the essential and highly conserved snare ( soluble n - ethylmaleimide - sensitive factor attachment protein receptor ) proteins are the molecular machines that drive membrane fusion , which represents the final step in every trafficking pathway . the neuronal snare complex , consisting of vamp2 bound to the synaptic vesicle ( v - snare ) and syntaxin1a and snap25 on the target membrane ( t - snare ) , has been extensively studied and serves as a model system for understanding complex assembly and snare - mediated membrane fusion . a progressive zippering model was proposed for snare assembly , which suggests that the snare motifs from vamp2 , syntaxin1a and snap25 , zipper directionally from their membrane - distal n - terminal domains ( ntds ) to their c - terminal domains ( ctds ) into a four - helical coiled - coil bundle . interactions extend through the membrane - proximal linker domains ( lds ) and the transmembrane domains ( tmds ) helices of snares . a switch that activates the t - snare exists in ntd assembly of snare complex . ( c ) activation of the t - snare requires snares to assemble at least to layer 1 . ( d ) layer 1 is required for vn to bind tightly with the t - snare . here we demonstrate for the first time that even in the absence of any regulatory protein , a half - zippered snare complex represents a functional intermediate in a two - step folding process , and this intrinsic property of snares provides a molecular basis that supports the models put forward for the function of complexin . we show distinct functions for n- and c - terminal snare zippering , namely , prestructuring the t - snare and driving fusion , respectively . the full length t - snare complex , which includes full length , wild - type mouse his6-snap25 and rat syn1a , was produced by expression of the polycistronic plasmid ptw34 in the bl-21 gold ( de3 ) escherichia coli bacterial strain and purified as described before . the soluble t - snare complex , made of the cytoplasmic domain of rat syntaxin 1a ( residues 1265 ) and mouse his6-snap25 ( residues 1206 ) , was produced by coexpression of pjm57 and pjm72 plasmids in the bl-21 gold ( de3 ) escherichia coli bacterial strain and purified as described before . the plasmids for the vn variants were produced by cloning the n - terminus of vamp2 of various lengths into a pcdfduet-1 vector containing gst - prescission - vn ( containing mouse vamp2 n - terminal residues ) . the plasmids for the tm - vc variants were produced by cloning the c - terminus of vamp2 of various lengths into a pet sumo vector containing n - terminal his6 tag . cytosolic t - snare ( cdt ) of various concentrations was then added to each labeled vn peptide ( 200 nm ) . to obtain the kinetics and thermodynamics parameters , we consider the following binding reaction : the kinetics equation is1where kon is the on - rate , koff is the off - rate , and [ v ] , [ t ] , and [ vt ] are the concentrations of vamp2 peptide , t - snare , and snare complex at time t , respectively . hence , eq 3 can be simplified as4combining eqs 2 and 4 , we obtain5to obtain kon , we performed a series of reactions that labeled vamp2 peptide binds to t - snare at various initial concentrations , t0 , and monitored the variation of a with t. for each t0 , we obtained the initial rate da / dt from the a versus t curve , then plotted ( da / dt)/(avt av ) versus t0 . let kd be the affinity constant , ap be the measured anisotropy at equilibrium , and [ v]p , [ t]p , and [ vt]p be the concentrations of vamp2 peptide , t - snare , and snare complex at equilibrium , respectively . then67solving eq 6 for [ vt]p , and then entering into eq 7 , we have8or9by changing the initial concentration of t - snare , t0 , while keeping v0 constant , one can obtain a curve of ap as a function of t0 . the model for complexin clamping is that complexin binds the half - assembled , intermediate snare complex and arrests further zippering and that upon clamp release , the zippering of the c - terminal portions provides sufficient energy to drive fusion ( figure 1a ) . however , fusion was restored when covalently separated n - terminal portion of the v - snare ( vn ) was added . these results show that ( i ) the c - terminal ( ctd - ld - tmd ) assembly of snares provides sufficient energy needed to drive fusion whereas the energy from the n - terminal assembly has no direct contribution to fusion and ( ii ) n - terminal assembly with the t - snare is a prerequisite to enable fusion driven by ctd - ld - tmd assembly . ntd binding switches the t - snare to an activated state that must be reached before fusion can occur . therefore , the process of snare - mediated fusion can be functionally divided into two distinct and sequential steps , and the fusion in the positive control with full length snares follows the same two - step pattern : the ntd of snares assembles first and activates the t - snare , after which the c - termini assemble to drive fusion . this shows that ( i ) c - terminal assembly of snares is rapid and not rate - limiting and that ( ii ) the n - terminal assembly with the t - snare is the rate - limiting factor . the binding curves in figure 1d show a similar transition regarding the sequence of vn : vn , vn , and vn bound tightly to the t - snare , and the binding constants for these peptides are virtually identical ( 70 to 100 nm ) ; however , vn and vn exhibited a dramatic reduction in affinity , with affinity constants of 10 m , showing that truncations of layer 1 of vamp2 lead to a large loss of binding to the t - snare . only upon the binding of vn or longer versions is the t - snare switched on for fusion . to investigate the molecular basis for the n - terminal activation , we compared the crystal structures of a postfusion , fully zippered snare complex and a partially assembled , half - zippered complex . the partially assembled snare complex mimicked an intermediate folding state with a vn peptide , vn , which was reconstituted into a complex with the complete t - snare motifs . the crystal structure of the resulting half - zippered snare complex ( snare ) showed that surprisingly the snare motifs of syntaxin and snap25 almost entirely adopt an -helical conformation ( figure 2a ) : even though the c - terminal half of the complex only consists of three helices , they still display exactly the same four - helix bundle configuration as in the fully zippered complex . previous studies have shown that the binary syntaxin / snap25 t - snare complex is unstructured in its c - terminal portion , implying together with our data that binding of vamp2 ntd to the t - snare triggers a binary switch that propagates the four - helix bundle geometry to the t - snare c - terminal domain ( a ) binding of vn prestructures the c - terminus of the t - snare , as seen in a comparison of the crystal structures of the postfusion snare complex ( left ) and a partially assembled complex ( right ) . the partially assembled snare complex contains a peptide vn binding to the t - snare motif , and similar to the postfusion complex , it also shows four - helix structure on both n- and c- termini , even though only three helices are present on its c - terminus . the transition from the partially unstructured t - snare to the folded snare core complex can be monitored using circular dichroism ( cd ) where the higher helical content of the ternary complex leads to a strong increase in ellipticity ( figure 2b ) . importantly , the truncated snare complex adopts a state of intermediate folding / helicity more similar to the snare core than the t - snare . taken together , our observations show that activation of fusion , peptide binding , and induction of helicity are all coupled in an all - or - nothing transition , revealing that the molecular basis of this binary switch is the structuring of the c - terminal domain of the t - snare . specifically , binding of vn induces the three helices of the t - snare ctd to adopt the same configuration as in the fully assembled , postfusion complex , resulting in a preformed binding site for the fourth helix , the c - terminal domain of vamp2 . the n - terminal activation of the snares profoundly impacts the assembly and fusion on their c - termini . to accurately quantify this effect , we used fluorescence anisotropy measurements and monitored the rate of assembly of a soluble vc peptide , vc ( which includes vamp2 residues 5894 , layers + 1 to + 8 plus linker domain ) , with soluble , cytosolic portion of the t - snare in the presence and absence of vn in real time ( figure 3a and figure 3b , supporting information figures s6 and s7 ) . in the absence of vn , rate of binding of vc to the t - snare is slow ( kon = ( 9 1 ) 10 m s ) and thermodynamically less favorable ( kd = 352 60 nm ) , which corresponds to a free energy of 14.9 0.2 kbt , where kb is the boltzmann constant . when the t - snare was switched on upon binding vn , the kinetics of c - terminal domain assembly was increased 70 times ( now kon = ( 6 1 ) 10 m s ) and affinity of vc was increased by 30-fold to kd = 12 2 nm , corresponding to a free energy 18.3 0.2 kbt . structuring of the t - snare facilitates c - terminal assembly both energetically and kinetically . fluorescence anisotropy experiments were performed to monitor the binding process of vc ( vamp2 residues 5894 ) to the cytosolic t - snare at various concentrations with and without prebound vn peptide in real time . ( a ) vn binding to the t - snare improves binding affinity of vc . ( b ) vn binding to the t - snare increases the on - rate of c - terminal assembly . the on - rate of vc assembling with preactivated t - snare is also rapid , with kon = ( 6 1 ) 10 m s. considering the concentration of snares between two docked membranes is 1 mm , the on - rate becomes 10 s ( time constant of 1 ms ) , which is very close to the rate measured by optical tweezers . under physiological conditions , vc and vn are located within a single molecule , and after vn prebinds the t - snare , c - terminal assembly will be an intramolecular binding , making the local concentration of vc even higher ( probably 0.1 m ) , and thus , the reaction will be even faster than the kinetics we measured here . hence the rapid c - terminal assembly of the snares should be capable of driving fusion at the time scale required in synaptic vesicle fusion . therefore , structuring of the t - snare c - terminal domain accelerates assembly of vc both energetically and kinetically by lowering the entropy of the t - snare as well the activation barrier of assembly , which is the mechanism underlying activation of fusion . when the t - snares were preassembled with vn ( figure 4a , supporting information figures s9 ) or with vn ( supporting information figures s10 and s11 ) to make sure that the t - snare was in the on state , the fusion capability of these vc - liposomes also exhibited an all - or - nothing behavior . membrane fusion ultimately requires the assembly of t- and v - snares into a four - helix bundle which brings the membranes into close proximity and triggers bilayer merging . formation of cis - snare complex was proposed to occur through continuous and progressive zippering from n - termini to c - termini and to culminate in a release of energy to drive membrane fusion . our data show that functionally , a two - step sequential zippering pathway is required in membrane fusion , and each step has its specific and distinct function ( figure 5 ) . the first step is characterized by docking and t - snare structuring and requires t- and v - snares to zipper to at least layer 1 ( assembly of layer 7 to layer 2 or shorter can barely dock the v - snare to the t - snare because binding of vn or shorter versions to the t - snare is extremely weak , with affinities 10 m ) . the long - range effect of vn binding on the structure of the t - snare c - terminus can best be explained by an induced - fit conformational transition of the t - snare from a triple - helix to a four - helix bundle configuration . the second step defines actual fusion , where t- and v - snares assemble from layer + 1 to the transmembrane domain and energy generated from this step of zippering is used to overcome the fusion barrier . these findings are intrinsic properties of the snares ; in vivo , regulatory proteins such as synaptotagmin may help stalk formation and pore opening . first the n - termini of snares assemble to at least layer 1 and switch the t - snare into fusion - ready conformation ( docking and structuring ) . the docking and structuring step is the rate - limiting step . in the conventional n - to - c zippering model , the very n - termini of the snares we show that n - terminal assembly has to reach the middles layers ( around layer 1 ) to induce a dramatic transition that ( i ) achieves a much higher binding affinity than that in the n - terminal layers , ( ii ) introduces a significant structural change in the t - snare , and ( iii ) facilitates c - terminal zippering of the snare complex . proposed a molecular mechanism for this finding , suggesting that binding of vc to the t - snare displaced the n - terminal regulatory domain of syntaxin and opened up the t - snare . however , in the current view of the folding pathway of the snares , because of the topological constraints , the n - termini zipper first , then followed by c - terminal assembly . here we use vn peptides to prebind the t - snare and liposome - reconstituted vc to initiate fusion . this design perfectly matches the folding pathway of the snares because it is completely viable that physiologically full length vamp2 uses its n - terminal portion to bind and prestructure the t - snare and then further zippers up its c - terminal portion with the t - snare to drive fusion . in our experiments , when flt - liposomes were prebound with vn ( supporting information figure s10 ) , the rate of fusion with flv - liposomes was about half the rate of the positive control ( fusion between flt - liposomes and flv - liposomes in the absence of vn ) , the result was inhibition , which is consistent with these reports . however , when flt - liposomes were prebound with vn ( supporting information figure s9 ) , the rate of fusion with flv - liposomes was about twice the rate of the positive control , the result was activation . there are two factors affecting the fusion rate : ( i ) prestructuring of the t - snare and ( ii ) overlap of residues between vn peptide and flv - liposomes . supporting information table the significance of the two - step assembly pathway becomes apparent in the context of regulatory proteins that influence fusion rates . complexin has been suggested to promote t- and v - snare interaction by binding with its central domain to a groove formed by vamp2 and syntaxin , while its accessory domain binds the t - snare to block progression of fusion . the n - terminal switch allows recruiting complexin and creation of a clamped state , as both interactions occur with the half - zippered snare complex . a further physiologically meaningful intermediate pause in the snare folding pathway can not exist after the action of complexin switch occurs , which indicates that the c - terminal zippering of the snares may happen as a single event . these results suggest that it is unlikely that there is another relevant intermediate state in the c - terminal assembly step of the snares . therefore , this switchlike , two - step folding pathway plays a critical role under physiological conditions and the half - zippered snare complex represents a previously unrecognized important intermediate stage of the snare assembly .

in 1951 , sir william dameshek postulated the concept of myeloproliferative disorders and furthermore ascribed their development to a hitherto of undiscovered stimulus . the past half - century has since brought light to the cryptic stimulus believed to drive these disabling neoplasms . developing from a host of myelostimulatory mutations , myeloproliferative neoplasms ( mpns ) including polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and myelofibrosis ( mf ) propagate through an evolving cascade of inflammatory conduits well documented to inflict dramatic symptomatology and impair quality of life . great gains have been made in our understanding of how these disrupted signaling pathways coalesce into dysregulated synthesis of cytokines , chemokines , and reactive species that ultimately induce symptoms . in this paper , we discuss the role inflammation plays in mpn pathobiology , disease advancement , and symptom development . the burdensome symptom profile is arguably the most recognizable feature of the mpn disease process and in itself may contribute to reduced life expectancy , as observed in myelofibrosis risk scoring [ 1 , 2 ] . prominent symptoms include fatigue ( 92.7% ) , early satiety ( 61.9% ) , abdominal pain ( 45.9% ) , abdominal discomfort ( 53.2% ) , inactivity ( 60.5% ) , headache ( 48.3% ) , concentration problems ( 61.7% ) , dizziness ( 55.2% ) , numbness ( 61.3% ) , insomnia ( 65.4% ) , sad mood ( 62.7% ) , sexuality problems ( 57.9% ) , cough ( 46.4% ) , night sweats ( 56.4% ) , itching ( 52.6% ) , bone pain ( 48.5% ) , fever ( 20.2% ) , weight loss ( 34.2% ) , and impaired quality of life ( 84.2% ) . the mpn symptom burden has been closely examined for its impact on patient daily living through the mpn landmark survey . this study systematically surveyed 813 mpn patients and discovered that mpn symptoms negatively impacted work hours , number of sick days taken , the need for medical disability and/or early retirement , and overall activities of daily living . patients additionally described feeling anxious and worried about their conditions ( mf , 91% ; pv , 78% ; et , 74% ) which in turn compromised overall quality of life ( mf , 81% ; pv , 66% ; et , 57% ) . adding to the complexity is the recent revelation that mpn symptoms indeed promote the development of other symptoms . an investigation of the symptom of insomnia revealed that the complaint correlates closely with most other mpn related symptoms and functional domains . a similar study investigating correlations with mpn - related sexuality complaints found that this symptom also correlated with other mpn symptoms ( insomnia , depression / sad mood , night sweats , and qol ) , as well as emotional , cognitive , and social domains of functioning . it has been well recognized that the prevalence and severity of symptoms differ by mpn subtype . however , more recent studies have demonstrated that significant heterogeneity exists even within mpn subtypes . a prospective evaluation of 1470 mpn patients discovered the presence of five clusters in pv and et , respectively , and four clusters in mf . symptom clusters in et and pv differed by clinical variables including age , language , gender , the presence of laboratory abnormalities , spleen size , history of hemorrhage , and myeloproliferative neoplasm symptom assessment form total symptom score ( mpn - saf tss ) value . notably , neither pv nor et clusters differed by risk scores suggesting symptomatology likely presents independent of disease stage and risk scoring tools should not be applied as surrogate measurements of disease severity . in mf , clusters differed by a variety of clinical variables as well as risk scores ( dipss ) with increasing degrees of symptomatology correlating with higher risk score categories . recent efforts have aimed to analyze the scope and extent of mpn symptoms in a systematic format . the first investigation was completed in 2007 as a self - reported internet survey of 1179 mpn patients . this revealing study showed that fatigue , pruritus , bone pain , fevers , and weight loss led to restricted participation in physical and social functions and furthermore that available treatment regimens including androgens , steroids , hydroxyurea , and erythropoiesis - stimulating agents not only failed to improve the symptom burden but paradoxically contributed to its development . this survey served as a benchmark for the development of three mpn - specific pro tools : mf - saf , mpn - saf , and mpn - saf tss / mpn-10 . from these instruments , below we discuss these tools individually ( table 1 ) . the myelofibrosis symptom assessment form ( mf - saf ) was created in 2009 and served as the first validated mpn patient reported outcome ( pro ) tool to be made available for clinical and trial settings . a 20-item instrument , the survey attempted to capture the most common symptoms within myelofibrosis and content included issues related to catabolic / proliferative symptoms , quality of life , fatigue , and splenomegaly - associated issues . questions were constructed in a yes , no , or 0 ( absent ) to 10 ( worst imaginable ) scale . the tool proved useful in the open label phase ii trial of the jak2 inhibitor , ruxolitinib . the myeloproliferative neoplasm symptom assessment form ( mpn - saf ) was developed two years later in efforts to capture the symptoms within pv and et as well as mf . this survey included the items present within the mf - saf , along with questions related to microvasculature complications such as headaches , concentration problems , lightheadedness , dizziness , vertigo , numbness / tingling , and sexual dysfunction . this expanded version was structured in a similar format to the mf - saf and proved beneficial in evaluation of a variety of novel targeted compounds including ruxolitinib , ly2784544 , sar302503 , vorinostat , and pegylated interferon [ 1113 ] . the myeloproliferative neoplasm symptom assessment form total symptom score ( mpn - saf tss ; mpn-10 ) is an abbreviated version of the mpn - saf containing the 10 most symptomatic and pertinent items . this tool allows for rapid administration in clinical and trial formats and has replaced the mpn - saf in most settings . the survey has been successfully cross - validated against the eortc qlq - c30 and is available in a variety of languages including english , italian , german , french , mandarin , arabic , spanish , dutch , swedish , portuguese , japanese , hebrew , and czech . in healthy individuals , the inflammatory cascade is driven by a delicate interplay between cellular responses and neurohormonal stimulatory factors / cytokines . although the initial inciting event has yet to be clearly elucidated , all mpn disorders arise from genetic defects within pluripotent stem cell populations that accumulate over the disease course . jak2v617f , a member of the janus kinase signal transduction pathway , was the first recognized mutation inherent to the mpn population ( pv 96% , et 50% , and mf 50% ) . the role the janus kinase cascade ( including jak2 , jak2 , jak3 , and tyk2 ) plays in the signaling of inflammatory cytokines is well documented and profound . as jaks are essential to the signaling of surface growth factor receptors and cytokines bereft of intrinsic kinase activity , constitutive activation , as observed in jak2v617f , induces unregulated signaling of stat transcription factors with resultant cellular growth and propagation . stat3 , in particular , is linked closely with cancer development via activation of immunomodulatory cytokines ( il-6 , il-10 , and il-17 ) , growth factors ( fgf , vegf ) , and matrix metalloproteinases . these products further induce positive autofeedback through the jak / stat pathway , perpetuating cellular malignant potential . in general , cytokines ( interleukins , interferons , and soluble growth factors ; definitions in table 2 ) are important regulators of cellular processes , particularly those involving immunomodulatory activities , cellular growth angiogenesis , and migration . cytokine dysregulation is believed to be associated with other mutations observed within mpns ( idh1/2 , tet2 ) but requires additional investigation . chronic inflammation has been hypothesized to play a supportive role in oncogenesis given its promotion of genomic instability through dna mutations and epigenetic changes , prevention of tumor immune surveillance , and encouragement of clonal evolution [ 17 , 20 , 21 ] . mpn cells ( leukocytes , platelets ) with inherent hypersensitivity to cytokines and or growth factors respond in a proliferative fashion with resultant production of more stimulatory factors . as chronic inflammatory conditions , mpn disorders revolve around a perpetual cycle of dna damage , cellular remodeling , and subsequent fibrosis . this process has been a topic of great interest , especially as it relates to the heterozygous clinical presentation of mpn patients . the relationship between chronic inflammation and mpn symptom development has also been a topic of recent interest . an evaluation of abnormal cytokine expression within myelofibrosis determined that primary myelofibrosis ( pmf ) patients had significantly increased levels of il-1b , il-1ra , il-2r , il-6 , il-8 , il-10 , il-12 , il-13 , and il-15 and tnf-1 , g - csf , ifn- , mip-1 , mip-1 , hgf , inf--ip , and vegf in addition to reduced ifn- levels . il-2r , il-12 , il-15 , and ip-10 were independently predictive of inferior survival . il-2r and il-12 were associated with transfusion needs and hgf , mig , and il-1ra were associated with marked splenomegaly . evaluation of the association between cytokines and mf symptoms was undertaken in 2013 through an ad hoc analysis of 309 mf patients during the blinded phase of the comfort-1 trial evaluating ruxolitinib against placebo . changing levels of five cytokines was significantly associated with change in the mpn - saf tss when controlled for arm , visit - by - arm interaction , age , sex , and body mass index ( bmi ) . , it has previously been shown within other malignancies to promote angiogenesis , induce leukocyte chemotaxis / activation , and stimulate cellular reproduction . a recent study determined il-8 to be associated with elevated levels of circulating blasts and the presence of constitutional symptoms . in polycythemia vera , patients demonstrate increased levels of il-1ra , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12 , il-13 , ifn- , gm - csf , mcp-1 , mip-1 , mip-1 , hgf , ip-10 , mig , mcp-1 , pdgf - bb , tnf- , ifn- , and vegf [ 25 , 26 ] . pv patients also had significantly elevated levels of il-7 , gm - csf , mip-1 , ip-10 , mig , eotaxin , ifn- , and vegf in comparison to primary mf patients . in addition , hemoglobin count correlated with il-4 and mcp-1 , hematocrit count correlated with tnf- and mcp-1 , lymphocyte count correlated with il-6 and tnf-1 , and jak2v617f mutation status correlated with tnf-1 and pdgf - bb . an analysis of et patients determined this population to have elevated levels of il-1b , il-4 , il-6 , il-8 , il-10 , il-12 , hgf , gm - csf , ifn- , mcp-1 , pdgf - bb , tnf- , and vegf . interestingly , il-4 , il-8 , gm - csf , ifn- , mcp-1 , pdgf - bb , and vegf appeared to be significantly higher in et patients when compared to pv populations and may serve as useful markers to distinguish the two disorders . also within et patients , polynuclear cell counts were found to correlate with hgf , il-6 , il-12 , mg - csf , and vegf whereas red cell counts correlated with pdgf - bb levels . jak2v617f positive status also correlated with pdgf - bb and tnf- . in comparing pv and et patients with vascular complications versus those without complications , no significant differences in cytokine levels however , in comparing pv and et patients with a history of vascular events , et patients have significantly increased levels of il-4 , il-8 , gm - csf , ifn- , mcp-1 , and vegf . interestingly , the specific combination of inflammatory markers appears to be as important as the type of factor present . in mf , the combination of tnf- and timp-1 has been shown to promote survival of cd34 + stem cells whereas the combination of atp and tnf- has been shown to reduce proliferation . for example , pentraxin and crp are well established to play a role in thrombosis and atherogenesis . these biomarkers have been associated with the development of major thrombotic events in pv and et . a recent study also identified low levels of il-12 in mpn patients with vascular complications . altered levels of pdgf , fgf , and vegfb have also been noted in stromal cells of patients with pv , et , and pmf suggesting proinflammatory cytokines promote bone marrow fibrosis which is well established to contribute to anemia and subsequently fatigue [ 29 , 30 ] . cytokines ( bmp-1 , bmp - r2 , bmp-6 , and bmp-7 ) may have a role in promoting the advancement of mpns from early to later stages . of special interest , the presence of specific gene mutations impacts the type and degree of cytokine expression . for instance , jak2v617f positive patients have significantly higher levels of il1b , il-8 , il-17a , and ifn versus triple - negative ( jak2 , mpl negative ) patients . much has yet to be learned about the role cytokines play in mpn symptom development . the growing availability of cost - sensitive cytokine profile testing has offered us what can best be recognized as preliminary data on this complex topic . below we discuss the available literature on specific mpn symptoms and their relationships to inflammation ( table 3 ) . fatigue is a common complaint among cancer patients , present within 3060% of the cancer population . the symptom is particularly prominent within mpns ( pv 85% , et 72% , and mf 84% ) , representing the most common symptom voiced regardless of subtype . mpn - fatigue has been shown to correlate closely with functional capacities . in a novel evaluation of mpn patient functionality , participants were found to perform an average of 25.1 metabolic equivalents ( mets ) , akin to scores observed in parkinson disease patients and dramatically lower than healthy controls ( 45.8 mets ) . a recent study identified positive associations between tnf- and postchemotherapy fatigue in women with breast cancer . anemia is a recognized contributor to this symptom and recent studies have demonstrated that cytokines play a critical role in the development and perpetuation of this comorbidity . il-1 , il-6 , and tnf were recently shown to promote deregulation of erythropoietin with resultant anemia in acute myelogenous leukemia ( aml ) and myelodysplastic syndromes ( mds ) [ 35 , 36 ] . cytokines have been shown to induce dysregulation of the hpa axis and promote a blunted stress response due to subtherapeutic cortisol production . in cancer , fatigue has been closely associated with depressed mood and increased levels of il-6 , a cytokine observed within mpns [ 38 , 39 ] . a survey of 1788 mpn patients confirmed that 32% have been seen or diagnosed with depression and 22.2% had received active treatment of mood disorder within the prior six months suggesting potential association in this population . the combination of cancer - related depression and fatigue also contributes to a sedentary lifestyle which further encourages a proinflammatory state that propagates symptoms . multiple randomized controlled trials have demonstrated that cancer - related fatigue may be reduced through aerobic physical activity , potentially through modulation of cytokine production . the mechanism has yet to be elucidated as intense physical activity has been shown to increase circulating levels of il-6 which subsequently stimulates the production of other anti - inflammatory cytokines including il-1ra and il-10 , and inhibit proinflammatory cytokines such as tnf-. the mpn fatigue project is an international effort performed in collaboration with the mpn forum aiming at evaluating the breadth and efficacy of current strategies targeting mpn fatigue . the study remains ongoing and includes evaluation of treatments such as sleep deprivation , dietary supplements , and exercise . abdominal - related complaints are common among mpn patients , largely attributable to splenomegaly , portal hypertension , mechanical obstruction , and splenic infarcts . an independent source of morbidity and mortality complaints related to the abdomen has included early satiety ( 76% ) , abdominal pain ( 63% ) , abdominal discomfort ( 72% ) , and weight loss ( 48% ) . however , cytokines may also play an important role in the development of this symptom . splenomegaly has been associated with expansion of the malignant clone from the bone marrow microenvironment to extramedullary sites including the spleen . tnf- , in particular , promotes clonal expansion in jak2v617f positive mpns [ 42 , 43 ] . the development of splenomegaly has also been associated with specific cytokines including mig , hgf , and il-1ra . however , interestingly , jak2/stat3 signaling has been shown to promote fibrosis , angiogenesis , and inflammation in the setting of portal hypertension , independent of the presence of malignancy suggesting that regional inflammation also plays a role in abdominal pain , whether or not cancer is present . thrombosis may result in a variety of abdominal complaints , particularly with et and pv . an evaluation of 244 consecutive pv and et patients demonstrated that patients within the highest crp protein tertile had the highest rate of major thrombotic events . similarly patients demonstrating the lowest pentraxin 3 levels had higher risks for major thrombotic events . of interest , values of hs - crp and ptx3 may also be exacerbated by cytokine - induced nerve hyperstimulation , both peripherally and centrally . animal studies have shown increased expression of tnf- , il-1 , and il-6 after nerve injury , cytokines all disproportionately high in the mpn population . in addition , inflammation and trauma have been shown to induce peripheral nerve cell release of inflammatory cytokines within the central nervous system via glial stimulation . the effects of cytokine - mediated pain were demonstrated in a recent study of patients with painful neuropathies where it was observed that this population had twofold higher levels of il-2 mrna and tnf- mrna in comparison to healthy controls . the direct impact these cytokines have on nerves within mpn populations has yet to be investigated . microvascular complaints typically refer to those symptoms that result from disease activity occurring at a capillary level . in the mpns , these symptoms may include headaches , concentration problems , lightheadedness , dizziness , vertigo , numbness / tingling , and sexual dysfunction . historically , neurocognitive disturbances have been attributed to cellular stasis and microthrombosis . proinflammatory cytokine production is believed to be a contributor to cognitive impairment in cancer patients via the disruption of neurohormonal signaling and impaired creation of neurotransmitters . these neurotransmitters include serotonin , dopamine , and norepinephrine , all of which are critical to functions involving homeostasis of sleep , mood , and memory [ 4749 ] . a recent analysis of patients treated with ruxolitinib in the comfort - ii trials identified rantes and pal1 levels to correlate with the complaints of insomnia . the role of inflammation in cognitive impairment has been intensely studied in both animal and human models . in an evaluation of il-6 deficient animals , injection of lipopolysaccharide ( lps , shown to inhibit memory and learning in animals ) failed to induce cognitive impairments suggesting il-6 plays a key role in interrupting the process of memory and learning . in human studies , elevated levels of il-1 , tnf- , il-6 , and crp were also linked to impaired memory and neurodegenerative disorders in the elderly [ 5153 ] . within hematological disorders , patients with elevated levels of il-6 were found to have worsened executive function . interestingly , aml and mds patients with higher levels of il-8 were found to have improved memory . a recent study of jak2v617f transgenic mice demonstrated increased number of mast cells in those with the pv phenotype . these mast cells represent a key source of prostaglandin , leukotriene , histamine , and tryptase , mediators of the inflammatory response with recognized ties to pruritus . a study evaluating symptoms of ruxolitinib treated patients within the comfort - ii trial determined that baseline pruritus was associated with lower ferritin levels , a surrogate marker of inflammation . pruritus , most prominent in pv patients ( 65% ) , may be tied to an inflammatory cellular response as well . basophils have been instigated as a primary mediator for symptom development and studies have demonstrated that the number of constitutively activated and hypersensitive circulating basophils is increased in pv , correlating with the degree of pruritus . recent studies using infrared thermography have documented mast cell degranulation due to temperature shifts with the release of pyrogenic factors such as interleukins , histamine , and leukotrienes [ 56 , 57 ] . both fevers and night sweats are recognized to be partially cytokine driven , typically by il-1 , il-2 , il-6 , tnf- , and ifn . within other malignancies , il-6 has been found to correlate with the presence of b symptoms which serve as a prognostic factor in chronic lymphocytic leukemia ( cll ) and hodgkin 's lymphoma [ 5860 ] . the development of mpn - associated weight loss is complex and relates to a variety of factors including splenomegaly , portal hypertension , and cancer - cachexia . the role of a cytokine - driven proinflammatory state as the nidus for cancer - cachexia is well supported by literature [ 61 , 62 ] . defined as a process of dysregulated carbohydrate and fat metabolism with ongoing skeletal muscle breakdown , the presence of cancer - cachexia is linked to a dismal survival rate in comparison to cancer patients lacking this feature . in cancer patients , tnf- has been shown to induce proteolysis of skeletal muscle and furthermore enhance the expression of genes related to enzymes in the ubiquitin - dependent proteolytic pathway . in a review of cytokine levels present in patients from the comfort - ii trial , weight loss was associated with lower leptin levels and high cd40l was associated with loss of appetite . whether these aberrant cytokines function to support cancer - cachexia or involve an alternative mechanism of action has yet to be investigated . recognizing the substantial impact chronic inflammation has on the mpn symptom burden , attentions have turned to therapies demonstrating efficacy in reducing cytokines . as in other malignancies , improving physical activity and reducing fat intake may reduce inflammatory cytokines and improve survival . in noncancer patients , increased physical activity has been shown to reduce tlr4 signaling and truncate release of inflammatory cytokines . in obese subjects , proinflammatory cytokines have also been shown to be released by white fat which may be subsequently removed through physical activity . in addition to evaluating the effects of sedentary living on the mpn symptom burden , the final phases of the mpn fatigue project involve the development of comprehensive patient activity programs which may have subsequent impact on cytokine - induced symptomatology . recognizing the role constitutive janus kinase signaling plays in inflammation , jak2 inhibition has become a rational target for preventing cytokine dysregulation . a recent study evaluated meaningful changes in cytokine expression following 24 months of ruxolitinib therapy in 63 high - risk mf patients . ruxolitinib was able to induce profound reductions in the expression of tnf- and mip-1 at both 4 weeks and 24 months . the expression of ige was also strongly reduced in almost all patients with direct impact on the amount of activated anti - inflammatory macrophages . a similar study utilized the functional assessment of cancer therapy - lymphoma ( fact - lym ) to evaluate symptoms of ruxolitinib treated patients within the comfort ii trial and compare them to changes in cytokine levels . ten symptoms including fever , weight loss , fatigue , loss of appetite , pain , itching , sleeping well , lack of energy , night sweats , and trouble sleeping were assessed at baseline and weeks 8 , 24 , and 48 . treatment with ruxolitinib led to improved items of itching , night sweats , and weight loss with subsequent reduction in numerous cytokines . loss of appetite improved over time and negatively correlated with decreases in il-1ra levels in ruxolitinib treated patients . the impact emerging jak2 inhibitors such as momelotinib and pacritinib will have on cytokines is of high interest as they progress through clinical trials . importantly , their limited hematological toxicities may be reflective of their selective inhibition of kinases . a recent investigation of pacritinib demonstrated that it selectively spares jak1 while inhibiting jak2 , jak2v617f , flt3 , and irak1 , an il-1 receptor kinase associated with the inflammatory response and suppression of normal hematopoiesis . whether inhibition of irak1 is of clinical significance from a symptomatic standpoint has yet to be investigated . other cellular signaling networks such as the pi3k - akt - mtor pathway impact cytokine development and represent novel targets for intervention . similarly , as antifibrosing agents , hedgehog inhibitors , hypomethylating agents , histone deacetylation inhibitors , and hsp90 inhibitors enter the treatment landscape , knowledge of their impact on inflammation is of great interest . though most data remains in early stages of investigation , knowledge gleaned from other malignancies has offered us potential mechanisms that explain these observed cytokine - symptom associations . however , how these markers differ between mpn subtypes , change with disease progression , and relate to transformation remain unknown . the expanded access to targeted agents has provided a platform by which cytokine signals may be inhibited early within the cascade , limiting their potential for toxicity . it is with great anticipation that we venture into this uncharted territory of cytokine - symptom associations and explore novel therapies hosting high potential for symptomatic benefit .

myeloproliferative neoplasms ( essential thrombocythemia , et ; polycythemia vera , pv ; myelofibrosis , mf ) are monoclonal malignancies associated with genomic instability , dysregulated signaling pathways , and subsequent overproduction of inflammatory markers . acknowledged for their debilitating symptom profiles , recent investigations have aimed to determine the identity of these markers , the upstream sources stimulating their development , their prevalence within the mpn population , and the role they play in symptom development . creation of dedicated patient reported outcome ( pro ) tools , in combination with expanded access to cytokine analysis technology , has resulted in a surge of investigations evaluating the potential associations between symptoms and inflammation . emerging data demonstrates clear relationships between individual mpn symptoms ( fatigue , abdominal complaints , microvascular symptoms , and constitutional symptoms ) and cytokines , particularly il-1 , il-6 , il-8 , and tnf-. information is also compiling on the role symptoms paradoxically play in the development of cytokines , as in the case of fatigue - driven sedentary lifestyles . in this paper , we explore the symptoms inherent to the mpn disorders and the potential role inflammation plays in their development .

1. Introduction 2. Characterization of MPN Symptoms 3. Origins of Inflammation in MPN Patients 4. Inflammation and MPN Symptom Development 5. Improving Symptoms by Targeting Cytokines 6. Conclusion

in 1951 , sir william dameshek postulated the concept of myeloproliferative disorders and furthermore ascribed their development to a hitherto of undiscovered stimulus . the past half - century has since brought light to the cryptic stimulus believed to drive these disabling neoplasms . developing from a host of myelostimulatory mutations , myeloproliferative neoplasms ( mpns ) including polycythemia vera ( pv ) , essential thrombocythemia ( et ) , and myelofibrosis ( mf ) propagate through an evolving cascade of inflammatory conduits well documented to inflict dramatic symptomatology and impair quality of life . great gains have been made in our understanding of how these disrupted signaling pathways coalesce into dysregulated synthesis of cytokines , chemokines , and reactive species that ultimately induce symptoms . in this paper , we discuss the role inflammation plays in mpn pathobiology , disease advancement , and symptom development . the burdensome symptom profile is arguably the most recognizable feature of the mpn disease process and in itself may contribute to reduced life expectancy , as observed in myelofibrosis risk scoring [ 1 , 2 ] . prominent symptoms include fatigue ( 92.7% ) , early satiety ( 61.9% ) , abdominal pain ( 45.9% ) , abdominal discomfort ( 53.2% ) , inactivity ( 60.5% ) , headache ( 48.3% ) , concentration problems ( 61.7% ) , dizziness ( 55.2% ) , numbness ( 61.3% ) , insomnia ( 65.4% ) , sad mood ( 62.7% ) , sexuality problems ( 57.9% ) , cough ( 46.4% ) , night sweats ( 56.4% ) , itching ( 52.6% ) , bone pain ( 48.5% ) , fever ( 20.2% ) , weight loss ( 34.2% ) , and impaired quality of life ( 84.2% ) . this study systematically surveyed 813 mpn patients and discovered that mpn symptoms negatively impacted work hours , number of sick days taken , the need for medical disability and/or early retirement , and overall activities of daily living . adding to the complexity is the recent revelation that mpn symptoms indeed promote the development of other symptoms . a similar study investigating correlations with mpn - related sexuality complaints found that this symptom also correlated with other mpn symptoms ( insomnia , depression / sad mood , night sweats , and qol ) , as well as emotional , cognitive , and social domains of functioning . symptom clusters in et and pv differed by clinical variables including age , language , gender , the presence of laboratory abnormalities , spleen size , history of hemorrhage , and myeloproliferative neoplasm symptom assessment form total symptom score ( mpn - saf tss ) value . recent efforts have aimed to analyze the scope and extent of mpn symptoms in a systematic format . this revealing study showed that fatigue , pruritus , bone pain , fevers , and weight loss led to restricted participation in physical and social functions and furthermore that available treatment regimens including androgens , steroids , hydroxyurea , and erythropoiesis - stimulating agents not only failed to improve the symptom burden but paradoxically contributed to its development . this survey served as a benchmark for the development of three mpn - specific pro tools : mf - saf , mpn - saf , and mpn - saf tss / mpn-10 . the myelofibrosis symptom assessment form ( mf - saf ) was created in 2009 and served as the first validated mpn patient reported outcome ( pro ) tool to be made available for clinical and trial settings . a 20-item instrument , the survey attempted to capture the most common symptoms within myelofibrosis and content included issues related to catabolic / proliferative symptoms , quality of life , fatigue , and splenomegaly - associated issues . questions were constructed in a yes , no , or 0 ( absent ) to 10 ( worst imaginable ) scale . this survey included the items present within the mf - saf , along with questions related to microvasculature complications such as headaches , concentration problems , lightheadedness , dizziness , vertigo , numbness / tingling , and sexual dysfunction . this expanded version was structured in a similar format to the mf - saf and proved beneficial in evaluation of a variety of novel targeted compounds including ruxolitinib , ly2784544 , sar302503 , vorinostat , and pegylated interferon [ 1113 ] . the myeloproliferative neoplasm symptom assessment form total symptom score ( mpn - saf tss ; mpn-10 ) is an abbreviated version of the mpn - saf containing the 10 most symptomatic and pertinent items . this tool allows for rapid administration in clinical and trial formats and has replaced the mpn - saf in most settings . the survey has been successfully cross - validated against the eortc qlq - c30 and is available in a variety of languages including english , italian , german , french , mandarin , arabic , spanish , dutch , swedish , portuguese , japanese , hebrew , and czech . although the initial inciting event has yet to be clearly elucidated , all mpn disorders arise from genetic defects within pluripotent stem cell populations that accumulate over the disease course . jak2v617f , a member of the janus kinase signal transduction pathway , was the first recognized mutation inherent to the mpn population ( pv 96% , et 50% , and mf 50% ) . the role the janus kinase cascade ( including jak2 , jak2 , jak3 , and tyk2 ) plays in the signaling of inflammatory cytokines is well documented and profound . as jaks are essential to the signaling of surface growth factor receptors and cytokines bereft of intrinsic kinase activity , constitutive activation , as observed in jak2v617f , induces unregulated signaling of stat transcription factors with resultant cellular growth and propagation . stat3 , in particular , is linked closely with cancer development via activation of immunomodulatory cytokines ( il-6 , il-10 , and il-17 ) , growth factors ( fgf , vegf ) , and matrix metalloproteinases . in general , cytokines ( interleukins , interferons , and soluble growth factors ; definitions in table 2 ) are important regulators of cellular processes , particularly those involving immunomodulatory activities , cellular growth angiogenesis , and migration . cytokine dysregulation is believed to be associated with other mutations observed within mpns ( idh1/2 , tet2 ) but requires additional investigation . chronic inflammation has been hypothesized to play a supportive role in oncogenesis given its promotion of genomic instability through dna mutations and epigenetic changes , prevention of tumor immune surveillance , and encouragement of clonal evolution [ 17 , 20 , 21 ] . as chronic inflammatory conditions , mpn disorders revolve around a perpetual cycle of dna damage , cellular remodeling , and subsequent fibrosis . this process has been a topic of great interest , especially as it relates to the heterozygous clinical presentation of mpn patients . the relationship between chronic inflammation and mpn symptom development has also been a topic of recent interest . an evaluation of abnormal cytokine expression within myelofibrosis determined that primary myelofibrosis ( pmf ) patients had significantly increased levels of il-1b , il-1ra , il-2r , il-6 , il-8 , il-10 , il-12 , il-13 , and il-15 and tnf-1 , g - csf , ifn- , mip-1 , mip-1 , hgf , inf--ip , and vegf in addition to reduced ifn- levels . il-2r , il-12 , il-15 , and ip-10 were independently predictive of inferior survival . il-2r and il-12 were associated with transfusion needs and hgf , mig , and il-1ra were associated with marked splenomegaly . changing levels of five cytokines was significantly associated with change in the mpn - saf tss when controlled for arm , visit - by - arm interaction , age , sex , and body mass index ( bmi ) . , it has previously been shown within other malignancies to promote angiogenesis , induce leukocyte chemotaxis / activation , and stimulate cellular reproduction . a recent study determined il-8 to be associated with elevated levels of circulating blasts and the presence of constitutional symptoms . in polycythemia vera , patients demonstrate increased levels of il-1ra , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12 , il-13 , ifn- , gm - csf , mcp-1 , mip-1 , mip-1 , hgf , ip-10 , mig , mcp-1 , pdgf - bb , tnf- , ifn- , and vegf [ 25 , 26 ] . an analysis of et patients determined this population to have elevated levels of il-1b , il-4 , il-6 , il-8 , il-10 , il-12 , hgf , gm - csf , ifn- , mcp-1 , pdgf - bb , tnf- , and vegf . interestingly , il-4 , il-8 , gm - csf , ifn- , mcp-1 , pdgf - bb , and vegf appeared to be significantly higher in et patients when compared to pv populations and may serve as useful markers to distinguish the two disorders . also within et patients , polynuclear cell counts were found to correlate with hgf , il-6 , il-12 , mg - csf , and vegf whereas red cell counts correlated with pdgf - bb levels . in comparing pv and et patients with vascular complications versus those without complications , no significant differences in cytokine levels however , in comparing pv and et patients with a history of vascular events , et patients have significantly increased levels of il-4 , il-8 , gm - csf , ifn- , mcp-1 , and vegf . interestingly , the specific combination of inflammatory markers appears to be as important as the type of factor present . in mf , the combination of tnf- and timp-1 has been shown to promote survival of cd34 + stem cells whereas the combination of atp and tnf- has been shown to reduce proliferation . these biomarkers have been associated with the development of major thrombotic events in pv and et . altered levels of pdgf , fgf , and vegfb have also been noted in stromal cells of patients with pv , et , and pmf suggesting proinflammatory cytokines promote bone marrow fibrosis which is well established to contribute to anemia and subsequently fatigue [ 29 , 30 ] . cytokines ( bmp-1 , bmp - r2 , bmp-6 , and bmp-7 ) may have a role in promoting the advancement of mpns from early to later stages . of special interest , the presence of specific gene mutations impacts the type and degree of cytokine expression . for instance , jak2v617f positive patients have significantly higher levels of il1b , il-8 , il-17a , and ifn versus triple - negative ( jak2 , mpl negative ) patients . much has yet to be learned about the role cytokines play in mpn symptom development . below we discuss the available literature on specific mpn symptoms and their relationships to inflammation ( table 3 ) . the symptom is particularly prominent within mpns ( pv 85% , et 72% , and mf 84% ) , representing the most common symptom voiced regardless of subtype . anemia is a recognized contributor to this symptom and recent studies have demonstrated that cytokines play a critical role in the development and perpetuation of this comorbidity . il-1 , il-6 , and tnf were recently shown to promote deregulation of erythropoietin with resultant anemia in acute myelogenous leukemia ( aml ) and myelodysplastic syndromes ( mds ) [ 35 , 36 ] . in cancer , fatigue has been closely associated with depressed mood and increased levels of il-6 , a cytokine observed within mpns [ 38 , 39 ] . a survey of 1788 mpn patients confirmed that 32% have been seen or diagnosed with depression and 22.2% had received active treatment of mood disorder within the prior six months suggesting potential association in this population . the mechanism has yet to be elucidated as intense physical activity has been shown to increase circulating levels of il-6 which subsequently stimulates the production of other anti - inflammatory cytokines including il-1ra and il-10 , and inhibit proinflammatory cytokines such as tnf-. the mpn fatigue project is an international effort performed in collaboration with the mpn forum aiming at evaluating the breadth and efficacy of current strategies targeting mpn fatigue . the study remains ongoing and includes evaluation of treatments such as sleep deprivation , dietary supplements , and exercise . abdominal - related complaints are common among mpn patients , largely attributable to splenomegaly , portal hypertension , mechanical obstruction , and splenic infarcts . an independent source of morbidity and mortality complaints related to the abdomen has included early satiety ( 76% ) , abdominal pain ( 63% ) , abdominal discomfort ( 72% ) , and weight loss ( 48% ) . however , cytokines may also play an important role in the development of this symptom . tnf- , in particular , promotes clonal expansion in jak2v617f positive mpns [ 42 , 43 ] . the development of splenomegaly has also been associated with specific cytokines including mig , hgf , and il-1ra . however , interestingly , jak2/stat3 signaling has been shown to promote fibrosis , angiogenesis , and inflammation in the setting of portal hypertension , independent of the presence of malignancy suggesting that regional inflammation also plays a role in abdominal pain , whether or not cancer is present . thrombosis may result in a variety of abdominal complaints , particularly with et and pv . animal studies have shown increased expression of tnf- , il-1 , and il-6 after nerve injury , cytokines all disproportionately high in the mpn population . in addition , inflammation and trauma have been shown to induce peripheral nerve cell release of inflammatory cytokines within the central nervous system via glial stimulation . the effects of cytokine - mediated pain were demonstrated in a recent study of patients with painful neuropathies where it was observed that this population had twofold higher levels of il-2 mrna and tnf- mrna in comparison to healthy controls . in the mpns , these symptoms may include headaches , concentration problems , lightheadedness , dizziness , vertigo , numbness / tingling , and sexual dysfunction . proinflammatory cytokine production is believed to be a contributor to cognitive impairment in cancer patients via the disruption of neurohormonal signaling and impaired creation of neurotransmitters . the role of inflammation in cognitive impairment has been intensely studied in both animal and human models . in human studies , elevated levels of il-1 , tnf- , il-6 , and crp were also linked to impaired memory and neurodegenerative disorders in the elderly [ 5153 ] . these mast cells represent a key source of prostaglandin , leukotriene , histamine , and tryptase , mediators of the inflammatory response with recognized ties to pruritus . a study evaluating symptoms of ruxolitinib treated patients within the comfort - ii trial determined that baseline pruritus was associated with lower ferritin levels , a surrogate marker of inflammation . basophils have been instigated as a primary mediator for symptom development and studies have demonstrated that the number of constitutively activated and hypersensitive circulating basophils is increased in pv , correlating with the degree of pruritus . both fevers and night sweats are recognized to be partially cytokine driven , typically by il-1 , il-2 , il-6 , tnf- , and ifn . within other malignancies , il-6 has been found to correlate with the presence of b symptoms which serve as a prognostic factor in chronic lymphocytic leukemia ( cll ) and hodgkin 's lymphoma [ 5860 ] . the development of mpn - associated weight loss is complex and relates to a variety of factors including splenomegaly , portal hypertension , and cancer - cachexia . the role of a cytokine - driven proinflammatory state as the nidus for cancer - cachexia is well supported by literature [ 61 , 62 ] . in a review of cytokine levels present in patients from the comfort - ii trial , weight loss was associated with lower leptin levels and high cd40l was associated with loss of appetite . recognizing the substantial impact chronic inflammation has on the mpn symptom burden , attentions have turned to therapies demonstrating efficacy in reducing cytokines . as in other malignancies , improving physical activity and reducing fat intake may reduce inflammatory cytokines and improve survival . in noncancer patients , increased physical activity has been shown to reduce tlr4 signaling and truncate release of inflammatory cytokines . in addition to evaluating the effects of sedentary living on the mpn symptom burden , the final phases of the mpn fatigue project involve the development of comprehensive patient activity programs which may have subsequent impact on cytokine - induced symptomatology . recognizing the role constitutive janus kinase signaling plays in inflammation , jak2 inhibition has become a rational target for preventing cytokine dysregulation . the expression of ige was also strongly reduced in almost all patients with direct impact on the amount of activated anti - inflammatory macrophages . a similar study utilized the functional assessment of cancer therapy - lymphoma ( fact - lym ) to evaluate symptoms of ruxolitinib treated patients within the comfort ii trial and compare them to changes in cytokine levels . ten symptoms including fever , weight loss , fatigue , loss of appetite , pain , itching , sleeping well , lack of energy , night sweats , and trouble sleeping were assessed at baseline and weeks 8 , 24 , and 48 . treatment with ruxolitinib led to improved items of itching , night sweats , and weight loss with subsequent reduction in numerous cytokines . importantly , their limited hematological toxicities may be reflective of their selective inhibition of kinases . a recent investigation of pacritinib demonstrated that it selectively spares jak1 while inhibiting jak2 , jak2v617f , flt3 , and irak1 , an il-1 receptor kinase associated with the inflammatory response and suppression of normal hematopoiesis . similarly , as antifibrosing agents , hedgehog inhibitors , hypomethylating agents , histone deacetylation inhibitors , and hsp90 inhibitors enter the treatment landscape , knowledge of their impact on inflammation is of great interest . however , how these markers differ between mpn subtypes , change with disease progression , and relate to transformation remain unknown . the expanded access to targeted agents has provided a platform by which cytokine signals may be inhibited early within the cascade , limiting their potential for toxicity .

nitroarene dioxygenases are members of the naphthalene family of rieske nonheme iron dioxygenases , which are able to oxidize a variety of aromatic compounds through direct incorporation of atmospheric oxygen into the substrate , leading to the formation of a cis - dihydrodiol . apart from cis - dihydroxylation , rieske dioxygenases also catalyze monohydroxylation , desaturation , sulfoxidation , o- and n - dealkylation , and amine oxidation . the broad substrate specificity and versatility of rieske dioxygenases examples of their application in large - scale biosynthesis include production of indigo and generation of chiral precursors of drugs , such as indinavir sulfate . being involved in bacterial metabolism , rieske enzymes initiate degradation of many environmental contaminants , and thus remain viable targets for bioremediation platforms . nitroarene dioxygenases are uniquely able to oxidize the aromatic ring of nitroarene compounds , resulting in the elimination of the nitro group , which facilitates further metabolization . nitrobenzene dioxygenase ( nbdo ) , in particular , is the only nitroarene dioxygenase that can catalyze the denitration of all mono- and dinitrotoluenes . nbdo , like other rieske dioxygenases , is a three - component system that consists of an nadh - dependent flavoprotein reductase , a rieske [ 2fe-2s ] ferredoxin , and an 33 terminal oxygenase . during the catalytic cycle two electrons are transferred one at a time from the reductase to the [ 2fe-2s ] cluster in ferredoxin and subsequently to the rieske [ 2fe-2s ] cluster and mononuclear iron(ii ) center located in the terminal oxygenase component , where the reaction takes place . the oxygenase component is an 33 heterohexamer with a mushroom - shaped quaternary structure , in which subunits containing the active site and rieske domains bear catalytic function , while subunits , which are located more than 10 from the active sites , are believed to have a purely structural function . the active site domain in each subunit hosts a high - spin mononuclear fe bound to two histidines and a bidentate aspartate residue , forming the 2-his-1-carboxylate facial triad motif found in many mononuclear nonheme iron(ii ) oxygenases . on the other hand , the rieske domain contains a rieske type [ 2fe-2s ] cluster coordinated by two cysteine and two histidine residues ( figure 1 ) . schematic representation of the interface between the mononuclear iron center located in the active site of one subunit and the rieske domain from a neighboring subunit connected by hydrogen bonds through asp203 residue . some of the possible hydrogen bond interactions involved in the contact are indicated . within a single subunit , these two domains are separated by a distance of around 45 , which is too far for an effective electron transfer . in a tight trimer formed by three subunits , however , the rieske [ 2fe2s ] cluster and mononuclear iron(ii ) center from two adjacent subunits are only 12 apart and are connected through a conserved asp residue possibly serving as a route for the transfer of the two electrons from nad(p)h to the active site during the catalytic cycle . the most thoroughly studied representative of rieske dioxygenases is naphthalene dioxygenase ( ndo ) , with which nbdo shares 80% sequence identity . crystal structures of free ndo , as well as complexes along the reaction pathway have been solved and serve as the prototype for the general dioxygen activation and substrate oxidation mechanism in rieske dioxygenases . as it was shown in a single turnover study of ndo , an isolated oxygenase component is capable of cis - dihydroxylation of an aromatic substrate , provided the rieske cluster is in the reduced state . reduction of the rieske cluster and formation of the enzyme substrate complex triggers side - on binding of o2 to the mononuclear iron(ii ) and its subsequent activation . through extensive studies on ndo , including crystallographic data , single turnover and peroxide shunt experiments , and theoretical calculations , a ferric ( hydro)peroxo complex formed upon dioxygen reduction has been identified as a key reaction intermediate . however , the possibility of a high - spin iron - oxo intermediate formed upon oxygen oxygen bond cleavage being the oxidant that eventually attacks the substrate has also been indicated and found support , especially in studies of ndo reactivity using probe molecules . oxygen intermediate oxidizing the substrate and the versatility of reactions catalyzed by rieske dioxygenases have prompted numerous experimental and theoretical studies of their catalytic mechanism . an understanding of the cis - dihydroxylation mechanism of rieske dioxygenases is particularly valuable in the development of bioremediation measures and protein engineering of dioxygenases to modify selectivity and enhance efficiency . substrate specificity of rieske dioxygenases is attributed to specific interactions between the substrate and active site residues . hence , there exists an apparent need for a detailed investigation of the specific interactions taking place in the active site of these enzymes , in hopes of understanding the structural basis for their activity toward different compounds . in particular , the activity of nitroarene dioxygenases toward nitroaromatic compounds is attributed to the presence of a polar asn residue forming a hydrogen bond with the nitro group of the substrate , positioning it for ring oxidation . moreover , nitroarene dioxygenases catalyze both aromatic cis - dihydroxylation and monohydroxylation of alkyl side chains , with the predominant reaction dependent on the binding position of the substrate . it is thus important to consider the protein environment in modeling these reactions , which can be accomplished by means of biomolecular simulations of the enzymes of interest . while combined quantum mechanical / molecular mechanical ( qm / mm ) studies have been carried out on enzymes that also have the 2-his-1-carboxylate facial triad motif , there are currently no published force field parameters specifically developed for this moiety , and only partial atomic charges for the rieske cluster in cytochrome bc1 complex , which also has one fe atom bound to histidine residues and the other bound to cysteine residues , have been determined and combined with charmm force field parameters . in this work , we present a classical molecular dynamics simulation of the oxygenase component of the nbdo system in explicit water environment using the amber ff99sb force field , including parameters developed for the mononuclear iron center and rieske cluster . results of the molecular simulation provide information regarding the structure and dynamics of the enzyme in aqueous solution and specifically give insight into key hydrogen - bonding interactions within the active site region , including protein water interactions in the substrate binding pocket . the interface between the rieske cluster and mononuclear iron center is also analyzed and described in terms of hydrogen bonds formed between the two domains . additionally , the results of the simulation are compared with available experimental data to estimate the performance of the new parameters developed for the metal centers of the protein . since the residues coordinating the rieske cluster and mononuclear iron are completely conserved within rieske dioxygenases , the parameters derived herein might be applied to studying other members of this family of enzymes . the crystal structure of nitrobenzene dioxygenase in complex with nitrobenzene ( pdb i d : 2bmq ) consisting of a single dimer was used as the initial structure and transformed into an 33 heterohexamer by generating symmetry - related copies of monomers using coot . the enzyme was assumed to be in its oxidized form , with dioxygen reduced to the form of a side - on bound hydro(peroxo ) moiety . therefore , the two water molecules coordinated to fe in the active site ( hoh2227 and hoh2401 ) were removed and hydro(peroxo ) ligand was placed on the face of the iron center through an overlay with the o2 adduct of the ndo - indole complex ( pdb i d : 1o7n ) . hydrogen atoms of the protein residues were added using leap , and their standard protonation states were verified using propka 3.1 . the system was then soaked in a truncated octahedral box of 26735 tip3p water molecules extending to at least 15 from the protein atoms . the ff99sb force field was used to describe the protein , together with general amber force field ( gaff ) parameters for nitrobenzene . the metal centers of the protein were parametrized based on the bonded plus electrostatics model approach following the procedure described by peters et al . sulfur cluster and mononuclear iron center , [ 2fe-2s ] ( s = 0 ) and [ fe ooh ] ( s = 5/2 ) , respectively , as this is the reactive state of the enzyme . ( a description of the parametrization method and the derived partial charges and force field parameters are included in supporting information ) . the system , prepared as described above , was first subjected to 500 steps of steepest descent and 500 steps of conjugate gradient minimization , with the protein held fixed by using position restraints with a force constant of 500 kcal mol . this was followed by an additional 2500 steps of steepest descent and 2500 steps of conjugate gradient minimization . the system was then heated to the target temperature of 300 k for a period of 20 ps under constant volume periodic boundary conditions ( nvt ) , with weak positional restraints applied to the protein atoms ( force constant of 10 kcal mol ) . subsequently , approximately 40 ns of constant pressure and temperature simulation ( npt ) was carried out to equilibrate the system , which was followed by 30 ns of production simulation performed under the same conditions . an average pressure of 1 atm was maintained by using isotropic position scaling with a relaxation time of 2 ps . a cutoff of 10 was used for nonbonded interactions and long - range electrostatic interactions were treated by means of the particle mesh ewald ( pme ) method . all bonds involving hydrogen were constrained by the shake method , and the time step for numerical integration was 2 fs . the simulation results were analyzed using the ptraj program in the amber11 package and vmd . the presence of hydrogen bonds was analyzed based on a geometry criterion where a given h - bond was considered to be formed or disrupted at a cutoff distance of 3.6 between the donor and acceptor atom and a cutoff bond angle of 120. occupancy is calculated as the percent of time the hydrogen bond is formed over the trajectory . the stability of the overall nbdo structure throughout the simulation was investigated via root - mean - square deviations ( rmsd ) of backbone and all heavy atoms of the enzyme with respect to the x - ray structure ( figure 2 ) . the rmsd values reached plateau after approximately 30 ns of the simulation , indicating that the system reached its equilibrium . the average values of 1.07 for the backbone and 1.51 for the heavy atom rmsd show that we did not observe significant deviation of the overall enzyme structure from the x - ray data . time evolution of the root - mean - square deviation from the crystallographic structure for backbone ( dotted line ) and all heavy atoms ( solid line ) of nbdo during simulation . the geometries of individual and subunits do not undergo substantial changes along the trajectory , which is reflected by small values of root - mean - square deviations between the rms - fitted average structures of the and subunits and their corresponding crystal structures ( see table s11 in supporting information ) . even smaller structural changes are observed for the active site residues , with the largest heavy atom rmsd of 0.9 obtained for the active site located in the second subunit . we have also investigated whether there exist any differences between single subunits in the and trimers , and between the three active sites . as might be expected from the overall stability of the enzyme structure , the average structures of the three and three subunits are very similar ( figure 3 ) , with calculated rmsd values of less than 1 for the backbone atoms of the rms - fitted average structures ( see table s12 in supporting information ) . superposition of the average structures of the three subunits ( a ) , three subunits ( b ) , and three active sites ( c ) calculated from the last 30 ns of the trajectory . red , blue , and orange colored structures correspond to the subunit ( or active site ) 1 , 2 , and 3 , respectively . the average geometries of the three active sites are also similar to each other , with rmsd values ranging from 0.2 to 0.5 , and from 0.4 to 0.8 for the backbone and all heavy atoms , respectively ( see table s13 in supporting information ) . the biggest rmsd values are observed between active site 2 and the other two active sites . an overlay of the average structures presented in figure 3c indicates that the structural difference causing the increase of the rmsd value lies in a slight change of the position of the phe222 and asn295 side chains , which occurs around the 47th ns of the simulation . however , since it is not accompanied by any other significant change in the active site geometry and the position of any of the surrounding residues is not altered , we believe it not to be particularly meaningful here . the flexibility of the active site residues was also investigated in terms of b - factors calculated for heavy atoms of the active site according to the relationship:1where ri2 is the atomic positional variance of each heavy atom of residue i. the average ( mass - weighted ) b - factors computed for each residue in the three active sites indicate rather low flexibility of these residues ( for details , see figure s2 in supporting information ) , with the mean value for all active sites oscillating around 8.7 per residue . as might be expected , the largest dynamic flexibility is observed for the substrate , the b - factor of which was calculated to range from 20.8 to 33.0 in the three active sites . b - factors were also used to investigate the dynamics of a lid covering the entrance to the substrate pocket , formed by the loops between 8 and 13 ( residues 211 to 238 ) and between 13 and 9 ( residues 238 to 265 ) domains . the values calculated for the main chain c atoms range from 9.9 to 33.4 ( for details , see figure s3 in supporting information ) , with the most flexible fragment located between val221 and glu237 . regardless of high flexibility , the mobile part of the loop maintains a closed conformation throughout the simulation , which was confirmed by monitoring the distance between the glu234 and val221 c atoms . the average distance of 11.2 0.7 shows that the position of the loop does not change relative to the crystal structure ( dglu234-val221 = 11.3 ) during the simulation , blocking the entrance to the active site while the substrate is present . as already pointed out , the overall geometry of the active site observed during the simulation does not differ significantly from the crystal structure . moreover , we observe a high degree of similarity between subunits of a particular type and between the three active sites in the simulated nbdo structure . therefore , in the remainder of this paper , we will discuss the results obtained for active site 1 only . the residues forming a substrate pocket do not exhibit large positional fluctuations , which are reflected by small b - factors . the position of the substrate undergoes larger fluctuations , compared to the amino acid side chains , but nevertheless , its orientation remains rather stable . mean distances between the reacting carbon atoms of nitrobenzene and the oxygen atoms of the iron oxygen complex are 3.27 0.24 ( c1o ) and 3.46 0.27 ( c2o1 ) , with the latter value slightly larger than 3.02 found in the starting structure for the simulation . this small change in the distance between the o1 and c2 atoms during the simulation reflects a shift in the substrate position relative to the fe ooh moiety . the dihedral angle formed between the o and o1 atoms and the c1c2 double bond of nitrobenzene changed from 39.8 in the starting structure to 0.4 in the average structure of the active site obtained from the simulation , which is the expected parallel orientation of the substrate relative to the activated dioxygen required by the cis - stereospecificity of substrate dihydroxylation . another important aspect of substrate positioning in the active site of nbdo , namely , formation of a hydrogen bond with asn258 , will be discussed later in this paper . the geometry of the mononuclear iron center located in the active site was monitored during the simulation and mean distances between the fe atom and its three ligands , as well as iron dioxygen distances , are compared with the x - ray data ( table 1 ) . mean values together with standard deviations for the md simulation and values obtained from x - ray crystallography are presented . dioxygen position determined through an overlay with naphthalene dioxygenase crystal structure ( see computational methods for details ) . the distances between the fe ion and his206 , his211 and asp360 are slightly smaller than in the nbdo crystal structure , while in the average geometry of the iron oxygen moiety , we observe slightly elongated bonds compared to the geometry of this complex adopted from the crystal structure of ndo . the average geometry of the rieske center located in the subunit adjacent to the subunit containing the active site was also analyzed . the mean binding distances of the four fe ligands and the average geometry parameters of the [ 2fe-2s ] cluster are presented in table 2 . similarly to the mononuclear iron center , the geometry of the iron sulfur cluster from the md simulation agrees very well with the experimental x - ray structure . the binding distances between the iron and sulfur atoms , and the ligating residues are well reproduced , and the [ 2fe-2s ] cluster maintains the form of a flat rhombic cluster , as found in the crystal structure . see figure 1 for atom numbering . interactions between the rieske cluster and the surrounding residues involve hydrogen bonds from the main chain nitrogen of arg82 ( 44.8% occupancy ) to the s1 sulfide ion , and from the main chain nitrogen of trp104 ( 24.3% occupancy ) to the s2 ion ( figure 4 ) . other possible hydrogen bonds between the sulfide ions and the neighboring main chain atoms of his102 and gly103 were detected with occupancies of less than 5% . all four ligands of the iron atoms form hydrogen bonds in the second coordination shell . main chain nitrogen atoms of cys79 and cys99 form hydrogen bonds with main chain oxygens of lys84 and trp104 , respectively , with 100% occupancy each . additionally , cys99 binds through the main chain oxygen with the main chain nitrogen of gly103 ( 84.7% occupancy ) . both iron - coordinating histidines keep hydrogen - bond contacts with the residues located in the neighboring subunit . his81 interacts with the carboxylic oxygens of glu408 , with 22.5 and 98.1% occupancy , and his102 forms hydrogen bonds with carboxyl group of asp203 , with 70.5 and 80.1% occupancy for the two oxygen atoms . interactions between the rieske cluster and the surrounding residues , together with the most highly populated hydrogen bonds formed between the four ligands of the iron atoms and their second coordination shell . as already mentioned in the introduction , the catalytic center of rieske dioxygenases , including nbdo , is formed at the interface of two neighboring subunits by the active site containing mononuclear iron located in one subunit and the [ 2fe-2s ] rieske cluster from the adjacent subunit . these two components are connected through a conserved aspartic acid residue , which is believed to be the route for electron transfer from the rieske cluster to the nonheme mononuclear iron during the catalytic cycle . the role of this residue in the catalytic process was first suggested based on the crystal structure of ndo , where it was found to form the most direct pathway for an efficient electron transfer . this hypothesis was later confirmed in a site - directed mutagenesis study of ndo , in which it was shown that replacement at position 205 severely decreases the activity of the enzyme , and thus , asp205 is likely the major pathway for the electron transfer in rieske dioxygenases . in the crystal structure of nbdo , the [ 2fe-2s ] rieske cluster and mononuclear iron center are connected by asp203 , which is hydrogen bonded through the carbonyl oxygen atom to his206 and through one of the carboxyl oxygen atoms to his102 . we have monitored these hydrogen bond contacts during the simulation , together with other possible hydrogen bonds that might be formed by asp203 with the histidine ligands of the two connected metal centers . figure 5 lists the analyzed hydrogen bonds together with their percentage occupancies and shows the change of the occupancy of a given bond along the trajectory . as can be seen , indeed , the major h - bond chain connecting the two centers is formed by n of his206 in the active site with the carbonyl oxygen of asp203 ( 96.1% occupancy ) and by n of his102 in the rieske cluster with either of the two carboxyl oxygen atoms of asp203 , which are involved in the bond exchangeably ( 80.1% and 70.5% occupancy for the od2 and od1 atoms , respectively ) . while the asp203-his102 contact is limited to the od2(od1)-n hydrogen bond , other possible h - bonds are formed between asp203 and his206 with significant occupancy . these include hydrogen bonds between the carboxyl oxygen of asp203 and the main chain nitrogen of his206 or n of his206 , and less significant contact between the main chain atoms of the two residues . from the time evolution of the percentage occupancy shown in figure 5 , it is easily seen that asp203(carboxyl)-his206(side chain ) and asp203(carboxyl)-his206(main chain ) hydrogen contacts are formed exchangeably , in a manner obviously coupled with the asp203(carboxyl)-his102 bonds , maintaining the hydrogen bond interaction between the carboxyl group of the aspartate and both his206 and his102 residues throughout the simulation . the possible charge transfer channels formed between the rieske cluster and the nonheme iron center thus include the his102:n-asp203:od1(od2 ) asp203:o - his206:n and his102:n-asp203:od1(od2 ) asp203:od2(od1)-his206:n(n ) routes . percentage occupancy of hydrogen bond contacts formed through asp203 between the active site mononuclear iron center and the rieske cluster from an adjacent subunit . right - hand side of the picture shows time evolution of the occupancy of the listed hydrogen bonds . hydrogen bonds formed by the active site residues in nbdo include intramolecular backbone backbone , backbone side chain , and side chain side chain hydrogen bonds , as well as h - bonding interactions between the active site amino acids and water molecules located in their vicinity . the most highly populated bonds formed in the active site are indicated in figure 6 . hydrogen - bond pattern found in the active site of nbdo , including hydrogen bonds formed between the active site residues and water molecules . figure presents only those hydrogen bonds for which the calculated occupancy was greater than 10% , and the value of the occupancy of each hydrogen bond is given . the most functionally important residues and a hydrogen bond chain found at the entrance to the active site ( see text for details ) are presented in bold . majority of the 17 residues lining the substrate pocket in nbdo are hydrophobic . thus , understandably , water molecules are not present inside the substrate cavity , but rather tend to surround it , as can be clearly seen from a volumetric map of water density around the active site of nbdo ( figure 7 ) . the water molecules located closest to the substrate pocket form hydrogen bonds with the side chains of polar residues or backbone atoms of several active site amino acids . there is also visible water density at the entrance to the active site , in close vicinity to the iron - dioxygen complex . this density represents the presence of two water molecules forming a short hydrogen bond chain connecting dioxygen with the asn199 residue , which will be described in detail below . volumetric map of water density ( solid surface ) around the active site of nbdo ( residues in licorice representation ) . many of the hydrophobic residues located in the substrate pocket are involved in persistent backbone hydrogen - bonding interactions . the most highly occupied of these bonds ( occupancy 95% ) include backbone hydrogen bonds between asn295 and gly249 , phe293 and phe251 , ala345 and ser349 . for most of the carbonyl oxygen atoms of the active site residues , backbone nitrogen atoms are the only h - bond partners . an exception to this is the asp203 residue , which interacts with both backbone and side chain nitrogens of his206 , and ala345 , which forms a hydrogen bond with the hydroxyl oxygen of ser349 ( occupancy 10.3% ) . on the other hand , the backbone oxygen of phe200 is not involved in an h - bond interaction with any of the backbone amine groups but forms a hydrogen bond with the side chain of asn295 with a high occupancy of 91.1% . the most important hydrogen - bonding interaction occurring in the active site , from the perspective of substrate specificity of nbdo , is the hydrogen bond formed between the side chain of asn258 and nitro group of the substrate . the formation of this bond is believed to be responsible for the proper positioning of the nitroaromatic substrate for oxidation . as it was shown in a site - directed mutagenesis study of nbdo , replacement of asparagine at this position by valine , which eliminates the said hydrogen bond , results in formation of nitrobenzyl alcohols as the major product ( as much as 99% ) , rather than catechols , from mononitroarene substrates . according to the md simulation results presented here , the amide group of asn258 donates one of its hydrogen atoms to form an h - bond with either of the two oxygens of the substrate nitro group with an occupancy of 50% each , so that the hydrogen bond between asn258 and nitrobenzene is effectively formed throughout the whole simulation . this result explains the position of the substrate observed in the simulation , with the c1c2 double bond parallel to the attacking dioxygen and thus agrees with the role of asn258 proposed in literature . simultaneously to forming an h - bond with the nitro group , the amide group of asn258 is also interacting through a second hydrogen atom with a water molecule ( 96% ) . the backbone oxygen and side chain carbonyl oxygen of asn258 also form hydrogen bonds with water molecules and neighboring ser349 ( see figure 6 ) . an interesting observation regarding other hydrogen bonds formed within the active site is that hydrogen bond interaction involving the carboxyl oxygens of asp203 , serving as a link between the mononuclear iron center and the rieske cluster , are not limited to forming h - bonds with his206 and his102 residues , which were discussed before . additionally , asp203 interacts with water , and with the side chain of asn199 . as for asn199 itself , it forms hydrogen bonds with water molecules residing at the entrance to the active site , which are at the same time interacting with the peroxide moiety of the iron - dioxygen complex . the total occupancy for the feooh water and asn199water hydrogen bonds is 81% and 77.6% respectively , with feooh serving as a donor , and the carbonyl oxygen of asn199 as acceptor in hydrogen bonding interactions with any water molecule . closer inspection reveals , however , that there are , in fact , two specific water molecules with which feooh and asn199:od are almost exclusively interacting throughout the simulation . these two water molecules are exchangeably acting as a proton acceptor in feooh water interaction ( 62.6% and 18.2% occupancy ) and a proton donor in the asn199:od water interaction ( 41.2% and 36.3% occupancy ) , being at the same time connected with each other through a water water hydrogen bond ( 77.2% occupancy ) ( for details regarding water this way , a short hydrogen bond chain is formed ( see bottom of figure 6 ) , which involves the peroxide moiety bound to mononuclear iron and thus might be of potential importance to the catalytic process involving its attack on the substrate . we have performed a 70 ns - long classical molecular dynamics simulation of nbdo , a representative of the naphthalene family of rieske dioxygenases , using the amber force field together with a new set of force field parameters developed for the description of the active site mononuclear iron center and iron sulfur rieske cluster . the md simulation was carried out in explicit water environment under npt ensemble for a heterohexameric model of the oxidized form of the enzyme with nitrobenzene bound in the active site and ( hydro)peroxide moiety attached to the mononuclear iron . simulation results were analyzed with the focus on the comparison of the overall structure and key geometrical parameters with x - ray data , investigation of the hydrogen - bonding interactions occurring within the active site and rieske cluster domains , as well as at the interface of the two metal centers , and role of the solvent molecules in the substrate binding pocket . the overall structure of nbdo in aqueous solution is stable and does not undergo significant structural changes along the simulated trajectory . no structural differences are observed between the individual subunits within the 3 and 3 trimers , as well as between the three active sites formed in the heterohexamer . the average b - factors indicate rather low flexibility of the active site residues , with the highest values obtained for the substrate . a highly flexible loop covering the entrance to the active site remains in a closed conformation throughout the simulation . in general , the structure of the enzyme observed during the simulation shows very small deviation from the x - ray data . the newly developed parameters reproduce very well the geometry of the mononuclear iron center and rieske cluster and may potentially be used in simulations of other rieske dioxygenases . the hydrogen bond analysis showed that the mononuclear iron center and [ 2fe-2s ] cluster are connected through several hydrogen bonds formed between carbonyl and carboxyl oxygen atoms of the asp203 residue and his ligands of the two metal centers along the entire trajectory . interaction of the substrate with the active site residues is limited to the formation of a hydrogen bond with the asn258 residue , which is effectively formed throughout the whole simulation and positions the substrate for the reaction with the activated dioxygen . finally , water molecules are not in general present inside the hydrophobic substrate pocket . however , there exists a short hydrogen bond chain at the entrance to the active site , which involves two water molecules , asn199 residue and the ( hydro)peroxo ligand bound to fe ion and might thus be potentially important for the catalytic processes involving this moiety .

molecular dynamics simulation of the oxygenase component of nitrobenzene dioxygenase ( nbdo ) system , a member of the naphthalene family of rieske nonheme iron dioxygenases , has been carried out using the amber force field combined with a new set of parameters for the description of the mononuclear nonheme iron center and iron sulfur rieske cluster . simulation results provide information on the structure and dynamics of nitrobenzene dioxygenase in an aqueous environment and shed light on specific interactions that occur in its catalytic center . the results suggest that the architecture of the active site is stabilized by key hydrogen bonds , and asn258 positions the substrate for oxidation . analysis of protein water interactions reveal the presence of a network of solvent molecules at the entrance to the active site , which could be of potential catalytic importance .

Introduction Computational Methods Results and Discussion Conclusions

nitroarene dioxygenases are members of the naphthalene family of rieske nonheme iron dioxygenases , which are able to oxidize a variety of aromatic compounds through direct incorporation of atmospheric oxygen into the substrate , leading to the formation of a cis - dihydrodiol . nitrobenzene dioxygenase ( nbdo ) , in particular , is the only nitroarene dioxygenase that can catalyze the denitration of all mono- and dinitrotoluenes . nbdo , like other rieske dioxygenases , is a three - component system that consists of an nadh - dependent flavoprotein reductase , a rieske [ 2fe-2s ] ferredoxin , and an 33 terminal oxygenase . the oxygenase component is an 33 heterohexamer with a mushroom - shaped quaternary structure , in which subunits containing the active site and rieske domains bear catalytic function , while subunits , which are located more than 10 from the active sites , are believed to have a purely structural function . schematic representation of the interface between the mononuclear iron center located in the active site of one subunit and the rieske domain from a neighboring subunit connected by hydrogen bonds through asp203 residue . in a tight trimer formed by three subunits , however , the rieske [ 2fe2s ] cluster and mononuclear iron(ii ) center from two adjacent subunits are only 12 apart and are connected through a conserved asp residue possibly serving as a route for the transfer of the two electrons from nad(p)h to the active site during the catalytic cycle . reduction of the rieske cluster and formation of the enzyme substrate complex triggers side - on binding of o2 to the mononuclear iron(ii ) and its subsequent activation . through extensive studies on ndo , including crystallographic data , single turnover and peroxide shunt experiments , and theoretical calculations , a ferric ( hydro)peroxo complex formed upon dioxygen reduction has been identified as a key reaction intermediate . substrate specificity of rieske dioxygenases is attributed to specific interactions between the substrate and active site residues . hence , there exists an apparent need for a detailed investigation of the specific interactions taking place in the active site of these enzymes , in hopes of understanding the structural basis for their activity toward different compounds . in particular , the activity of nitroarene dioxygenases toward nitroaromatic compounds is attributed to the presence of a polar asn residue forming a hydrogen bond with the nitro group of the substrate , positioning it for ring oxidation . while combined quantum mechanical / molecular mechanical ( qm / mm ) studies have been carried out on enzymes that also have the 2-his-1-carboxylate facial triad motif , there are currently no published force field parameters specifically developed for this moiety , and only partial atomic charges for the rieske cluster in cytochrome bc1 complex , which also has one fe atom bound to histidine residues and the other bound to cysteine residues , have been determined and combined with charmm force field parameters . in this work , we present a classical molecular dynamics simulation of the oxygenase component of the nbdo system in explicit water environment using the amber ff99sb force field , including parameters developed for the mononuclear iron center and rieske cluster . results of the molecular simulation provide information regarding the structure and dynamics of the enzyme in aqueous solution and specifically give insight into key hydrogen - bonding interactions within the active site region , including protein water interactions in the substrate binding pocket . the interface between the rieske cluster and mononuclear iron center is also analyzed and described in terms of hydrogen bonds formed between the two domains . since the residues coordinating the rieske cluster and mononuclear iron are completely conserved within rieske dioxygenases , the parameters derived herein might be applied to studying other members of this family of enzymes . the crystal structure of nitrobenzene dioxygenase in complex with nitrobenzene ( pdb i d : 2bmq ) consisting of a single dimer was used as the initial structure and transformed into an 33 heterohexamer by generating symmetry - related copies of monomers using coot . the enzyme was assumed to be in its oxidized form , with dioxygen reduced to the form of a side - on bound hydro(peroxo ) moiety . therefore , the two water molecules coordinated to fe in the active site ( hoh2227 and hoh2401 ) were removed and hydro(peroxo ) ligand was placed on the face of the iron center through an overlay with the o2 adduct of the ndo - indole complex ( pdb i d : 1o7n ) . the ff99sb force field was used to describe the protein , together with general amber force field ( gaff ) parameters for nitrobenzene . ( a description of the parametrization method and the derived partial charges and force field parameters are included in supporting information ) . red , blue , and orange colored structures correspond to the subunit ( or active site ) 1 , 2 , and 3 , respectively . the average geometries of the three active sites are also similar to each other , with rmsd values ranging from 0.2 to 0.5 , and from 0.4 to 0.8 for the backbone and all heavy atoms , respectively ( see table s13 in supporting information ) . an overlay of the average structures presented in figure 3c indicates that the structural difference causing the increase of the rmsd value lies in a slight change of the position of the phe222 and asn295 side chains , which occurs around the 47th ns of the simulation . however , since it is not accompanied by any other significant change in the active site geometry and the position of any of the surrounding residues is not altered , we believe it not to be particularly meaningful here . the flexibility of the active site residues was also investigated in terms of b - factors calculated for heavy atoms of the active site according to the relationship:1where ri2 is the atomic positional variance of each heavy atom of residue i. the average ( mass - weighted ) b - factors computed for each residue in the three active sites indicate rather low flexibility of these residues ( for details , see figure s2 in supporting information ) , with the mean value for all active sites oscillating around 8.7 per residue . b - factors were also used to investigate the dynamics of a lid covering the entrance to the substrate pocket , formed by the loops between 8 and 13 ( residues 211 to 238 ) and between 13 and 9 ( residues 238 to 265 ) domains . the average distance of 11.2 0.7 shows that the position of the loop does not change relative to the crystal structure ( dglu234-val221 = 11.3 ) during the simulation , blocking the entrance to the active site while the substrate is present . as already pointed out , the overall geometry of the active site observed during the simulation does not differ significantly from the crystal structure . the position of the substrate undergoes larger fluctuations , compared to the amino acid side chains , but nevertheless , its orientation remains rather stable . the dihedral angle formed between the o and o1 atoms and the c1c2 double bond of nitrobenzene changed from 39.8 in the starting structure to 0.4 in the average structure of the active site obtained from the simulation , which is the expected parallel orientation of the substrate relative to the activated dioxygen required by the cis - stereospecificity of substrate dihydroxylation . another important aspect of substrate positioning in the active site of nbdo , namely , formation of a hydrogen bond with asn258 , will be discussed later in this paper . the geometry of the mononuclear iron center located in the active site was monitored during the simulation and mean distances between the fe atom and its three ligands , as well as iron dioxygen distances , are compared with the x - ray data ( table 1 ) . the average geometry of the rieske center located in the subunit adjacent to the subunit containing the active site was also analyzed . similarly to the mononuclear iron center , the geometry of the iron sulfur cluster from the md simulation agrees very well with the experimental x - ray structure . interactions between the rieske cluster and the surrounding residues involve hydrogen bonds from the main chain nitrogen of arg82 ( 44.8% occupancy ) to the s1 sulfide ion , and from the main chain nitrogen of trp104 ( 24.3% occupancy ) to the s2 ion ( figure 4 ) . his81 interacts with the carboxylic oxygens of glu408 , with 22.5 and 98.1% occupancy , and his102 forms hydrogen bonds with carboxyl group of asp203 , with 70.5 and 80.1% occupancy for the two oxygen atoms . interactions between the rieske cluster and the surrounding residues , together with the most highly populated hydrogen bonds formed between the four ligands of the iron atoms and their second coordination shell . as already mentioned in the introduction , the catalytic center of rieske dioxygenases , including nbdo , is formed at the interface of two neighboring subunits by the active site containing mononuclear iron located in one subunit and the [ 2fe-2s ] rieske cluster from the adjacent subunit . this hypothesis was later confirmed in a site - directed mutagenesis study of ndo , in which it was shown that replacement at position 205 severely decreases the activity of the enzyme , and thus , asp205 is likely the major pathway for the electron transfer in rieske dioxygenases . in the crystal structure of nbdo , the [ 2fe-2s ] rieske cluster and mononuclear iron center are connected by asp203 , which is hydrogen bonded through the carbonyl oxygen atom to his206 and through one of the carboxyl oxygen atoms to his102 . figure 5 lists the analyzed hydrogen bonds together with their percentage occupancies and shows the change of the occupancy of a given bond along the trajectory . as can be seen , indeed , the major h - bond chain connecting the two centers is formed by n of his206 in the active site with the carbonyl oxygen of asp203 ( 96.1% occupancy ) and by n of his102 in the rieske cluster with either of the two carboxyl oxygen atoms of asp203 , which are involved in the bond exchangeably ( 80.1% and 70.5% occupancy for the od2 and od1 atoms , respectively ) . these include hydrogen bonds between the carboxyl oxygen of asp203 and the main chain nitrogen of his206 or n of his206 , and less significant contact between the main chain atoms of the two residues . the possible charge transfer channels formed between the rieske cluster and the nonheme iron center thus include the his102:n-asp203:od1(od2 ) asp203:o - his206:n and his102:n-asp203:od1(od2 ) asp203:od2(od1)-his206:n(n ) routes . percentage occupancy of hydrogen bond contacts formed through asp203 between the active site mononuclear iron center and the rieske cluster from an adjacent subunit . hydrogen bonds formed by the active site residues in nbdo include intramolecular backbone backbone , backbone side chain , and side chain side chain hydrogen bonds , as well as h - bonding interactions between the active site amino acids and water molecules located in their vicinity . figure presents only those hydrogen bonds for which the calculated occupancy was greater than 10% , and the value of the occupancy of each hydrogen bond is given . the most functionally important residues and a hydrogen bond chain found at the entrance to the active site ( see text for details ) are presented in bold . thus , understandably , water molecules are not present inside the substrate cavity , but rather tend to surround it , as can be clearly seen from a volumetric map of water density around the active site of nbdo ( figure 7 ) . the water molecules located closest to the substrate pocket form hydrogen bonds with the side chains of polar residues or backbone atoms of several active site amino acids . there is also visible water density at the entrance to the active site , in close vicinity to the iron - dioxygen complex . for most of the carbonyl oxygen atoms of the active site residues , backbone nitrogen atoms are the only h - bond partners . on the other hand , the backbone oxygen of phe200 is not involved in an h - bond interaction with any of the backbone amine groups but forms a hydrogen bond with the side chain of asn295 with a high occupancy of 91.1% . the most important hydrogen - bonding interaction occurring in the active site , from the perspective of substrate specificity of nbdo , is the hydrogen bond formed between the side chain of asn258 and nitro group of the substrate . the formation of this bond is believed to be responsible for the proper positioning of the nitroaromatic substrate for oxidation . according to the md simulation results presented here , the amide group of asn258 donates one of its hydrogen atoms to form an h - bond with either of the two oxygens of the substrate nitro group with an occupancy of 50% each , so that the hydrogen bond between asn258 and nitrobenzene is effectively formed throughout the whole simulation . an interesting observation regarding other hydrogen bonds formed within the active site is that hydrogen bond interaction involving the carboxyl oxygens of asp203 , serving as a link between the mononuclear iron center and the rieske cluster , are not limited to forming h - bonds with his206 and his102 residues , which were discussed before . as for asn199 itself , it forms hydrogen bonds with water molecules residing at the entrance to the active site , which are at the same time interacting with the peroxide moiety of the iron - dioxygen complex . the total occupancy for the feooh water and asn199water hydrogen bonds is 81% and 77.6% respectively , with feooh serving as a donor , and the carbonyl oxygen of asn199 as acceptor in hydrogen bonding interactions with any water molecule . these two water molecules are exchangeably acting as a proton acceptor in feooh water interaction ( 62.6% and 18.2% occupancy ) and a proton donor in the asn199:od water interaction ( 41.2% and 36.3% occupancy ) , being at the same time connected with each other through a water water hydrogen bond ( 77.2% occupancy ) ( for details regarding water this way , a short hydrogen bond chain is formed ( see bottom of figure 6 ) , which involves the peroxide moiety bound to mononuclear iron and thus might be of potential importance to the catalytic process involving its attack on the substrate . we have performed a 70 ns - long classical molecular dynamics simulation of nbdo , a representative of the naphthalene family of rieske dioxygenases , using the amber force field together with a new set of force field parameters developed for the description of the active site mononuclear iron center and iron sulfur rieske cluster . the md simulation was carried out in explicit water environment under npt ensemble for a heterohexameric model of the oxidized form of the enzyme with nitrobenzene bound in the active site and ( hydro)peroxide moiety attached to the mononuclear iron . simulation results were analyzed with the focus on the comparison of the overall structure and key geometrical parameters with x - ray data , investigation of the hydrogen - bonding interactions occurring within the active site and rieske cluster domains , as well as at the interface of the two metal centers , and role of the solvent molecules in the substrate binding pocket . the average b - factors indicate rather low flexibility of the active site residues , with the highest values obtained for the substrate . a highly flexible loop covering the entrance to the active site remains in a closed conformation throughout the simulation . the newly developed parameters reproduce very well the geometry of the mononuclear iron center and rieske cluster and may potentially be used in simulations of other rieske dioxygenases . the hydrogen bond analysis showed that the mononuclear iron center and [ 2fe-2s ] cluster are connected through several hydrogen bonds formed between carbonyl and carboxyl oxygen atoms of the asp203 residue and his ligands of the two metal centers along the entire trajectory . interaction of the substrate with the active site residues is limited to the formation of a hydrogen bond with the asn258 residue , which is effectively formed throughout the whole simulation and positions the substrate for the reaction with the activated dioxygen . however , there exists a short hydrogen bond chain at the entrance to the active site , which involves two water molecules , asn199 residue and the ( hydro)peroxo ligand bound to fe ion and might thus be potentially important for the catalytic processes involving this moiety .

the m5.1 la habra earthquake occurred on 28 march 2014 at a depth of ~5.85 km ( 33.9225n , 117.9352w ) beneath suburban la habra at the northeastern margin of the los angeles basin [ wright , 1991 ] in southern california . global positioning system ( gps ) geodesy and interferometric synthetic aperture radar ( insar ) show that the northern los angeles region is shortening at a rate of 4.5 1 mm / yr in a northsouth direction [ argus et al . , the style of tectonic deformation in the region is influenced by northwest trending rightlateral strikeslip faults associated with the peninsular ranges and san andreas plate boundary fault system , and northsouth shortening along north dipping thrust faults , often associated with oblique leftlateral motion and eastwest trending folds of the transverse ranges [ yeats , 2004 ] . seismic hazard assessments for the region have focused on major faults such as the strikeslip newportinglewood fault that caused the destructive 1933 mw 6.4 long beach earthquake [ hauksson and gross , 1991 ] and the blind thrust system that generated the 1994 m6.7 northridge earthquake [ walls et al . , the la habra earthquake sequence occurred between the rightlateral strikeslip whittier fault and the puente hills thrust fault , above a regional decollement [ yang and hauksson , 2011 ] . a surprising amount of damage occurred , despite the moderate main shock magnitude and peak ground accelerations of 0.7 g and 0.35 g northeast and southeast of and near the epicenter ( usgs : peak ground acceleration for la habra earthquake , http://earthquake.usgs.gov/earthquakes/shakemap/sc/shake/15481673/stationlist.html#sce.13883 ) . more than a dozen water mains broke in la habra and fullerton alone [ tully and casiano , 2014 ] , and widespread damage to infrastructure in orange county exceeded $ 12 m [ wiskol , 2014 ] . we documented 13 water main breaks , a gas line break , and numerous pavement cracks associated with the earthquake sequence . most of these were within a 6 km radius of the epicenter and mostly occurred outside of the earthquake zone of seismicity with about half of the water main breaks occurring in the west coyote hills to the south ( figure 1 ) . red dots show locations of the m5.1 main shock , m4.1 aftershock , and m5.4 chino earthquake . relocated aftershocks are shown as green dots . modeled faults are shown in brown with the heavier reddish brown line denoting the bottom of the fault and labeled with italics . water main breaks are shown as blue dots , a gas line break as a red open circle , an observed fault kink band near trojan way as a triangle , and road cracks as red crosses . the 5.85 km deep main shock was followed by a relatively shallow aftershock sequence extending upward from a depth of 7 km to approximately 3 km below the communities of la habra , fullerton , and brea . the northeast trend of aftershocks is consistent with one of the focal mechanism solutions , indicating main shock rupture of a northeast striking , northwest dipping leftlateral oblique thrust fault . the largest aftershock , m4.1 , occurred on 29 march 2014 at a depth of 6.38 km ( 33.9563n , 117.8970w ) . both events were widely felt in southern california , with residents reporting maximum main shock instrumental intensity of vii in the la habra and brea epicentral area . the relatively strong shaking and largearea extent of reported motion are consistent with the shallow depth of the sequence . we determined displacements that occurred as a result of the 28 march 2014 m 5.1 la habra earthquake using gps and uninhabited aerial vehicle synthetic aperture radar ( uavsar ) measurements . both the gps and uavsar measurements show a broader pattern of deformation than would be expected from a m5.1 earthquake . we used daily gps positions produced by the university of nevada , reno , within a 50 km radius of the la habra earthquake to estimate offsets from the event [ blewitt et al . , 2013 ] . we averaged daily positions for these 32 stations for 15 days before the event and differenced those from 14 day averages after the event . the gps results ( figure 2 ) show a pattern of northsouth shortening and westward motion aligned with the center of the zone of convergence , which extends about 35 km north and 20 km south of the la habra earthquake epicenter . the band of westward extrusion is about 40 km wide , extending 25 km west and 15 km east of the epicenter . displacements at the time of the la habra earthquake for gps stations within a 50 km radius of the epicenter ( red circle around black dot near snhs ) . the m5.1 main shock , m4.1 aftershock , and m5.4 chino hills earthquake are noted by red circles around a black dot . since 2009 , we have collected measurements from nasa 's uavsar l band radar instrument to monitor deformation across the los angeles region . the los angeles region was selected in part to test earthquake forecasting methodology [ rundle et al . , 2002 , 2003 ; tiampo et al . , 2002 ; holliday et al . , 2005 , 2007 ] , which indicated a high probability of a > m5.0 earthquake at the front of the transverse ranges in the chino hills area . north and south looking uavsar lines were flown before the earthquake on 22 january 2014 . the north looking line was remeasured 3 days after the earthquake on 31 march 2014 , and the south looking line was remeasured a week after the event on 4 april 2014 . the uavsar repeat pass interferometry ( rpi ) products show uplift that is consistent with the location of the main shock beneath the town of la habra ( figure 3 ) . the results also show considerable aseismic northward horizontal deformation with minor uplift in the west coyote hills , south of the relocated seismicity . a small narrow band of shortening was also observed with uavsar , and confirmed with on the ground field observations , at the trojan way kink band , nearly one fault dimension southwest of the main rupture . the inset location map shows southern california with the 2007 forecast for likely earthquakes shown as red areas . ( top left ) northward looking lines with 08521 covering la habra to the south and 08523 covering the san gabriel valley to the north . ( top right ) the southward looking lines with 26522 covering la habra and 26524 covering the san gabriel valley . the discontinuity across the tracks is due to the ground elevation or look angle varying by 40 across the swath due to the aircraft flying at a much lower elevation than a spacecraft . uavsar data can have large errors on the scale of several km from unmodeled aircraft motion and troposphere error , but it is very precise over a few hundred meters . the results of the image pairs we show here were for lines flown with opposite looks hours apart on 22 january 2014 , and 4 days apart following the earthquake and troposphere errors would not be correlated . the results near the epicenter are consistent with spaceborne insar images of the event [ fielding et al . , 2015 ] . the uavsar measurements were optimally oriented to detect the northward motion of the west coyote hills and shortening of the trojan way kink band . the northerly tracks of the spaceborne radar assets were subparallel to the direction of motion of those two features and as a result were not sensitive to motion in the northerly direction . we inverted the gps and uavsar measurements with numerous starting nominal fault parameters to determine the structures responsible for the observed deformation field . our best fit model was built up by inverting for deformation on individual structures in subregions identified by surface deformation gradients in the gps or uavsar data . because all of the modeled structures are buried the data are best fit by the following deformation sources , with parameters shown in table 1 . leftlateral oblique thrust motion is associated with the main shock and bounds the southeast margin of the aftershock zone . movement occurred on shallow lowangle northward thrusts in the west coyote hills and on a fairly extensive but lowslip northeast striking northwest dipping oblique leftlateral fault crossing the chino hills ( figures 1 , 2 , 3 and table 1 ) . shallow deformation also occurred in the san gabriel valley and can best be explained by a pair of northeast striking leftlateral shear zones . shallow lowangle motion of the upper sediments can also explain the motion , but that solution is not as robust . the suite of faults is located within an intricate structural zone where deformation from regional strikeslip and thrust systems overlaps . the modeled faults represent the general characteristics of the active structures , with the styles and locations noted emerging in the majority of the inversions . modeled structures and slip that produce the observed deformation field based on inversions of gps and uavsar observationsa the faults in the table represent the general characteristics of the structures responsible for the deformation . total summed moment of all the modeled segments is 6.761023 dyne cm . the location of the earthquake main shock and aftershock zone is northwest of but subparallel to the coyote hills segment of puente hills thrust fault [ shaw et al . , 2002 ; pratt et al . , the location , orientation , and mechanism of the modeled main shock fault ( table 1 ) are coincident with the approximate location of a tear fault in the coyote hills ( ch ) segment of the puente hills fault inferred by shaw et al . connecting thrust ramps beneath the west and east coyote hills oil fields . modeling reveals the presence of gently dipping shallow planes in the west coyote hills , west coyote 1 , and west coyote 2 ( table 1 ) , which caused northward horizontal deformation of the hills . the deformation can be seen in the interferograms as color gradients associated with the west coyote hills modeled structures ( figures 3 and 4 ) . combining the north and south looks enables estimation of horizontal and vertical motions ( figure 4 ) . the combined looks indicate about 80 mm of northward horizontal motion with a slight amount of uplift totaling 510 mm . the dips in the points along the horizontal profile at about 0.3 , 0.7 , and 1.1 km correlate with roads , suggesting that the roads restrained the northward motion at the surface . the shallow depth of our modeled faults , west coyote 1 and west coyote 2 , suggests that they are associated with a prominent shallow dipping structure identified on seismic reflection images by pratt et al . as possibly an unconformity at 0.21.0 km depth , or they are consistent with the bedding planes indicated on the map in figure 3 of pratt et al . . these structures are linked with fold growth and movement of the deeper puente hills thrust . ( top left ) a blue line marks the south to north profile shown in the plots . ( top right ) the line of sight observations show opposite change in range between the ground and the instrument . the two looks were combined to produce estimates of ( bottom left ) horizontal and ( bottom right ) vertical motion . there is controversy about whether a blind thrust fault , such as the puente hills thrust , or a strikeslip fault poses greater hazard to the metropolitan region . limitations of the data and models preclude us from accurately determining this ; however , myers et al . estimate a longterm slip rate of 1.3 mm / yr on this section of the puente hills thrust . approximately 32 mm of displacement should have accumulated since the 1989 whittier narrows earthquake , which is roughly consistent with the shallow slip that we estimated . the overlying sediments that are less compliant than deeper sediments may have relieved accumulated strain that is still present on the puente hills thrust . shallow seismic reflection profiles along trojan way in the west coyote hills [ pratt et al . , 2002 ; shaw et al . , 2002 ; leon et al . , a 9 m high surface scarp across trojan way has been interpreted as the surface expression of an active synclinal axial surface , or kink band , consistent with uplift and folding on the underlying coyote hills segment of the puente hills thrust [ pratt et al . , this kink band is 9 km southwest of the main shock epicenter but shows activation in the uavsar repeat pass interferometry in the 22 january to 31 march 2014 pair . our postseismic field investigation identified a zone of cracks that correlates with uavsar fringes , which show 4 cm of ground range change of the ground toward the instrument . about 10 km farther west , a narrow < 145 m anticline has been identified in the geologic data as an upward termination of the puente hills blind thrust fault [ dolan et al . , 2003 ] . the orientation of our measured deformation trends more northeasterly than this eastnortheast structure but is consistent with the arcuate expression of the faults and other structures . the modeled northeast striking shear zones in the san gabriel valley and chino hills ( figure 2 and table 1 ) are part of a series of incompletely mapped active leftlateral oblique faults in a structurally complex zone . the modeled faults extend northeast from mapped segments of the raymond , elysian park , and puente hills faults . recent moderate magnitude leftlateral strikeslip mechanism earthquakes on this system include the 2008 mw 5.4 chino hills earthquake ( 33.95n , 117.76w ) , which is coincident with the modeled chino hills structure , the 1988 mw 4.9 pasadena earthquake on the raymond fault , the 1988 and 1990 ml 4.6 and ml 5.2 upland earthquakes on the buried san jose fault , and the 1991 m5.6 sierra madre earthquake on the clamshellsawpit fault . notable northeast striking alignments of seismicity include the yorba linda [ yeats , 2004 ] and the fontana [ hauksson and jones , 1991 ] seismicity trends , which are subparallel to the san gabriel and chino shear zones revealed by our inversion of uavsar and gps data . the fontana seismicity trend aligns with the epicenter of the 2008 chino hills earthquake and projects southwestward into the coyote hills segment of the puente hills thrust . our results are consistent with northsouth shortening and westward escape of the crust near los angeles . the la habra earthquake that occurred at the northeastern margin of the los angeles basin reflects a broader pattern of northsouth shortening and westward escape of the upper crust [ walls et al . , this area marks the transition between two tectonic regimes where regional rightlateral shear is accommodated by major northwest trending faults of the peninsular ranges , and northsouth shortening is accommodated by north dipping thrust faults and eastwest trending folds of the transverse ranges ( hauksson and jones , 1991 ; seeber and armbruster , 1995 ; yeats , 2004 ; yang and hauksson , 2011 ) . the 2014 m5.1 la habra earthquake was a small reflection of a larger episode of deformation that occurred concurrently with the earthquake . we used the modeled fault parameters ( table 1 ) and the southern california earthquake center ( scec ) velocity 4 model to calculate the rigidity from vs and density at the center of each modeled structure [ kohler et al . , 2003 ] to estimate the geodetic moment , which is the area of the fault multiplied by slip on the fault and rigidity [ kanamori , 1978 ] . the seismic moment of this m5.1 earthquake is 5.52 10 dyne cm , which is 82% of the total observed geodetic moment of 6.76 10 dyne cm . the geodetic and seismic moment of the mainshock ruptures match , but the total geodetic moment is equivalent to an m5.2 earthquake . the earthquake main shock accounts for 100% of the geodetic moment estimated for the main shock fault alone . the observed ground deformation could represent release of accumulated tectonic strain or be the result of dynamic shaking along weaker structures than the surrounding area . if it is related to release of accumulated strain , then it provides an estimate for the amount of strain accumulated at depth that has not yet been released . using the observed slip and modeled structures as a guide , we estimate the potential for a future earthquake if these structures were to rupture from the base of the seismogenic zone to the base of the modeled faults ( figure 5 , inset ) . the results for a potential earthquake range from m6.1 for a seismic moment release of 82% of the total release and a seismogenic depth of 15 km to a m6.3 event for 100% of the release occurring seismically and a 20 km depth of the base of the seismogenic zone . paleoearthquake evidence suggests the puente hills blind thrust has produced earthquakes in the mw 7.27.5 range in the last 11,000 years [ dolan et al . , 2003 ] . reach similar conclusions that the upper compliant sediments relieve accumulated strain during moderate to large earthquakes : the uppermost few hundred meters of rock fails in oscillating dynamic friction beneath whittier narrows [ roten et al . , 2014 ] , and the ambient faultnormal stress relaxes during the process over time accommodating the shallow tectonic strain [ sleep , 2015 ] . repeated strong shaking keeps the shallow ambient stress at low levels . mori and abercrombie find that deeper earthquakes are more likely to grow into large earthquakes , which also suggests that strain is relieved aseismically or more regularly in the upper crust . gutenbergrichter relation for a 100 km radius circle from the la habra earthquake epicenter since the 1994 m6.7 northridge earthquake . n = total number of m>3 earthquakes since the last m>6 earthquake within the circle , which was the january 17 , 1994 northridge event . table shows the probably of occurrence of events of different magnitude of different time frames . seismicity data are from the advanced national seismic system ( 2015 , http://www.quake.geo.berkeley.edu/anss/catalogsearch.html ) and u.s . geological survey ( 2015 , http://earthquake.usgs.gov/earthquakes/states/events/1952_07_21.php ) . inset shows a cross section illustrating the concept of slip in the upper sediments releasing shallow accumulated strain while the lower fault segment remains locked . the gutenbergrichter relation for a 100 km radius circle around the la habra earthquake epicenter for events beginning just after the 1994 m6.7 northridge earthquake shows a deficiency of earthquakes m > 5 ( figure 5 ) , which is consistent with our analysis of the geodetic data . the deficit of earthquakes having ~ m5 and larger can be seen relative to the scaling line . the b value shown here is consistent with b values for southern california determined by mori and abercrombie for earthquakes > 9 km depth . for the gutenbergrichter relation to be completed , this deficit must eventually be filled with large earthquakes , up to m6.2 , which is consistent with the above analysis . we assign a probability to these large earthquakes using a weibull distribution [ weibull , 1951 ] and the assumption that over long times and large regions the gutenbergrichter magnitudefrequency relation is linear [ rundle et al . , 2012 ; the calculated probability for a m 6 earthquake within a circle of radius 100 km , and over the 3 years following 1 april 2015 , is 35% . for a m 5 earthquake within a circle of radius 100 km , and over the 3 years following 1 april 2015 , the probability is 99.9% . our results indicate that significant ground deformation and infrastructure damage can occur beyond the epicentral region of a moderate earthquake near los angeles . identifying specific structures most likely to be responsible for future earthquakes is difficult for this intricate network of active faults and presence of weak slip planes . the observed widespread and largely aseismic slip may be because the puente hills thrust and related faults are structurally immature [ dolan and haravitch , 2014 ] . geodetic imaging of active structures , however , can be used to identify the full extent of slip and provide a timeindependent means of estimating a lower bound of future earthquake potential . in the la habra and puente hills area observed here , the lower bound for a potential earthquake is m6.16.3 . andrea donnellan , scott hensley , john rundle , and jay parker conceived the uavsar experiment over the los angeles area . lisa grant ludwig led the geological interpretation and participated in the fieldwork with andrea donnellan . scott hensley leads the uavsar project including development of the instrument and processing of the data . jay parker estimated the gps offsets of the earthquake and developed software tools for inverting the gps and uavsar observations . marlon pierce and jun wang led the development of the computational infrastructure for analyzing , inverting , and visualizing the results presented in this paper . john rundle led the estimate of earthquake potential and probability based on the seismic data . following publication of this article , rob graves brought to our attention that several of the reported seismic moments were calculated incorrectly . and a future m 66.7 earthquake was changed to a future m 6.16.3 earthquake.in table 1 caption , the total summed moment was changed to 6.76 x 10 from 2.05 x 10.in table 1 : a new row on rigidity was added . the moments were revised as follows : 1.64 x 10 to 5.48 x 10 ; 4.02 x 10 to 7.42 x 10 ; 1.72 x 10 to 3.17 x 10 ; 1.82 x 10 to 5.48 x 10 ; 2.08 x 10 to 7.07 x 10 ; and 8.10 x 10 to 1.26 x 10 . the magnitudes ( mw ) were similarly revised as follows : 5.4 to 5.1 ; 3.7 to 3.2 ; 3.5 to 3.0 ; 4.8 to 4.5 ; 4.8 to 4.5 ; and 3.9 to 3.4.in section 5 , deeper locked faults have the potential to produce a large earthquake vp was changed to vs in the first paragraph. the end of the first paragraph in this section originally read the seismic moment of a m 5.1 earthquake is 24% of the total observed geodetic moment of 2.05 x 10 dyne cm , which is equivalent to a m 5.5 earthquake . the earthquake mainshock accounts for 30% of the geodetic moment estimated for the mainshock fault alone , suggesting that a vast majority of deformation generated during the event occurred aseismically , both locally and regionally. in the second paragraph , . . 24% of the total release and a seismogenic depth of 15 km , to a m 6.7 event was changed to . . . 82% of the total release and a seismogenic depth of 15 km , to a m 6.3 event . . . in the last sentence of the paper , the lower bound for a potential earthquake was changed from m 6.16.7 to m 6.16.3.figure 5 has been replaced so the axis is read as ( n ) , not ( gr frequency ) . the convention was to distinguish our resetting the relationship after the northridge earthquake rather than the full gr relationship for the entire catalog.the following sentence was added to the caption for figure 5 : n = total number of m>3 earthquakes since the last m>6 earthquake within the circle , which was the january 17 , 1994 northridge event.the final sentence of the acknowledgments was added . in the abstract , 24% of the total geodetic moment was changed to 82% . . and a future m 66.7 earthquake was changed to a future m 6.16.3 earthquake . in table 1 caption , the total summed moment was changed to 6.76 x 10 from 2.05 x 10 . in table 1 the moments were revised as follows : 1.64 x 10 to 5.48 x 10 ; 4.02 x 10 to 7.42 x 10 ; 1.72 x 10 to 3.17 x 10 ; 1.82 x 10 to 5.48 x 10 ; 2.08 x 10 to 7.07 x 10 ; and 8.10 x 10 to 1.26 x 10 . the magnitudes ( mw ) were similarly revised as follows : 5.4 to 5.1 ; 3.7 to 3.2 ; 3.5 to 3.0 ; 4.8 to 4.5 ; 4.8 to 4.5 ; and 3.9 to 3.4 . in section 5 , deeper locked faults have the potential to produce a large earthquake vp was changed to vs in the first paragraph . the seismic moment of a m 5.1 earthquake is 24% of the total observed geodetic moment of 2.05 x 10 dyne cm , which is equivalent to a m 5.5 earthquake . the earthquake mainshock accounts for 30% of the geodetic moment estimated for the mainshock fault alone , suggesting that a vast majority of deformation generated during the event occurred aseismically , both locally and regionally . in the second paragraph , . . . 24% of the total release and a seismogenic depth of 15 km , to a m 6.7 event was changed to . . . 82% of the total release and a seismogenic depth of 15 km , to a m 6.3 event . . in the last sentence of the paper , the lower bound for a potential earthquake was changed from m 6.16.7 to m 6.16.3 . figure 5 has been replaced so the axis is read as ( n ) , not ( gr frequency ) . the convention was to distinguish our resetting the relationship after the northridge earthquake rather than the full gr relationship for the entire catalog . n = total number of m>3 earthquakes since the last m>6 earthquake within the circle , which was the january 17 , 1994 northridge event .

abstracttectonic motion across the los angeles region is distributed across an intricate network of strikeslip and thrust faults that will be released in destructive earthquakes similar to or larger than the 1933 m6.4 long beach and 1994 m6.7 northridge events . here we show that los angeles regional thrust , strikeslip , and oblique faults are connected and move concurrently with measurable surface deformation , even in moderate magnitude earthquakes , as part of a fault system that accommodates northsouth shortening and westerly tectonic escape of northern los angeles . the 28 march 2014 m5.1 la habra earthquake occurred on a northeast striking , northwest dipping leftlateral oblique thrust fault northeast of los angeles . we present crustal deformation observation spanning the earthquake showing that concurrent deformation occurred on several structures in the shallow crust . the seismic moment of the earthquake is 82% of the total geodetic moment released . slip within the unconsolidated upper sedimentary layer may reflect shallow release of accumulated strain on stilllocked deeper structures . a future m6.16.3 earthquake would account for the accumulated strain . such an event could occur on any one or several of these faults , which may not have been identified by geologic surface mapping .

The 2014M 5.1 La Habra Earthquake Geodetic Measurements Indicate a Broad Pattern of Crustal Deformation Several Structures Produce the Observed Deformation Shallow Deformation Reflects Strain Accumulation on Deeper Faults Deeper Locked Faults Have the Potential to Produce a Large Earthquake Author Contributions Erratum

the m5.1 la habra earthquake occurred on 28 march 2014 at a depth of ~5.85 km ( 33.9225n , 117.9352w ) beneath suburban la habra at the northeastern margin of the los angeles basin [ wright , 1991 ] in southern california . global positioning system ( gps ) geodesy and interferometric synthetic aperture radar ( insar ) show that the northern los angeles region is shortening at a rate of 4.5 1 mm / yr in a northsouth direction [ argus et al . , the style of tectonic deformation in the region is influenced by northwest trending rightlateral strikeslip faults associated with the peninsular ranges and san andreas plate boundary fault system , and northsouth shortening along north dipping thrust faults , often associated with oblique leftlateral motion and eastwest trending folds of the transverse ranges [ yeats , 2004 ] . seismic hazard assessments for the region have focused on major faults such as the strikeslip newportinglewood fault that caused the destructive 1933 mw 6.4 long beach earthquake [ hauksson and gross , 1991 ] and the blind thrust system that generated the 1994 m6.7 northridge earthquake [ walls et al . , the la habra earthquake sequence occurred between the rightlateral strikeslip whittier fault and the puente hills thrust fault , above a regional decollement [ yang and hauksson , 2011 ] . a surprising amount of damage occurred , despite the moderate main shock magnitude and peak ground accelerations of 0.7 g and 0.35 g northeast and southeast of and near the epicenter ( usgs : peak ground acceleration for la habra earthquake , http://earthquake.usgs.gov/earthquakes/shakemap/sc/shake/15481673/stationlist.html#sce.13883 ) . more than a dozen water mains broke in la habra and fullerton alone [ tully and casiano , 2014 ] , and widespread damage to infrastructure in orange county exceeded $ 12 m [ wiskol , 2014 ] . we documented 13 water main breaks , a gas line break , and numerous pavement cracks associated with the earthquake sequence . most of these were within a 6 km radius of the epicenter and mostly occurred outside of the earthquake zone of seismicity with about half of the water main breaks occurring in the west coyote hills to the south ( figure 1 ) . red dots show locations of the m5.1 main shock , m4.1 aftershock , and m5.4 chino earthquake . modeled faults are shown in brown with the heavier reddish brown line denoting the bottom of the fault and labeled with italics . the 5.85 km deep main shock was followed by a relatively shallow aftershock sequence extending upward from a depth of 7 km to approximately 3 km below the communities of la habra , fullerton , and brea . the northeast trend of aftershocks is consistent with one of the focal mechanism solutions , indicating main shock rupture of a northeast striking , northwest dipping leftlateral oblique thrust fault . the largest aftershock , m4.1 , occurred on 29 march 2014 at a depth of 6.38 km ( 33.9563n , 117.8970w ) . both events were widely felt in southern california , with residents reporting maximum main shock instrumental intensity of vii in the la habra and brea epicentral area . the relatively strong shaking and largearea extent of reported motion are consistent with the shallow depth of the sequence . we determined displacements that occurred as a result of the 28 march 2014 m 5.1 la habra earthquake using gps and uninhabited aerial vehicle synthetic aperture radar ( uavsar ) measurements . we used daily gps positions produced by the university of nevada , reno , within a 50 km radius of the la habra earthquake to estimate offsets from the event [ blewitt et al . the gps results ( figure 2 ) show a pattern of northsouth shortening and westward motion aligned with the center of the zone of convergence , which extends about 35 km north and 20 km south of the la habra earthquake epicenter . displacements at the time of the la habra earthquake for gps stations within a 50 km radius of the epicenter ( red circle around black dot near snhs ) . the m5.1 main shock , m4.1 aftershock , and m5.4 chino hills earthquake are noted by red circles around a black dot . since 2009 , we have collected measurements from nasa 's uavsar l band radar instrument to monitor deformation across the los angeles region . the los angeles region was selected in part to test earthquake forecasting methodology [ rundle et al . , 2005 , 2007 ] , which indicated a high probability of a > m5.0 earthquake at the front of the transverse ranges in the chino hills area . the north looking line was remeasured 3 days after the earthquake on 31 march 2014 , and the south looking line was remeasured a week after the event on 4 april 2014 . the uavsar repeat pass interferometry ( rpi ) products show uplift that is consistent with the location of the main shock beneath the town of la habra ( figure 3 ) . the results also show considerable aseismic northward horizontal deformation with minor uplift in the west coyote hills , south of the relocated seismicity . a small narrow band of shortening was also observed with uavsar , and confirmed with on the ground field observations , at the trojan way kink band , nearly one fault dimension southwest of the main rupture . the results of the image pairs we show here were for lines flown with opposite looks hours apart on 22 january 2014 , and 4 days apart following the earthquake and troposphere errors would not be correlated . the results near the epicenter are consistent with spaceborne insar images of the event [ fielding et al . the uavsar measurements were optimally oriented to detect the northward motion of the west coyote hills and shortening of the trojan way kink band . the northerly tracks of the spaceborne radar assets were subparallel to the direction of motion of those two features and as a result were not sensitive to motion in the northerly direction . our best fit model was built up by inverting for deformation on individual structures in subregions identified by surface deformation gradients in the gps or uavsar data . because all of the modeled structures are buried the data are best fit by the following deformation sources , with parameters shown in table 1 . leftlateral oblique thrust motion is associated with the main shock and bounds the southeast margin of the aftershock zone . movement occurred on shallow lowangle northward thrusts in the west coyote hills and on a fairly extensive but lowslip northeast striking northwest dipping oblique leftlateral fault crossing the chino hills ( figures 1 , 2 , 3 and table 1 ) . shallow deformation also occurred in the san gabriel valley and can best be explained by a pair of northeast striking leftlateral shear zones . the suite of faults is located within an intricate structural zone where deformation from regional strikeslip and thrust systems overlaps . the modeled faults represent the general characteristics of the active structures , with the styles and locations noted emerging in the majority of the inversions . modeled structures and slip that produce the observed deformation field based on inversions of gps and uavsar observationsa the faults in the table represent the general characteristics of the structures responsible for the deformation . total summed moment of all the modeled segments is 6.761023 dyne cm . the location of the earthquake main shock and aftershock zone is northwest of but subparallel to the coyote hills segment of puente hills thrust fault [ shaw et al . , the location , orientation , and mechanism of the modeled main shock fault ( table 1 ) are coincident with the approximate location of a tear fault in the coyote hills ( ch ) segment of the puente hills fault inferred by shaw et al . modeling reveals the presence of gently dipping shallow planes in the west coyote hills , west coyote 1 , and west coyote 2 ( table 1 ) , which caused northward horizontal deformation of the hills . the dips in the points along the horizontal profile at about 0.3 , 0.7 , and 1.1 km correlate with roads , suggesting that the roads restrained the northward motion at the surface . the shallow depth of our modeled faults , west coyote 1 and west coyote 2 , suggests that they are associated with a prominent shallow dipping structure identified on seismic reflection images by pratt et al . there is controversy about whether a blind thrust fault , such as the puente hills thrust , or a strikeslip fault poses greater hazard to the metropolitan region . approximately 32 mm of displacement should have accumulated since the 1989 whittier narrows earthquake , which is roughly consistent with the shallow slip that we estimated . , this kink band is 9 km southwest of the main shock epicenter but shows activation in the uavsar repeat pass interferometry in the 22 january to 31 march 2014 pair . our postseismic field investigation identified a zone of cracks that correlates with uavsar fringes , which show 4 cm of ground range change of the ground toward the instrument . about 10 km farther west , a narrow < 145 m anticline has been identified in the geologic data as an upward termination of the puente hills blind thrust fault [ dolan et al . the modeled northeast striking shear zones in the san gabriel valley and chino hills ( figure 2 and table 1 ) are part of a series of incompletely mapped active leftlateral oblique faults in a structurally complex zone . the modeled faults extend northeast from mapped segments of the raymond , elysian park , and puente hills faults . recent moderate magnitude leftlateral strikeslip mechanism earthquakes on this system include the 2008 mw 5.4 chino hills earthquake ( 33.95n , 117.76w ) , which is coincident with the modeled chino hills structure , the 1988 mw 4.9 pasadena earthquake on the raymond fault , the 1988 and 1990 ml 4.6 and ml 5.2 upland earthquakes on the buried san jose fault , and the 1991 m5.6 sierra madre earthquake on the clamshellsawpit fault . notable northeast striking alignments of seismicity include the yorba linda [ yeats , 2004 ] and the fontana [ hauksson and jones , 1991 ] seismicity trends , which are subparallel to the san gabriel and chino shear zones revealed by our inversion of uavsar and gps data . our results are consistent with northsouth shortening and westward escape of the crust near los angeles . the la habra earthquake that occurred at the northeastern margin of the los angeles basin reflects a broader pattern of northsouth shortening and westward escape of the upper crust [ walls et al . , this area marks the transition between two tectonic regimes where regional rightlateral shear is accommodated by major northwest trending faults of the peninsular ranges , and northsouth shortening is accommodated by north dipping thrust faults and eastwest trending folds of the transverse ranges ( hauksson and jones , 1991 ; seeber and armbruster , 1995 ; yeats , 2004 ; yang and hauksson , 2011 ) . the 2014 m5.1 la habra earthquake was a small reflection of a larger episode of deformation that occurred concurrently with the earthquake . , 2003 ] to estimate the geodetic moment , which is the area of the fault multiplied by slip on the fault and rigidity [ kanamori , 1978 ] . the seismic moment of this m5.1 earthquake is 5.52 10 dyne cm , which is 82% of the total observed geodetic moment of 6.76 10 dyne cm . the geodetic and seismic moment of the mainshock ruptures match , but the total geodetic moment is equivalent to an m5.2 earthquake . the earthquake main shock accounts for 100% of the geodetic moment estimated for the main shock fault alone . the observed ground deformation could represent release of accumulated tectonic strain or be the result of dynamic shaking along weaker structures than the surrounding area . if it is related to release of accumulated strain , then it provides an estimate for the amount of strain accumulated at depth that has not yet been released . using the observed slip and modeled structures as a guide , we estimate the potential for a future earthquake if these structures were to rupture from the base of the seismogenic zone to the base of the modeled faults ( figure 5 , inset ) . the results for a potential earthquake range from m6.1 for a seismic moment release of 82% of the total release and a seismogenic depth of 15 km to a m6.3 event for 100% of the release occurring seismically and a 20 km depth of the base of the seismogenic zone . paleoearthquake evidence suggests the puente hills blind thrust has produced earthquakes in the mw 7.27.5 range in the last 11,000 years [ dolan et al . , 2014 ] , and the ambient faultnormal stress relaxes during the process over time accommodating the shallow tectonic strain [ sleep , 2015 ] . repeated strong shaking keeps the shallow ambient stress at low levels . mori and abercrombie find that deeper earthquakes are more likely to grow into large earthquakes , which also suggests that strain is relieved aseismically or more regularly in the upper crust . gutenbergrichter relation for a 100 km radius circle from the la habra earthquake epicenter since the 1994 m6.7 northridge earthquake . n = total number of m>3 earthquakes since the last m>6 earthquake within the circle , which was the january 17 , 1994 northridge event . inset shows a cross section illustrating the concept of slip in the upper sediments releasing shallow accumulated strain while the lower fault segment remains locked . the gutenbergrichter relation for a 100 km radius circle around the la habra earthquake epicenter for events beginning just after the 1994 m6.7 northridge earthquake shows a deficiency of earthquakes m > 5 ( figure 5 ) , which is consistent with our analysis of the geodetic data . for the gutenbergrichter relation to be completed , this deficit must eventually be filled with large earthquakes , up to m6.2 , which is consistent with the above analysis . , 2012 ; the calculated probability for a m 6 earthquake within a circle of radius 100 km , and over the 3 years following 1 april 2015 , is 35% . our results indicate that significant ground deformation and infrastructure damage can occur beyond the epicentral region of a moderate earthquake near los angeles . identifying specific structures most likely to be responsible for future earthquakes is difficult for this intricate network of active faults and presence of weak slip planes . the observed widespread and largely aseismic slip may be because the puente hills thrust and related faults are structurally immature [ dolan and haravitch , 2014 ] . in the la habra and puente hills area observed here , the lower bound for a potential earthquake is m6.16.3 . andrea donnellan , scott hensley , john rundle , and jay parker conceived the uavsar experiment over the los angeles area . jay parker estimated the gps offsets of the earthquake and developed software tools for inverting the gps and uavsar observations . marlon pierce and jun wang led the development of the computational infrastructure for analyzing , inverting , and visualizing the results presented in this paper . john rundle led the estimate of earthquake potential and probability based on the seismic data . following publication of this article , rob graves brought to our attention that several of the reported seismic moments were calculated incorrectly . and a future m 66.7 earthquake was changed to a future m 6.16.3 earthquake.in table 1 caption , the total summed moment was changed to 6.76 x 10 from 2.05 x 10.in table 1 : a new row on rigidity was added . the magnitudes ( mw ) were similarly revised as follows : 5.4 to 5.1 ; 3.7 to 3.2 ; 3.5 to 3.0 ; 4.8 to 4.5 ; 4.8 to 4.5 ; and 3.9 to 3.4.in section 5 , deeper locked faults have the potential to produce a large earthquake vp was changed to vs in the first paragraph. the end of the first paragraph in this section originally read the seismic moment of a m 5.1 earthquake is 24% of the total observed geodetic moment of 2.05 x 10 dyne cm , which is equivalent to a m 5.5 earthquake . the earthquake mainshock accounts for 30% of the geodetic moment estimated for the mainshock fault alone , suggesting that a vast majority of deformation generated during the event occurred aseismically , both locally and regionally. 24% of the total release and a seismogenic depth of 15 km , to a m 6.7 event was changed to . 82% of the total release and a seismogenic depth of 15 km , to a m 6.3 event . in the last sentence of the paper , the lower bound for a potential earthquake was changed from m 6.16.7 to m 6.16.3.figure 5 has been replaced so the axis is read as ( n ) , not ( gr frequency ) . the convention was to distinguish our resetting the relationship after the northridge earthquake rather than the full gr relationship for the entire catalog.the following sentence was added to the caption for figure 5 : n = total number of m>3 earthquakes since the last m>6 earthquake within the circle , which was the january 17 , 1994 northridge event.the final sentence of the acknowledgments was added . in the abstract , 24% of the total geodetic moment was changed to 82% . and a future m 66.7 earthquake was changed to a future m 6.16.3 earthquake . the seismic moment of a m 5.1 earthquake is 24% of the total observed geodetic moment of 2.05 x 10 dyne cm , which is equivalent to a m 5.5 earthquake . the earthquake mainshock accounts for 30% of the geodetic moment estimated for the mainshock fault alone , suggesting that a vast majority of deformation generated during the event occurred aseismically , both locally and regionally . 24% of the total release and a seismogenic depth of 15 km , to a m 6.7 event was changed to . 82% of the total release and a seismogenic depth of 15 km , to a m 6.3 event . in the last sentence of the paper , the lower bound for a potential earthquake was changed from m 6.16.7 to m 6.16.3 . the convention was to distinguish our resetting the relationship after the northridge earthquake rather than the full gr relationship for the entire catalog . n = total number of m>3 earthquakes since the last m>6 earthquake within the circle , which was the january 17 , 1994 northridge event .

lifelong premature ejaculation ( ll - pe ) is defined as a male sexual dysfunction characterized by ejaculation which always or nearly always occurs before or within about one minute of vaginal penetration and the inability to delay ejaculation on all or nearly all vaginal penetrations and negative personal consequences , such as distress , bother , frustration , and/or the avoidance of sexual intimacy . the organs involved in the emission phase comprise the epididymis , vas deferens , seminal vesicles , prostate gland , prostatic urethra , and bladder neck . the organs participating in the expulsion phase include the bladder neck and urethra , as well as the pelvic striated muscles . the central ejaculatory neural circuit comprises spinal and cerebral areas that form a highly interconnected network . the sympathetic , parasympathetic , and somatic spinal centers , under the influence of sensory genital and cerebral stimuli integrated and processed at the spinal cord level , act in synergy to command physiologic events occurring during ejaculation . a wide number of neurotransmitters , including serotonin ( 5-ht ) , dopamine , oxytocin , gamma - aminobutyric acid ( gaba ) , adrenaline , acetylcholine , and nitric oxide ( no ) , have been shown to be involved in the regulation of ejaculation . since 1984 , the nonselective alpha - blocker phenoxybenzamine was demonstrated to be able to improve premature ejaculation in humans . subsequent animal studies demonstrated that the effect of selective alpha blockade is obtained by inhibiting the contractile response of the rat seminal vesicle to electrical nerve stimulation . further evidence indicates that the contractility of the human seminal vesicle is under the control of the no - cgmp pathway , thus giving a rationale for the use of no donors in the pharmacotherapy of pe . the baseline pulse amplitude at fingertip level is highly dependent on digital blood flow and sympathetic tone , as is evidenced by a marked reduction in digital pulse amplitude after the administration of phenylephrine , an alpha - adrenergic vasoconstrictor agent . measurement of peripheral vasodilator response with a fingertip pulse amplitude tonometry ( pat ) device is emerging as a useful method for assessing vascular function [ 6 , 7 ] . in response to hyperemic flow , digital pulse amplitude increases , a response that has been shown to depend in part on no synthesis . augmentation of pulse amplitude in the finger with hyperemia is a complex response to ischemia and reflects both changes in digital flow and digital microvessels dilation and is blunted by the presence of increased sympathetic tone . in this study , we investigated the pulse amplitude hyperemic response within the first 60 seconds in men with lifelong pe without vascular risk factors as a possible marker of sympathetic overtone and compared it with age - matched normal subjects . then , in a randomized , double - blind , placebo - controlled crossover study , we investigated the effects of vardenafil fixed dose on reactive hyperemia index ( rhi ) variations and on intravaginal ejaculatory latency time ( ielt ) . vardenafil nave men aged 1840 years were included if they met the essm definition of ll - pe , as a persistent or recurrent ejaculation with minimal sexual stimulation before , upon , or shortly after penetration and before the subject wishes it . patients were included if they had a score of 11 of the premature ejaculation diagnostic tool , a 5-item questionnaire ( scored from 0 to 4 according to progressive severity of ejaculatory dysfunction ) to identify men who may have a problem with ejaculating too soon during sexual activity . they were entered into a 4-week run - in period , during which a diary of all sexual activity was filled . subjects who reported at least one intercourse episode per week and ielt 1 minute at stopwatch in 90% of intercourse attempts during the run - in period were enrolled and randomized to receive vardenafil or placebo for 8 weeks in the double - blind , placebo - controlled crossover trial ( figure 1 ) . ielt was defined as the time elapsed between penetration and ejaculation , and an ejaculation occurred before penetration was assigned an ielt of 0 min . patients had to remain in a stable , single - partner relationship and have at least one sexual intercourse episode per week throughout the treatment period . patients were excluded if they always experienced ejaculation prior to penetration or had ielt 1 minute in 90% of intercourse attempts . patients were further excluded if they had a history of ed ( score of < 22 on erectile function domain of the iief ) or other ejaculatory dysfunctions to limit the possibility of a response to vardenafil as a result of treating comorbid moderate - to - severe ed . the erectile function domain of iief is a 5-item questionnaire ( iief5 ) that investigates the presence of erectile disturbances ( scored from 1 to 5 for each item ) according to the capacity to obtain and maintain an erection successful for sexual intercourse along with the evaluation of confidence and satisfaction with sexual life . patients were also excluded if they used condoms or masturbated before sexual intercourse for purposes of decreasing penile sensitivity or used any other treatment for pe . patients were excluded if they had a history of vascular disease including stroke , myocardial infarction , unstable angina , or life - threatening arrhythmias within the past 6 months or were using organic nitrates or cytochrome p450 inhibitors . moreover , screening for hyperthyroidism ( tsh , ft3 , and ft4 ) , hypogonadism ( total testosterone ) , prolactin disorders ( prl ) , and prostate smears for detecting acute or subacute prostatitis were carried out before the study entry . prl and t were measured with chemiluminescent microparticle immunoassay ( cmia , architect system ) ( abbott laboratories , il , usa ) , with detection limits of 0.05 u / l 0.07 u / l and 0.6 ng / ml and 0.28 nmol / l , respectively ; intra- and interassay coefficients of variation for our laboratory were 3.5 and 4.2% at 5.8 ng / ml ( prl ) and 2.3 and 3.8% at 13.7 nmol / l ( t ) . serum concentrations of tsh , ft3 , and ft4 were determined by chemiluminescent microparticle immunoassay ( cmia , architect system ) ( abbott laboratories , il , usa ) , with limits of detection of 0.0025 miu / l , 1.536 pmol / l , and 5.148 pmol / l , respectively . couples were instructed to attempt sexual intercourse four or more times during the 4-week baseline period and six or more times per month during the 8-week treatment period ( minimum of 24 h between doses of medication ) and to record ielt for the first event after each dose in the event log by the stopwatch technique . patients were randomly assigned within each stratum 1 : 1 to receive placebo or fixed - dose phosphodiesterase type-5 inhibitor ( pde5-i ) vardenafil ( 10 mg ) and were given 15 doses of study medication ( one dose was one tablet ; tablets in all groups were identical in appearance since the active principle was encapsulated ) each four weeks ; one dose was to be taken 30 min before anticipated sexual intercourse , and no more than one dose was allowed to be taken in a single day . also , couples were instructed not to use condoms or topical anesthetic cream , not to pause during intercourse , or to have interrupted intromission . furthermore , they were requested not to increase their intercourse frequency , and if intercourse took place more than once in a single session , only the first intercourse ielt was measured . treatment efficacy was assessed at 8 weeks . in order to identify anxiety in the psychological context of the examined subjects , the state - trait anxiety inventory ( stai ) test , a self - administered questionnaire , it consists of two different scales ( stai - x1 and stai - x2 ) of 20 items each , with multiple choice answers ( never , sometimes , often , and always ) . stai - x1 is directed at investigating the state anxiety and gives a transitory estimation of the emotional state , which varies in intensity and fluctuates in time as a function of the stressors impinging on the individual at the moment of starting the procedure . stai - x2 is directed at relatively stable individual differences in subjects who become anxious in different circumstances . psychometric tests were performed to identify the presence of state anxiety ( stai - xl ) and trait anxiety ( stai - x2 ) before and after each treatment . the threshold scores for stai questionnaires were chosen according to previously published methods ( normal range = 2844 and 2848 for the x1 and x2 form , resp . ) . the primary endpoint of the study was to evaluate differences in rhi responses between men with ll - pe and controls and to evaluate differences in rhi responses after vardenafil or placebo in men with ll - pe . in addition , differences between anxiety scores at baseline and after different treatments were evaluated . ielt was defined as the mean duration of intercourse attempts since the last clinic visit ( past 4 weeks ) for which intravaginal ejaculation was reported ; ejaculation occurring before penetration was assigned an ielt of 0 minutes . during the screening visit , participants and partners received instructions on the ielt measurement technique , in which partners were to activate the supplied stopwatch on vaginal penetration during sexual intercourse and to stop the stopwatch on either intravaginal ejaculation or withdrawal without ejaculation . the time noted on the stopwatch at this point was recorded as the duration of sexual intercourse until ejaculation or withdrawal . each patient underwent peripheral arterial tonometry ( pat ) , a newly developed proprietary technology for noninvasively measuring the magnitude and dynamics of arterial tone changes in peripheral arterial beds . digital pulse amplitude was measured , with a pat device ( endo - pat2000 , itamar medical , caesarea , israel ) , in the fasting state in the supine position and both hands on the same level in a comfortable , thermoneutral environment , comprising a pneumatic plethysmograph that applies uniform pressure to the surface of the distal finger ( fingers ii , iii , or iv ) of each hand ( same finger on both hands ) , allowing measurement of pulse volume changes in the finger . arterial flow was interrupted for 5 min by a cuff placed on a proximal forearm at whichever occlusion pressure would be higher : 200 mmhg or 60 mmhg plus systolic blood pressure . pulse amplitude was recorded electronically in both fingers and analyzed by a computerized , automated algorithm ( owned by itamar medical ) that provided the average pulse amplitude for each 30-second interval after forearm cuff deflation up to 4 min . the hyperemic response ( called the pat ratio ) was expressed as the natural logarithm of the ratio of after deflation to baseline pulse amplitude in the hyperemic finger divided by the same ratio in the contralateral finger that served as control and expressed in percentage . we tested for differences between treatment groups by using ancova . for each 30-second interval , pat response to hyperemia is calculated as the natural logarithm transformation of pat ratio at 90- to 120-second postdeflation time period as follows : ln[(xh90120/xh120150)/(xc90120/xc120150 ) ] ; for our study , the pat ratio was modified , and calculated at 30- to 60-second postdeflation time period as follows : ln[(xh3060/xh6090)/(xc3060/xc6090 ) ] with h denoting hyperemic finger , x being the pulse amplitude , and with c denoting the control finger . a multiple regression analysis was performed for stai - x1 and stai - x2 against the variation of pat ratio . statistical analysis was performed using the computer statistical package spss/10.0 ( spss , chicago , il , usa ) and sas/6.4 ( sas institute cary , nc , usa ) . all patients were seen with their partners and interviewed about their sexual activity and patient 's ejaculation function . in total , 20 patients completed the whole randomized trial study . there were no statistical differences in patients ' characteristics at baseline at the time of randomization ( table 1 ) excepting for pedt scores and prolactin levels ( table 1 ) . at baseline , no differences in rhi values were found between groups ( figure 2(a ) ) when calculated as the natural logarithm transformation of pat ratio at 90- to 120-second after deflation ; when we calculate the differences in rhi , as the natural logarithm transformation of pat ratio at 30- to 60-second after deflation ( figure 2(b ) ) , a significant difference in the variation of rhi was found as suggested by delta_rhi calculation ( p < 0.001 , figure 2(c ) ) . all patients underwent pat evaluation either after one tablet assumption ( figure 3 ) or after 8-week on - demand treatment ( figure 4 ) . no differences in rhi values were found between groups ( figure 3(a ) ) when calculated as the natural logarithm transformation of pat ratio at 90- to 120-second after deflation ; when we calculate the differences in rhi , as the natural logarithm transformation of pat ratio at 30- to 60-second after deflation ( figure 3(b ) ) , a significant difference in the variation of rhi was found as suggested by delta_rhi calculation ( p < 0.01 , figure 3(c ) ) . also , after 8-weeks on - demand treatment , no differences in rhi values were found between groups ( data not shown ) ; when calculated as the natural logarithm transformation of pat ratio at 30- to 60-second after deflation , a significant difference in the variation of rhi was found as suggested by delta_rhi calculation in the treatment group only ( p < 0.01 , figure 4(a ) ) ; when delta_rhi was compared with placebo at the end of treatment , a significant difference between the two groups was found ( p < 0.01 , figure 4(b ) ) . interestingly , patients with higher delta_rhi at baseline showed greater decrease after vardenafil 8-week treatment ( p < 0.01 , figure 4(c ) ) . at baseline , stai - x1 scores were different between groups ( p < 0.0001 , figure 5(a ) ) . accordingly , also stai - x2 scores showed significant differences between groups ( p < 0.0001 , figure 5(b ) ) . after 8 weeks of treatment with vardenafil on - demand , a significant decrease in both stai - x1 and stai - x2 scores were found ( p < 0.001 ) which remained significant when compared with controls . interestingly , a direct relationship between delta_rhi and stai - x1 ( r2 = 0.55 , p < 0.001 ; figure 6(a ) ) and stai - x2 ( r2 = 0.59 , p < 0.001 ; figure 6(b ) ) was found . baseline ( geometric mean sd ) ielt for patients randomized to vardenafil or placebo was 0.6 0.3 minutes and 0.7 0.3 minutes , respectively ( data not shown ) . at the end of treatment , ielt time increased from 0.6 0.3 to 4.5 1.1 ( p < 0.01 , data not shown ) and from 0.7 0.3 to 0.9 1.0 ( ns ) , respectively . at the time of followup , patients who crossed over from placebo to vardenafil reported significant improvements in ielt ( from 0.9 1.0 to 2.0 0.9 min , p < 0.05 , data not shown ) , while on the other arm , a significant reduction in ielt was found compared with the end of study ( from 4.5 1.1 to 3.2 1.2 , p < 0.05 , data not shown ) ; however , this latter ielt was still superior to baseline ( p < 0.01 , data not shown ) . this represents a mean change per patient of 3.8 1.3 minutes for patients taking vardenafil and 0.2 0.3 minutes for patients taking placebo . thus , the magnitude of the increase in ielt compared with baseline was statistically significant ( p < 0.01 , data not shown ) . adverse events were significantly superior ( p < 0.01 ) with vardenafil ( versus placebo ) after 4 weeks of treatment and were headache ( 10% versus 1% ) , flushing ( 12% versus 0% ) , and dyspepsia ( 10% versus 1% ) , which tended to disappear at the end of the study ( data not shown ) . as far as we are aware , this is the first controlled study providing a possible explanation regarding the possible action of vardenafil on ejaculatory latency time . at baseline the finding that men with ll - pe have late vasodilator response at pat hyperemic response is very new . in fact , when we evaluate mean rhi scores , no difference between groups was found . indeed , a deeper evaluation of pat response within the first 60 seconds after induced shear - stress represents a new possible application of this technique . at the outset , we demonstrate a positive relationship between pat hyperemic response obtained within 60 seconds after vardenafil , treatment period and anxiety levels ; remarkably , no pat response to vardenafil was found in healthy controls . interestingly , ielt improved in men treated with vardenafil while no changes were found after placebo , as previously reported in a larger study by our group . sympathetic activation causes attenuation of the pat signal , indicative of vasoconstriction , coupled with pulse rate acceleration in addition to the typical changes reported by oximetry . this technique is widely used for the diagnostic approach to sleep apneas , by giving additional information on changes of sympathetic and parasympathetic tone during sleep . based on this knowledge , we applied this technique to men with ll - pe in order to identify differences in the autonomic control of peripheral vessels and to correlate them with the presence of anxiety . pat software employs algorithms based on weighting two features of the pat signal , each of which indicates sympathetic surge : amplitude attenuation ( reflecting vasoconstriction ) and pulse rate increase ( comparable to heart rate increase ) . the results presented in this paper , even if obtained in a small population , are consistent with the presence of sympathetic overtone as one of the possible causes of pe in this subset of young men . the significant modification of rhi ( expressed as delta rhi ) implies that vardenafil treatment was able to improve vasodilatation through a nitric - oxide-(no ) mediated pathway . given the improvements in ejaculatory function that have meaning for men with pe and their partners , the paucity of side - effects , and the fast onset of action , vardenafil may be offered as a treatment option in many men in which pe is associated with substantial psychological effects for example , interpersonal distress , decreased self - confidence , and relationship difficulties that play a major role in the pathogenesis of the disorder . indeed , psychological causes of pe , that is , increased performance anxiety , are well - known causes of pe and are usually treated with psychosexual therapies . furthermore , there is a real possibility for the motivated couple that the combination of pharmacotherapy with a pde5-i and behavioral techniques may yield greater improvements in ielt . the thoracolumbar sympathetic and the sacral parasympathetic and somatic spinal ejaculatory center ( onuf 's nucleus ) play a pivotal role in ejaculation because they integrate peripheral and central signals and send coordinated outputs to pelviperineal anatomic structures that allow a normal ejaculatory process to occur . the no / cgmp pathway seems to play a role in sexual behavior via a central effect [ 20 , 21 ] . some authors have demonstrated that no decreases central sympathetic output to the periphery via a cgmp - dependent mechanism or through interactions with other neurotransmitters . specifically , a decrease of sympathetic tone by no activity in the mpoa is related to inhibition of ejaculation . moreover , pde5i may increase sexual arousal by acting in the central nervous system , in part mediated by the activation of mesolimbic dopaminergic neurons . many lines of evidence support the presence and activity of the no / cgmp and no / camp signaling pathways in the vas deferens ( vd ) , smooth muscles , prostate , and urethra . thus , those pathways may be responsible for the peripheral effect of pde5i in the relaxation of penile corporal smooth muscles and could also affect smooth muscle in the vd , seminal vesicles ( svs ) , prostate , and urethra . a recent study demonstrated that the phasic contractile activity induced by means of electrical field stimulation of the sv tissue was most effectively inhibited by the pde4 inhibitor rolipram and the pde5 inhibitors sildenafil and vardenafil . these observations were consistent with the findings from the studies mentioned earlier , indicating that pde5 inhibitors can abolish the contractility of isolated human sv . overall , these findings are in agreement with both central and peripheral actions of vardenafil on sexual behavior and on ejaculation . the hormonal control of ejaculation and the pathophysiology of pe are still not fully understood . corona et al . reported that testosterone , prl , and tsh , significantly and independently contribute to the reported ielt variation in a large population of males complaining of sexual dysfunction . in particular , low prl and high testosterone levels were associated with a higher risk of pe , even after adjusting for confounding factors that include age , body mass index , medicaments , and smoking habit . prolactin levels have also been found to be positively correlated with reported ejaculatory latency ( from severe premature ejaculation to anejaculation ) , after excluding men with pathological hyperprolactinaemia and adjusting for ssri use . it must be emphasized that these associations were found in a population of patients without the specific use of the stopwatch method for the quantification of ielt , as it is evaluated in the present study where prl levels were higher in ll - pe group , but within the normal range . additionally , we can speculate that higher prl levels might be the result , not the cause , of the loss of ejaculatory control in our study population because of the presence of an elevated anxiety trait . there is suggestive evidence that men with pe are more likely to endorse questionnaire items indicating anxiety . given that pe may be psychogenic , at least in part , and possibly related to anxiety , while part of its definition is feelings of lack of ejaculatory control , bother , and distress ; there is considerable room for a placebo effect in all studies on the management of pe . in the present study , we observed an inconsistent placebo effect probably due to the design that considered an extension crossover period of time . we firstly demonstrated that pat hyperemic response was strongest in the 30- to 60-second interval after fingertip flow was restored . the logarithmic transformation of the pat ratio and selection of the 30- to 60-second time interval increased the overall association with the presence of a baseline constricted arterial tone , suggesting that this may be the optimal method for assessing the presence of sympathetic overactivity . the selected time period includes the portion of hyperemic response that has been previously shown to depend in part on no production . further studies in larger populations are needed to validate the possibility that the pat hyperemic response is decreased in men with ll - pe and to validate the use of pat as a diagnostic tool to identify possible responders to pde5-i in the clinical setting . in conclusion , fixed - dose vardenafil 10 mg is a safe and effective treatment in the absence of ed in young men with ll - pe and high anxiety scores . vardenafil effects on ielt and pat responses are encouraging ; even if this is a pilot study , this represents indirect evidence that neurobiological patterns of pe still need to be investigated . further larger population controlled studies to confirm the beneficial effects of vardenafil are warranted .

to elucidate whether adrenergic overtone is involved in the pathophysiology of men with lifelong ( ll ) premature ejaculation ( pe ) , we investigated differences in reactive hyperemia index ( rhi ) responses by using peripheral arterial tonometry ( pat ) . 20 men with ll - pe ( 1840 years ) were enrolled in an 8-week , double - blind , placebo - controlled , crossover study and compared with 10 age - matched controls without ll - pe . primary endpoints were pat modifications induced by vardenafil 10 mg on demand . secondary endpoints were the improvement in intravaginal ejaculatory latency time ( ielt ) as measured by the stopwatch technique and variations in anxiety scores at stai - x1 for state - anxiety and stai - x2 for trait - anxiety . at baseline , men with ll - pe showed higher rhi variation ( p < 0.001 ) , stai - x1 and stai x2 scores ( p < 0.0001 , resp . ) , and prolactin levels ( p < 0.05 ) compared with controls . vardenafil treatment markedly reduced rhi variation in men with ll - pe ( p < 0.01 ) when compared with placebo . mean changes in geometric ielt were higher after taking vardenafil ( 0.6 0.3 versus 4.5 1.1 min , p < 0.01 ) when compared with placebo . stai - x1 and stai - x2 scores fell within the normal range after treatment with vardenafil ( p < 0.01 ) . vardenafil was an effective treatment in men with ll - pe ; improvements of ielt may be due to increased no production which is able to reduce adrenergic overactivity and anxiety levels .

1. Introduction 2. Methods 3. Results 4. Discussion

lifelong premature ejaculation ( ll - pe ) is defined as a male sexual dysfunction characterized by ejaculation which always or nearly always occurs before or within about one minute of vaginal penetration and the inability to delay ejaculation on all or nearly all vaginal penetrations and negative personal consequences , such as distress , bother , frustration , and/or the avoidance of sexual intimacy . the organs involved in the emission phase comprise the epididymis , vas deferens , seminal vesicles , prostate gland , prostatic urethra , and bladder neck . the organs participating in the expulsion phase include the bladder neck and urethra , as well as the pelvic striated muscles . the central ejaculatory neural circuit comprises spinal and cerebral areas that form a highly interconnected network . the sympathetic , parasympathetic , and somatic spinal centers , under the influence of sensory genital and cerebral stimuli integrated and processed at the spinal cord level , act in synergy to command physiologic events occurring during ejaculation . a wide number of neurotransmitters , including serotonin ( 5-ht ) , dopamine , oxytocin , gamma - aminobutyric acid ( gaba ) , adrenaline , acetylcholine , and nitric oxide ( no ) , have been shown to be involved in the regulation of ejaculation . since 1984 , the nonselective alpha - blocker phenoxybenzamine was demonstrated to be able to improve premature ejaculation in humans . further evidence indicates that the contractility of the human seminal vesicle is under the control of the no - cgmp pathway , thus giving a rationale for the use of no donors in the pharmacotherapy of pe . measurement of peripheral vasodilator response with a fingertip pulse amplitude tonometry ( pat ) device is emerging as a useful method for assessing vascular function [ 6 , 7 ] . augmentation of pulse amplitude in the finger with hyperemia is a complex response to ischemia and reflects both changes in digital flow and digital microvessels dilation and is blunted by the presence of increased sympathetic tone . in this study , we investigated the pulse amplitude hyperemic response within the first 60 seconds in men with lifelong pe without vascular risk factors as a possible marker of sympathetic overtone and compared it with age - matched normal subjects . then , in a randomized , double - blind , placebo - controlled crossover study , we investigated the effects of vardenafil fixed dose on reactive hyperemia index ( rhi ) variations and on intravaginal ejaculatory latency time ( ielt ) . vardenafil nave men aged 1840 years were included if they met the essm definition of ll - pe , as a persistent or recurrent ejaculation with minimal sexual stimulation before , upon , or shortly after penetration and before the subject wishes it . patients were included if they had a score of 11 of the premature ejaculation diagnostic tool , a 5-item questionnaire ( scored from 0 to 4 according to progressive severity of ejaculatory dysfunction ) to identify men who may have a problem with ejaculating too soon during sexual activity . subjects who reported at least one intercourse episode per week and ielt 1 minute at stopwatch in 90% of intercourse attempts during the run - in period were enrolled and randomized to receive vardenafil or placebo for 8 weeks in the double - blind , placebo - controlled crossover trial ( figure 1 ) . ielt was defined as the time elapsed between penetration and ejaculation , and an ejaculation occurred before penetration was assigned an ielt of 0 min . patients were excluded if they had a history of vascular disease including stroke , myocardial infarction , unstable angina , or life - threatening arrhythmias within the past 6 months or were using organic nitrates or cytochrome p450 inhibitors . moreover , screening for hyperthyroidism ( tsh , ft3 , and ft4 ) , hypogonadism ( total testosterone ) , prolactin disorders ( prl ) , and prostate smears for detecting acute or subacute prostatitis were carried out before the study entry . prl and t were measured with chemiluminescent microparticle immunoassay ( cmia , architect system ) ( abbott laboratories , il , usa ) , with detection limits of 0.05 u / l 0.07 u / l and 0.6 ng / ml and 0.28 nmol / l , respectively ; intra- and interassay coefficients of variation for our laboratory were 3.5 and 4.2% at 5.8 ng / ml ( prl ) and 2.3 and 3.8% at 13.7 nmol / l ( t ) . serum concentrations of tsh , ft3 , and ft4 were determined by chemiluminescent microparticle immunoassay ( cmia , architect system ) ( abbott laboratories , il , usa ) , with limits of detection of 0.0025 miu / l , 1.536 pmol / l , and 5.148 pmol / l , respectively . couples were instructed to attempt sexual intercourse four or more times during the 4-week baseline period and six or more times per month during the 8-week treatment period ( minimum of 24 h between doses of medication ) and to record ielt for the first event after each dose in the event log by the stopwatch technique . patients were randomly assigned within each stratum 1 : 1 to receive placebo or fixed - dose phosphodiesterase type-5 inhibitor ( pde5-i ) vardenafil ( 10 mg ) and were given 15 doses of study medication ( one dose was one tablet ; tablets in all groups were identical in appearance since the active principle was encapsulated ) each four weeks ; one dose was to be taken 30 min before anticipated sexual intercourse , and no more than one dose was allowed to be taken in a single day . furthermore , they were requested not to increase their intercourse frequency , and if intercourse took place more than once in a single session , only the first intercourse ielt was measured . in order to identify anxiety in the psychological context of the examined subjects , the state - trait anxiety inventory ( stai ) test , a self - administered questionnaire , it consists of two different scales ( stai - x1 and stai - x2 ) of 20 items each , with multiple choice answers ( never , sometimes , often , and always ) . stai - x1 is directed at investigating the state anxiety and gives a transitory estimation of the emotional state , which varies in intensity and fluctuates in time as a function of the stressors impinging on the individual at the moment of starting the procedure . stai - x2 is directed at relatively stable individual differences in subjects who become anxious in different circumstances . psychometric tests were performed to identify the presence of state anxiety ( stai - xl ) and trait anxiety ( stai - x2 ) before and after each treatment . the threshold scores for stai questionnaires were chosen according to previously published methods ( normal range = 2844 and 2848 for the x1 and x2 form , resp . ) the primary endpoint of the study was to evaluate differences in rhi responses between men with ll - pe and controls and to evaluate differences in rhi responses after vardenafil or placebo in men with ll - pe . in addition , differences between anxiety scores at baseline and after different treatments were evaluated . during the screening visit , participants and partners received instructions on the ielt measurement technique , in which partners were to activate the supplied stopwatch on vaginal penetration during sexual intercourse and to stop the stopwatch on either intravaginal ejaculation or withdrawal without ejaculation . the time noted on the stopwatch at this point was recorded as the duration of sexual intercourse until ejaculation or withdrawal . each patient underwent peripheral arterial tonometry ( pat ) , a newly developed proprietary technology for noninvasively measuring the magnitude and dynamics of arterial tone changes in peripheral arterial beds . digital pulse amplitude was measured , with a pat device ( endo - pat2000 , itamar medical , caesarea , israel ) , in the fasting state in the supine position and both hands on the same level in a comfortable , thermoneutral environment , comprising a pneumatic plethysmograph that applies uniform pressure to the surface of the distal finger ( fingers ii , iii , or iv ) of each hand ( same finger on both hands ) , allowing measurement of pulse volume changes in the finger . the hyperemic response ( called the pat ratio ) was expressed as the natural logarithm of the ratio of after deflation to baseline pulse amplitude in the hyperemic finger divided by the same ratio in the contralateral finger that served as control and expressed in percentage . we tested for differences between treatment groups by using ancova . for each 30-second interval , pat response to hyperemia is calculated as the natural logarithm transformation of pat ratio at 90- to 120-second postdeflation time period as follows : ln[(xh90120/xh120150)/(xc90120/xc120150 ) ] ; for our study , the pat ratio was modified , and calculated at 30- to 60-second postdeflation time period as follows : ln[(xh3060/xh6090)/(xc3060/xc6090 ) ] with h denoting hyperemic finger , x being the pulse amplitude , and with c denoting the control finger . a multiple regression analysis was performed for stai - x1 and stai - x2 against the variation of pat ratio . there were no statistical differences in patients ' characteristics at baseline at the time of randomization ( table 1 ) excepting for pedt scores and prolactin levels ( table 1 ) . at baseline , no differences in rhi values were found between groups ( figure 2(a ) ) when calculated as the natural logarithm transformation of pat ratio at 90- to 120-second after deflation ; when we calculate the differences in rhi , as the natural logarithm transformation of pat ratio at 30- to 60-second after deflation ( figure 2(b ) ) , a significant difference in the variation of rhi was found as suggested by delta_rhi calculation ( p < 0.001 , figure 2(c ) ) . all patients underwent pat evaluation either after one tablet assumption ( figure 3 ) or after 8-week on - demand treatment ( figure 4 ) . no differences in rhi values were found between groups ( figure 3(a ) ) when calculated as the natural logarithm transformation of pat ratio at 90- to 120-second after deflation ; when we calculate the differences in rhi , as the natural logarithm transformation of pat ratio at 30- to 60-second after deflation ( figure 3(b ) ) , a significant difference in the variation of rhi was found as suggested by delta_rhi calculation ( p < 0.01 , figure 3(c ) ) . also , after 8-weeks on - demand treatment , no differences in rhi values were found between groups ( data not shown ) ; when calculated as the natural logarithm transformation of pat ratio at 30- to 60-second after deflation , a significant difference in the variation of rhi was found as suggested by delta_rhi calculation in the treatment group only ( p < 0.01 , figure 4(a ) ) ; when delta_rhi was compared with placebo at the end of treatment , a significant difference between the two groups was found ( p < 0.01 , figure 4(b ) ) . interestingly , patients with higher delta_rhi at baseline showed greater decrease after vardenafil 8-week treatment ( p < 0.01 , figure 4(c ) ) . at baseline , stai - x1 scores were different between groups ( p < 0.0001 , figure 5(a ) ) . accordingly , also stai - x2 scores showed significant differences between groups ( p < 0.0001 , figure 5(b ) ) . after 8 weeks of treatment with vardenafil on - demand , a significant decrease in both stai - x1 and stai - x2 scores were found ( p < 0.001 ) which remained significant when compared with controls . interestingly , a direct relationship between delta_rhi and stai - x1 ( r2 = 0.55 , p < 0.001 ; figure 6(a ) ) and stai - x2 ( r2 = 0.59 , p < 0.001 ; figure 6(b ) ) was found . baseline ( geometric mean sd ) ielt for patients randomized to vardenafil or placebo was 0.6 0.3 minutes and 0.7 0.3 minutes , respectively ( data not shown ) . at the end of treatment , ielt time increased from 0.6 0.3 to 4.5 1.1 ( p < 0.01 , data not shown ) and from 0.7 0.3 to 0.9 1.0 ( ns ) , respectively . at the time of followup , patients who crossed over from placebo to vardenafil reported significant improvements in ielt ( from 0.9 1.0 to 2.0 0.9 min , p < 0.05 , data not shown ) , while on the other arm , a significant reduction in ielt was found compared with the end of study ( from 4.5 1.1 to 3.2 1.2 , p < 0.05 , data not shown ) ; however , this latter ielt was still superior to baseline ( p < 0.01 , data not shown ) . this represents a mean change per patient of 3.8 1.3 minutes for patients taking vardenafil and 0.2 0.3 minutes for patients taking placebo . thus , the magnitude of the increase in ielt compared with baseline was statistically significant ( p < 0.01 , data not shown ) . adverse events were significantly superior ( p < 0.01 ) with vardenafil ( versus placebo ) after 4 weeks of treatment and were headache ( 10% versus 1% ) , flushing ( 12% versus 0% ) , and dyspepsia ( 10% versus 1% ) , which tended to disappear at the end of the study ( data not shown ) . as far as we are aware , this is the first controlled study providing a possible explanation regarding the possible action of vardenafil on ejaculatory latency time . at baseline the finding that men with ll - pe have late vasodilator response at pat hyperemic response is very new . indeed , a deeper evaluation of pat response within the first 60 seconds after induced shear - stress represents a new possible application of this technique . at the outset , we demonstrate a positive relationship between pat hyperemic response obtained within 60 seconds after vardenafil , treatment period and anxiety levels ; remarkably , no pat response to vardenafil was found in healthy controls . interestingly , ielt improved in men treated with vardenafil while no changes were found after placebo , as previously reported in a larger study by our group . based on this knowledge , we applied this technique to men with ll - pe in order to identify differences in the autonomic control of peripheral vessels and to correlate them with the presence of anxiety . the significant modification of rhi ( expressed as delta rhi ) implies that vardenafil treatment was able to improve vasodilatation through a nitric - oxide-(no ) mediated pathway . given the improvements in ejaculatory function that have meaning for men with pe and their partners , the paucity of side - effects , and the fast onset of action , vardenafil may be offered as a treatment option in many men in which pe is associated with substantial psychological effects for example , interpersonal distress , decreased self - confidence , and relationship difficulties that play a major role in the pathogenesis of the disorder . specifically , a decrease of sympathetic tone by no activity in the mpoa is related to inhibition of ejaculation . moreover , pde5i may increase sexual arousal by acting in the central nervous system , in part mediated by the activation of mesolimbic dopaminergic neurons . many lines of evidence support the presence and activity of the no / cgmp and no / camp signaling pathways in the vas deferens ( vd ) , smooth muscles , prostate , and urethra . thus , those pathways may be responsible for the peripheral effect of pde5i in the relaxation of penile corporal smooth muscles and could also affect smooth muscle in the vd , seminal vesicles ( svs ) , prostate , and urethra . a recent study demonstrated that the phasic contractile activity induced by means of electrical field stimulation of the sv tissue was most effectively inhibited by the pde4 inhibitor rolipram and the pde5 inhibitors sildenafil and vardenafil . the hormonal control of ejaculation and the pathophysiology of pe are still not fully understood . reported that testosterone , prl , and tsh , significantly and independently contribute to the reported ielt variation in a large population of males complaining of sexual dysfunction . in particular , low prl and high testosterone levels were associated with a higher risk of pe , even after adjusting for confounding factors that include age , body mass index , medicaments , and smoking habit . prolactin levels have also been found to be positively correlated with reported ejaculatory latency ( from severe premature ejaculation to anejaculation ) , after excluding men with pathological hyperprolactinaemia and adjusting for ssri use . it must be emphasized that these associations were found in a population of patients without the specific use of the stopwatch method for the quantification of ielt , as it is evaluated in the present study where prl levels were higher in ll - pe group , but within the normal range . additionally , we can speculate that higher prl levels might be the result , not the cause , of the loss of ejaculatory control in our study population because of the presence of an elevated anxiety trait . there is suggestive evidence that men with pe are more likely to endorse questionnaire items indicating anxiety . given that pe may be psychogenic , at least in part , and possibly related to anxiety , while part of its definition is feelings of lack of ejaculatory control , bother , and distress ; there is considerable room for a placebo effect in all studies on the management of pe . in the present study , we observed an inconsistent placebo effect probably due to the design that considered an extension crossover period of time . we firstly demonstrated that pat hyperemic response was strongest in the 30- to 60-second interval after fingertip flow was restored . the logarithmic transformation of the pat ratio and selection of the 30- to 60-second time interval increased the overall association with the presence of a baseline constricted arterial tone , suggesting that this may be the optimal method for assessing the presence of sympathetic overactivity . the selected time period includes the portion of hyperemic response that has been previously shown to depend in part on no production . further studies in larger populations are needed to validate the possibility that the pat hyperemic response is decreased in men with ll - pe and to validate the use of pat as a diagnostic tool to identify possible responders to pde5-i in the clinical setting . in conclusion , fixed - dose vardenafil 10 mg is a safe and effective treatment in the absence of ed in young men with ll - pe and high anxiety scores . further larger population controlled studies to confirm the beneficial effects of vardenafil are warranted .

cobalamins ( figure 1 ) consist of a six - coordinate low - spin co(iii ) ion that is ligated equatorially by the four nitrogens of a tetrapyrrole macrocycle , known as the corrin ring . the lower axial position is occupied by a nitrogen from the 5,6-dimethylbenzimidazole ( dmb ) base that is part of an intramolecular nucleotide loop bound to the corrin ring at c17 . at low ph , the coordinating nitrogen of the dmb group becomes protonated , which converts the cobalamins from their base - on state to their base - off form in which a solvent - derived water molecule now serves as the lower ligand . an excellent model of these base - off species at neutral ph is provided by cobinamides , which are naturally occurring cobalamin precursors that lack the terminal dmb group . chemical structure of nitrosylcobalamin ( nocbl ) , along with the numbering scheme used for the atoms in the corrin ring . colored in red is the pendant 5,6-dimethylbenzimidazole ( dmb ) group , which is absent in nitrosylcobinamide ( nocbi ) . the biologically active forms of cobalamin differ with respect to the identity of the variable upper axial ligand , with the best characterized forms being methylcobalamin ( mecbl ) and adenosylcobalamin ( adocbl ) . these molecules feature an organometallic bond between the co(iii ) ion and either a methyl group or an atp - derived 5-deoxyadenosyl group . in humans , mecbl serves as the cofactor of methionine synthase ( meth ) , involved in the synthesis of methionine from homocysteine , while adocbl is required by methylmalonyl - coa mutase ( mmcm ) for the isomerization of methylmalonyl - coa to succinyl - coa . experimental studies have revealed that both meth and mmcm are inhibited by nitric oxide ( no ) . while these enzymes are chemically unreactive toward no in their resting states , the co(ii)cbl intermediates formed during catalysis ( in the case of mmcm ) or cofactor reactivation ( meth ) are believed to be susceptible to reactions with this neutral radical species . in support of this assumption , in vitro studies have indicated that no reacts with co(ii)cbl on a microsecond time scale to yield nitrosylcobalamin ( nocbl ) , while in vivo studies in animals revealed that hydroxycobalamin supplementation can inhibit the physiological response to no , due to efficient no scavenging by the resulting co(ii)cbl formed in the cell . because nocbl does not support the catalytic activities of meth and mmcm , high cellular levels of no are expected to result in a buildup of homocysteine and methylmalonyl - coa , thereby causing disruption of the homocysteine metabolism and , possibly , the induction of methylmalonic aciduria . despite the wealth of experimental evidence supporting the formation of nocbl in vivo , the electronic structure of this species remains incompletely understood , in part because no is a redox - active ligand , thus making an oxidation state assignment for co ambiguous . using the enemark feltham formalism for metal nitrosyls , nocbl can be described as a { m no } type species , where the metal no fragment is treated as a single unit and is characterized by the total number of metal d and no * electrons . from a comparison to { m no } metalloporphyrin species , the co no fragment of nocbl is expected to adopt a bent geometry , with the no ligand exerting a strong trans influence . indeed , the crystal structure of nocbl obtained at 100 k shows a very long co n(dmb ) bond ( 2.322.35 ) , a short co no bond ( 1.911.94 ) , and a co n o bond angle of 120. these axial bond distances are consistent with structures reported for co(iii)cbl species featuring a strongly -donating upper ligand , thus supporting a co(iii)no description . additionally , the visible region of the electronic absorption ( abs ) spectrum of nocbl in aqueous solution at neutral ph is dominated by a broad asymmetric feature centered at 480 nm , which is characteristic of co(iii)cbl species . the dominant contributors to this abs feature of co(iii)cbl species are the so - called / bands that have been attributed to an electronic transition between corrin /*-based molecular orbitals . the energies of these bands have been shown to be sensitive to the electron - donating properties of the axial ligands . notably , in the case of nocbl , the peak position of the / bands ( 480 nm ) is similar to that of base - off alkylcobalamin species in which the lower axial dmb ligand is replaced by a water molecule ( e.g. , max 460 nm for mecbl at ph 2 ) . these observations suggest that the strongly -donating no ligand could promote complete dissociation of the dmb group under physiological conditions . however , detailed ph titration and nmr studies by van eldik and co - workers led to the identification of a pka value of 5.1 for the protonation and consequent dissociation of the dmb nitrogen , indicating that base - on nocbl is favored at neutral ph . yet , this pka value is only 0.5 ph units lower than that of the free nucleotide base ( pka of 5.56 in aqueous solution ) , suggesting that the co additional nmr studies by hassanin et al . led to the suggestion that at neutral ph , 33% of nocbl is present in the base - off form , with the remaining 67% being in the base - on form . to improve the current understanding of the electronic structures of nocbl in its base - on and base - off forms , we carried out abs , circular dichroism ( cd ) , magnetic cd ( mcd ) , and resonance raman ( rr ) spectroscopic studies of nocbl and nitrosylcobinamide ( nocbi ) . the spectroscopic data were analyzed within the framework of density functional theory ( dft ) and time - dependent - dft ( tddft ) calculations , employed previously with great success in computational studies of other co(iii)cbl and co(iii)cbi species . to identify the principal co ligand bonding interactions collectively , our results provide significant new insights into the spectral and electronic properties of nocbl and nocbi . additionally , by carrying out an analogous computational characterization of superoxocobalamin ( o2cbl ) , intriguing electronic structure differences between this { co o2 } species and nocbl were identified . aquacobalamin ( h2ocbl ) , dicyanocobinamide ( ( cn)2cbi ) , sodium borohydride ( nabh4 ) , potassium formate ( hcook ) , sodium nitrite ( nano2 ) , ascorbic acid , and copper tetrachloride ( cucl4 ) were purchased from sigma - aldrich and used as obtained . gaseous nitric oxide ( no ) was generated by the reaction of nano2 with ascorbic acid and aqueous cu(ii ) chloride under an argon ( ar ) atmosphere . nocbl was prepared by chemical reduction of 2 mm h2ocbl with a saturated solution of hcook under anaerobic conditions to yield co(ii)cbl , which was subsequently exposed to freshly prepared no gas for 2 h. to halt the reaction , the solution vials were purged with ar . nocbi was prepared according to the same procedure , except that in this case 2 mm diaquocobinamide ( ( h2o)2cbi ) was used as a precursor . ( h2o)2cbi was synthesized by the addition of nabh4 to an aqueous solution of ( cn)2cbi , as described in previous reports . the ph of the sample solutions was 7 unless otherwise indicated . a comparison of the electronic abs spectra of the resulting species to published spectra for nocbl and nocbi confirmed that the reactions went to completion ( see supporting information figure s1 for complete details ) . an electron paramagnetic resonance ( epr ) characterization of these samples revealed that less than 3% of co(ii)cbl remained in solution after no exposure ( see supporting information , figure s2 ) . up to 60% ( v / v ) degassed glycerol was added under anaerobic conditions to all samples used for low - temperature abs and mcd experiments to ensure glass formation upon freezing . isotopically enriched samples for rr experiments were prepared by the methods described above , except with the use of n - labeled nano2 ( 99% pure , cambridge isotope laboratories , inc . ) . frozen pellets were prepared by injecting small aliquots of fluid sample into a liquid n2 bath under an argon atmosphere . additional samples for rr studies were prepared by the addition of hcl to nocbl to reach a final ph value of < 2 . the room - temperature abs spectra of this low - ph nocbl species and nocbi were found to be superimposable , indicating that the former species was cleanly converted to its base - off form , as expected on the basis of the pka value of 5.1 reported for the ligating dmb nitrogen of nocbl . room - temperature cd and low - temperature abs , cd , and mcd spectra were acquired using a jasco j-715 spectropolarimeter in conjunction with an oxford instruments spectromag-4000 8 t magnetocryostat . all mcd spectra were obtained by taking the difference between spectra collected with the magnetic field aligned parallel and antiparallel to the light propagation axis to remove contributions from the natural cd and glass strain . room - temperature abs spectra were obtained with a cary 5e uv / vis spectrophotometer . the rr spectra were obtained upon excitation at 488.0 nm with a coherent i-305 ar ion laser and 40 mw of laser power at the sample . the scattered light was collected using a 135 backscattering arrangement , dispersed by an acton research triple monochromator ( equipped with 300 , 1200 , and 2400 grooves / mm gratings ) , and analyzed with a princeton instrument spec x : 100br deep depletion , back - thinned ccd camera . spectra were accumulated at 77 k by placing frozen pellets into a quartz finger dewar filled with liquid n2 . spectra of fluid solution samples , contained in sealed epr tubes under an argon atmosphere , were obtained with the same setup but by filling the finger dewar with an ice / water mixture . no spectroscopic changes attributable to photolytic or chemical degradation were observed during data collection . all rr spectra were baseline corrected using a piecewise line function to remove the broad nonresonant fluorescence contributions , and the intensities were normalized with respect to the features in the region between 1100 and 1400 cm . peak positions were calibrated against the 984 cm peak of a potassium sulfate standard as well as the ice peak at 228 cm for frozen samples or the water feature at 1637 cm for fluid solution samples . initial coordinates for the model of nocbl were obtained from the highest - resolution x - ray crystal structure reported by hassanin et al . in 2010 . these coordinates were also used as the basis for generating an initial model of nocbi , whereby the nucleotide loop was replaced by an h atom at the phosphate position and a water molecule was placed in the lower axial coordination site originally occupied by the dmb base ( see figure 1 ) . coordinates for the o2cbl model were obtained from the structure reported by hohenester et al . and used as is . because of the large number of atoms present in these species , smaller models were prepared for tddft excited - state and dft frequency calculations by removal of atoms that were not expected to contribute to the spectroscopic features observed experimentally . specifically , the corrin ring substituents were replaced by hydrogen atoms , except for the four methyl groups at the c1 , c2 , c5 , and c15 positions ( see figure 1 for the atom numbering scheme used ) . additionally , the two propionamide groups at c2 and c18 , along with the nucleotide loop at c7 in the case of nocbl , were replaced by methyl groups . this truncation scheme was adopted because the methyl groups at the c5 and c15 positions were shown to be crucial for an accurate treatment of the vibrational modes of the corrin ring , while the others were found to play a role in preventing excessive flattening of the corrin ring ( vide infra ) . to further increase the likelihood of obtaining realistic corrin fold angles , the entire dmb base was included in all nocbl models . full geometry optimizations of the complete and truncated cofactor models were performed with the amsterdam density functional ( adf ) 2012 suite of programs using the vosko wilk nusair ( vwn ) local - density approximation ( lda ) and the perdew , burke , and ernzerhof ( pbe ) gradient corrections for exchange and correlation . in each case , the triple with polarization ( tzp ) basis set was chosen , along with an integration constant of 5.0 and a small frozen core through 1s for c , n , and o and through 2p for co and p. the optimized models were subjected to frequency calculations to verify that a true energy minimum was found . in each case , only positive frequencies were obtained . for the computation of spectroscopic properties , the orca 2.8 software package developed by dr . f. neese was employed . while the optimized truncated models were used as - is , the full models were modified using the truncation scheme outlined above and subjected to partial geometry optimizations , whereby only the added h atoms were allowed to move . electronic transition energies and abs intensities were computed with the tddft method within the tamm the ahlrichs polarized split valence ( svp ) basis set was employed for all atoms , except for cobalt , the ligating nitrogens , and the oxygen from no , for which the triple- valence polarized ( tzvp ) basis was utilized . in each case , 40 excited states were computed by including all one - electron excitations among molecular orbitals within 3 hartree of the highest occupied molecular orbital / lowest unoccupied molecular orbital ( homo / lumo ) gap . to increase computational efficiency , the resolution of the identity ( ri ) approximation in conjunction with the sv / j auxiliary basis were employed in the evaluation of the coulomb term for all calculations . the tddft results were used as the basis for simulating abs and cd spectra , assuming that each electronic transition gives rise to a gaussian - shaped band with a full width at half maximum ( fwhm ) of v1/2 = 1250 cm . all calculated spectra were uniformly red - shifted by 2200 cm to facilitate a visual comparison with the experimental data . finally , off - resonance raman spectra for the fully optimized truncated models were computed with orca 2.8 , by evaluating the numerical frequencies and electronic polarizabilities of all normal modes using dft and the basis sets and functionals described above . the ground - state wave functions of the fully optimized models were analyzed further within the natural bond orbital ( nbo ) framework to assess the major bonding interactions , using the nbo5 interface as implemented in adf via the gennbo and adfnbo programs . the vwn / lda and the pbe gradient corrections for exchange and correlation , along with the tzp basis set ( without frozen core approximation ) , and an integration constant of 5.0 were used to compute the ground - state electron density . isosurface plots of all orbitals and electron density difference maps were generated with pymol using isodensity values of 0.03 au and 0.003 au , respectively . the low - temperature ( 4.5 k ) abs spectrum of nocbl in frozen solution exhibits partially resolved bands in the lower energy region with an intensity maximum near 20 000 cm , corresponding to the so - called / bands , along with a series of broad and more intense bands around 31 000 cm , historically referred to as the region ( figure 2 , top ) . this spectrum is very similar to those exhibited by alkyl - co(iii)cbl species , such as mecbl and adocbl , which were previously classified as unique abs spectra based primarily on the unusual appearance of the region . as in those latter spectra , the pair of features at 19 000 and 20 000 cm in the abs spectrum of nocbl also appear to correspond to the origin and first member of a vibrational progression in a totally symmetric breathing mode of the corrin ring . in support of these assignments , the corresponding features are of the same sign in both the cd and the mcd spectra of nocbl ( figure 2 , middle and bottom ) . the other bands in the visible region of the nocbl abs spectrum are due to at least one or more additional electronic transitions , as indicated by their opposite signs in the cd spectrum . consistent with its classification as a unique abs spectrum , several bands that arise from distinct electronic transitions can be identified in the region , including two similarly intense features centered at 31 000 cm with a low - energy shoulder at 28 000 cm . abs ( top ) , cd ( center ) , and 7 t mcd ( bottom ) spectra at various temperatures of nocbl . upon substitution of the dmb ligand of nocbl with a more weakly -donating water molecule in nocbi , a 1000 cm blue shift of the / bands is observed ( figures 3 , top ) . additionally , a positively signed feature at 18 500 cm appears in the cd spectrum of nocbi that has no counterpart in the nocbl spectrum , while the prominent negative feature at 19 000 cm decreases in intensity and shifts to higher energy ( figure 3 , middle ) . because the mcd spectra of nocbl and nocbi are essentially temperature - independent in the 4.5 to 50 k range ( supporting information , figures s4 and s5 ) , it can be concluded that both species possess diamagnetic ( s = 0 ) ground states , consistent with a co(iii)/no oxidation state assignment . previous studies of other co(iii ) corrinoids have revealed that the spectral changes in the / region that occur in response to a dmb h2o lower ligand substitution reflect a stabilization of the homo relative to the lumo , the extent of which depends on the -donating strength of the upper axial ligand . as the blue shift of the / bands from nocbl to nocbi ( 1000 cm ) is considerably smaller than the shift observed from mecbl to mecbi ( 2500 cm ) , no may appear to be a weaker -donating ligand than a methyl group . however , since the / bands of nocbl occur at higher energies than those of mecbl and all other alkylcobalamins , their small blue shifts from nocbl to nocbi could also be due to the fact that the dmb moiety is only weakly interacting with the co ion in the former species , as suggested by the unusually long co abs ( top ) , cd ( center ) , and 7 t mcd ( bottom ) spectra at various temperatures of nocbi , a spectroscopic model of base - off nocbl . intriguingly , while the abs and cd spectra of nocbi obtained at 4.5 and 300 k differ insignificantly with respect to band positions ( figure 3 , top and middle ) , the abs spectrum of nocbl collected in fluid solution at 300 k shows the / bands blue - shifted by 850 cm from their positions at 4.5 k ( figure 2 , top ) . significant temperature - dependent changes are also observed in the cd spectrum of nocbl , most notably a drastic decrease in the intensity of the lowest - energy , negatively signed feature at 18 000 cm and the appearance of a weak , positively signed feature at even lower energy ( figure 2 , middle ) . as a result , the room - temperature abs and cd spectra of nocbl quite closely resemble those of nocbi . collectively , these results suggest that by increasing the temperature from 4.5 to 300 k , the base - on to base - off equilibrium for nocbl changes , favoring dissociation of the n(dmb ) ligand at high temperatures . however , consistent with published nmr results , a sizable fraction of nocbl must remain in the base - on state even under ambient conditions , since the 300 k abs spectra of nocbl and nocbi are not superimposable ( see supporting information , figure s1 ) . yet , while the nmr data were interpreted to indicate that at room temperature 67% of the nocbl molecules are in the base - on state , the traces obtained by adding the 4.5 k abs or cd spectra of nocbl ( scaled by 0.67 ) and nocbi ( scaled by 0.33 ) differ from the 300 k abs and cd spectra of nocbl . n(dmb ) bond of base - on nocbl is longer at 300 k than it is at 4.5 k. the 77 k rr spectrum of nocbl obtained with laser excitation at 488.0 nm ( 20,490 cm ) shows strong enhancement of four features between 1450 and 1650 cm . on the basis of our recent study of the vibrational properties of vitamin b12 and its reduced forms , all of these features can be attributed to corrin - based vibrational modes . three of them ( termed s1s3 ) arise from totally symmetric modes ( assuming an effective cs symmetry , with the pseudo mirror plane being oriented along the co and c10 atoms and perpendicular to the corrin ring plane ) and are thus particularly strongly resonance enhanced , while the fourth ( as ) is associated with an asymmetric stretching mode . a more detailed analysis of these modes within the framework of our dft frequency calculations is provided below . a comparison of the low - energy regions of the rr spectra of nocbl and its nocbl isotopomer reveals an isotope - sensitive feature at 532 cm ( figure 4 , top ) , a region where the co no stretching and co subtraction of the nocbl from the nocbl trace yields a difference spectrum that shows an apparent shift of the 532 cm feature to 496 cm upon no no substitution . an analogous isotope - sensitive feature is present in the rr spectrum of base - off nocbl ( our model of nocbi , figure 4 , bottom traces ) , though in this case a much better - resolved difference spectrum is obtained . closer examination of this difference spectrum clearly discloses the presence of a shoulder on the low - energy side of the positive feature , suggesting that two isotope - sensitive modes actually occur in this region . indeed , a gaussian deconvolution of the rr spectra in figure 4 reveals that the vibrational modes of base - on and base - off nocbl at 515 and 532 cm shift to 500 and 521 cm , respectively , upon no no substitution ( see supporting information , figure s9 ) . in a previous rr study of nocbl , a single isotope - sensitive peak was observed at 514 cm that shifted to 496 cm upon no no substitution . however , the spectral resolution of these published data appears to be relatively low , as only two high - energy ( > 1,500 cm ) features associated with corrin - based modes could be identified , compared to the four features that are readily apparent in our spectra ( figure 5 ) . additionally , considering that mode coupling typically leads to lower isotope shifts than expected , the reported isotope shift of 18 cm , which largely exceeds the 14 cm decrease in vibrational frequency calculated using a harmonic oscillator model for a localized co low - energy region of rr spectra of nocbl and its no - enriched isotopomer in the base - on ( top ) and base - off ( bottom ) conformations , obtained at 77 k with 488 nm ( 20 491 cm ) laser excitation . a difference spectrum for each conformation is included below the two data sets to highlight the isotope - sensitive vibrational features . high - energy region of rr spectra of nocbl and its no - enriched isotopomer in the base - on and base - off conformations , obtained at 77 k with 488 nm ( 20 491 cm ) laser excitation . a difference spectrum for each conformation is included below the two data sets to highlight the isotope - sensitive vibrational features . the high similarity between the low - energy regions of the rr spectra of base - on and base - off nocbl ( figure 4 ) indicates that the co no bonding interaction is largely unperturbed by the dmb h2o lower - ligand substitution . consistent with this conclusion , the rr spectra of fluid solution samples of base - on and base - off nocbl are very similar to each other as well as to the corresponding 77 k spectra ( see supporting information , figures s7 and s8 ) , despite the thermochromism exhibited by nocbl . dft has been used by us with great success for the study of corrinoids in their co(iii ) , co(ii ) , and co(i ) oxidation states . while different functionals have been shown to provide variable descriptions of spectroscopic properties , careful evaluations of the computational results on the basis of experimental data have afforded a detailed understanding of the chemical and spectroscopic properties of a large number of different corrinoid species . from a recent study of mecbl , kozlowski and co - workers have suggested that pure gga functionals , rather than hybrid functionals , provide dft results more consistent with correlated wave function - based methods . furthermore , work by our group and others has revealed that complete cofactor models should be used to obtain accurate geometric structures of cobalamins , while truncated models can be used to compute various spectroscopic parameters . in light of these findings , we performed geometry optimizations of complete nocbl and nocbi species using the pure pbe functional . the resulting structures were then suitably truncated to predict the abs and cd spectra of these species using tddft , as well as to analyze their vibrational properties ( see methods for details ) . for comparison , we also carried out full geometry optimizations of these truncated models and calculated their abs and cd spectra . the most significant differences between the x - ray crystal structure and our dft - optimized model of nocbl include the co n(dmb ) bond distance and the folding of the corrin ring . while the crystal structure of nocbl shows an unusually long co n(dmb ) bond of 2.35 , our computation predicts this bond to be elongated by an additional 0.16 , to 2.51 ( see table 1 ) . a further elongation of this bond by 0.1 , along with a tilting of the dmb ring plane relative to the co n(dmb ) bond vector , occurred during the geometry optimization of our truncated model lacking the propionamide side chains . n(dmb ) bonds in our models may also stem from the neglect of intermolecular interactions that modulate the length of this bond in the solid state and in solution . n(dmb ) bond of nocbl is very weak and can thus be stretched , or potentially even broken , quite readily . distortions of the corrin ring can be assessed on the basis of the long - axis and short - axis fold angles , (la ) and (sa ) , respectively . (la ) is defined here as the angle between the plane containing the na , c4 , c5 , c6 , and nb atoms and the plane containing the nc , c14 , c15 , c16 , and nd atoms , while (sa ) corresponds to the angle between the planes containing the nd , c19 , c1 , and na atoms and the nb , c9 , c10 , c11 , and nc atoms ( see figure 1 for the atom numbering scheme used ) . as such , (la ) correlates with the amount of butterfly fold of the corrin ring and is thus particularly sensitive to the positioning or removal of the bulky dmb ligand . alternatively , (sa ) reflects the extent of ring ruffling due to steric constraints imposed on the propionamide and methyl groups on the a and d rings . as shown in table 1 , the (la ) values derived from the crystal structure and dft - optimized complete model of nocbl vary quite substantially , as expected from the sizable variation in the corresponding co in contrast , the (sa ) values are nearly identical , suggesting that the corrin ring conformation observed experimentally is minimally affected by crystal - packing effects and/or intermolecular h - bonding interactions . the fact that dft predicts both fold angles to decrease quite substantially upon removal of the propionamide side chains is consistent with our previous finding that distortions along the (la ) and (sa ) coordinates require very little energy . despite these modest discrepancies between the experimentally determined and dft - optimized nocbl structures , the metric parameters for the co n(o ) distances and co n o bond angles are identical to within 0.04 and < 1 , respectively . co bond distances reported for all other co(iii)cbl species , suggesting that this bond is exceptionally strong . although the computation appears to overestimate the n o bond distance by 0.05 , note that the value of 1.14 determined experimentally is surprisingly small , given that ( i ) the n o bond distance in nitric oxide is 1.15 and ( ii ) the no ligand in nocbl acquires a partial negative charge and thus increased -antibonding electron density ( vide infra ) . owing to the overall good agreement between the x - ray crystal structure and dft - optimized model of nocbl , a closer examination of the energy - minimized nocbi model is warranted . as expected , replacing the bulky dmb base with a water molecule in the lower axial position causes sizable changes to both of the corrin fold angles ( table 1 ) . o(h2 ) distance in our complete nocbi model is 3.58 , suggesting that the co ion resides in an effective five - coordinate ligand environment . although this distance shortens by 0.60 upon removal of the propionamide side chains , the lone pairs on oxygen in our truncated model are not actually oriented properly to engage in a bonding interaction with the co ion . a closer inspection of the complete nocbi model reveals that several of the propionamide side chains form a cage around the h2o molecule . the fact that removing these side chains causes a large rearrangement of the water molecule suggests a role of the propionamide groups in modulating the ligation of the lower ligand by imposing steric constraints and/or providing a solvent - protected pocket for the ligand . finally , as expected in light of the strong co no bonding interaction in nocbl , the co no unit is largely unaffected by the dmb h2o lower - ligand substitution ( table 2 , see nbo analysis for more details ) , with the most notable change being a small ( 0.02 ) decrease in the co n(dmb ) bonds in nocbl and mecbl , the base does play a small role in modulating the upper axial bonding interactions in these species . the computed abs spectrum for the nocbl model derived from the complete structure correctly predicts the major features observed experimentally , including the presence of two clusters of intense features near 20 000 cm and 31 000 cm , corresponding to the / region and the region , respectively ( figure 6 , top ) . the computed cd spectrum is also in reasonable agreement with our experimental spectrum , both exhibiting a derivative - shaped feature associated with two oppositely signed transitions centered at 20 000 cm and a series of features with alternating signs in the region ( supporting information , figure s10 , top ) . while the computed abs and cd spectra for the optimized truncated nocbl model are similar to those obtained with the modified complete model , the / bands are blue - shifted by 900 cm , and the lowest - energy , negatively signed feature in the cd spectrum is considerably weaker , which agrees less well with our experimental data ( supporting information , figure s11 ) . abs spectra of nocbl ( top ) and nocbi ( bottom ) collected at 4.5 k ( dotted lines ) superimposed on the td - dft results . the td - dft computed transitions ( vertical sticks ) were convoluted with gaussian bands with a constant fwhm of 1250 cm to obtain the predicted spectra plotted in dark blue for nocbl and in light blue for nocbi . in each case , the eddm for the -band transition is shown on the left , where regions of loss and gain of electron density are shown in gray and gold , respectively . the calculated spectra were uniformly red - shifted by 2200 cm to facilitate a direct comparison with the experimental results . upon dmb h2o lower - ligand substitution , the / bands in the tddft - computed abs and cd spectra blue shift by 3500 cm , and the negative cd feature at 20 000 cm decreases in intensity , causing an apparent blue shift of the derivative - shaped feature to 21 000 cm ( figure 6 , bottom , supporting information , figure s10 , bottom ) . as expected on the basis of the negligible co o(h2 ) bonding interactions in the two different nocbi models , the abs and cd spectra predicted for these species are almost identical ( supporting information , figure s12 ) . overall , the computed abs spectra for both nocbi models agree quite well with the experimental spectrum across the entire spectral range investigated , except for the presence of a small feature at 15 000 cm that is not observed experimentally . in contrast , the computed cd spectra only modestly reproduce the experimental spectrum , which is not unexpected because magnetic - dipole allowed transitions pose a significant challenge for tddft computations . while considerable differences are observed between the experimental abs , cd , and mcd spectra of nocbl and nocbi ( cf . figures 2 and 3 ) , the rr spectra obtained for these species show minimal differences with respect to the frequencies of the major corrin and co no - based modes ( vide supra ) . for this reason , and for computational practicality , the truncated nocbl and nocbi models were employed in our dft - assisted vibrational analysis . the dft - computed off - resonance raman spectra for these models exhibit four relatively intense features in the 14501650 cm region ( supporting information , figure s13 and s14 ) . a graphical representation of the corresponding normal modes of nocbi ( which are very similar to those of nocbl ) in terms of atomic displacement vectors is provided in figure 7 . the three modes that retain the approximate cs symmetry of nocbi are expected to be most strongly resonance - enhanced , because only totally symmetric modes can couple to electronic transitions and thus gain rr intensity via the franck even though dft methods typically overestimate vibrational frequencies , the agreement between the experimental and computed frequencies for these modes is excellent , thus permitting a straightforward assignment of the relevant vibrational features . specifically , the 1497 cm feature observed experimentally is assigned to the formally totally symmetric s1 mode predicted at 1506 cm , which involves nuclear motion primarily along the short axis of the corrin ring . alternatively , the 1541 and 1603 cm features in the experimental rr spectra are attributed to the s2 and s3 modes predicted at 1554 and 1613 cm , respectively . n stretching motion along the long axis of the corrin ring and symmetric methine stretching motion , respectively . the remaining feature observed at 1572 cm is then assigned to the antisymmetric as mode predicted at 1585 cm . while the large depolarization ratio ( 0.71 ) computed for as suggests that this mode should not be resonance - enhanced , it is predicted to carry significant off - resonance raman intensity and to entail large displacements of corrin ring atoms , thus facilitating intensity enhancement via herzberg teller coupling . overall , these assignments are consistent with our vibrational analysis recently carried out for cncbl and its reduced derivatives . eigenvector representations of the relevant normal modes for the truncated nocbi model , as obtained with dft . the computed frequency ( ) , isotope shift upon no no substitution ( n15 ) , depolarization ratio ( ) , off - resonance raman intensity ( i ) , and assignment are shown for each mode . our calculations for nocbi also predict two modes at 569 and 585 cm with no no isotope shifts of n15 = 7.3 and 6.0 cm , respectively . these predictions agree reasonably well with the frequencies ( 515 and 532 cm ) and isotope shifts ( n15 = 15 and 11 cm ) observed experimentally . both of these modes involve large atomic displacements along the co bending coordinates and are coupled out of phase and in phase , respectively , with corrin ring breathing modes . as such only one additional no no isotope - sensitive mode is computed at 1688 cm , corresponding to a relatively pure n while this mode is predicted to be strongly ir active , it carries negligible intensity in the computed off - resonance raman spectrum ( supporting information , figures s13 and s14 , respectively ) . this observation , as well as the lack of mechanical coupling between the n o and corrin ring stretches and the weak electronic coupling between the no moiety and corrin system ( see below ) predicted computationally are consistent with the absence of a feature attributable to the n o stretch in our experimental rr spectra of nocbl and nocbi . the good agreement between the experimental and computed abs , cd , and vibrational data presented above indicates that dft successfully models the salient bonding interactions present in nocbl and nocbi . for both species , the relative energies of the co 3d - based mos reflect the strongly - and weakly -donating nature of the tetradentate corrin ligand . among these orbitals ( see figure 8) , the co 3dx y - based mo ( # 158 in the case of nocbl ) is essentially nonbonding and thus lowest in energy , whereas the co 3dxy - based mo ( distributed over mos # 167 and # 168 due to mixing with another , energetically proximate mo ) is strongly -antibonding with respect to the co n(corrin ) bonds and therefore highest in energy ( note that the x and y axes are rotated by 45 about the z axis from their usual orientations due to the co intriguingly , the homo and lumo of nocbl derive mostly from co 3d and no *orbitals , in contrast to the case of alkylcobalamins ( e.g. , mecbl ) where these mos are primarily corrin /*-based . specifically , the homo of nocbl contains 28% co 3dz and 40% no * orbital contributions ( to differentiate between the two no * orbitals , the one with its lobes roughly parallel to the co n(o ) bond axis will be denoted as * and the other as * ) , making it strongly -bonding with respect to the co n(o ) bond ( see figure 8 for mo plots and supporting information , table s1 for compositions ) . alternatively , the lumo corresponds to the -antibonding combination of the co 3dyz and the no * orbitals ( 15% and 78% , respectively ) , with the bonding counterpart being considerably lower in energy ( mo # 162 ) . the dashed line separates occupied from empty orbitals . upon replacement of the axial dmb ligand of nocbl with a more weakly -donating water molecule in nocbi , mos with large co 3dz orbital contributions ( mos # 163 , # 164 , # 167 , and # 168 of nocbl , corresponding to mos # 128 , # 130 , # 133 , and # 134 of nocbi , see figure 9 ) are stabilized by 0.1 ev relative to the co 3dx y - based mo , which was chosen as the reference point because its composition remains essentially unchanged from nocbl to nocbi ( see supporting information , tables s1 and s2 ) . in particular , the co 3dz / no *-based homo of nocbi ( # 130 ) loses the -antibonding interaction with the lower axial ligand and gains contributions from the corrin 7 mo . this mixing causes a large stabilization of the corrin 7-based mo # 128 relative to the corrin 8-based mo # 132 of nocbi ( corresponding to mos # 163 and # 166 of nocbl ) . given the satisfactory agreement between the experimental and computed spectroscopic data of nocbl and nocbi , it is reasonable to assign the key abs features of these species within the framework of the dft - based mo descriptions as provided by the tddft results . because in the tddft formalism electronic transitions are described as linear combinations of one - electron excitations between occupied and virtual mos , it becomes difficult to identify the nature of a given transition in cases where multiple excitations make significant contributions . one approach to overcome this limitation involves computing electron difference density maps ( eddms ) , which provide a visual representation of the changes in electron density accompanying an electronic transition . on the basis of such eddms ( figure 6 ) , the electronic transitions producing the dominant contributions to the tddft - computed abs spectra can be assigned as shown in table 3 for nocbl and table 4 for nocbi . in the case of nocbl , the five lowest - energy transitions carrying significant abs intensity give rise to one broad feature in region i of the computed abs spectrum ( figure 6 , top ) , with two transitions ( involving excited states 12 and 15 ) having the largest oscillator strengths . on the basis of its eddm , transition 12 primarily entails a one - electron excitation from the corrin 7-based mo # 163 to the corrin 8-based mo # 166 , characteristic of the transition that is generally responsible for the / bands . the largest changes in electron density occur within the c9c10c11 fragment of the corrin ring , consistent with the strong rr enhancement of the s1 mode observed experimentally ( figure 5 ) . the eddms for the dominant transitions in regions ii ( excited states 21 , 23 , and 26 ) and iii ( excited state 37 ) display large changes in electron density around the dmb and no ligands , due to one - electron excitations from mos with significant orbital contributions from the dmb group . lastly , regions iv and v are dominated by several intense corrin * transitions that also cause a moderate electron density redistribution at the co center as a result of the sizable co 3d orbital character in the donor and acceptor mos . the prediction of numerous intense features in the region of the computed abs spectrum , as opposed to a single , relatively sharp band as is usually observed for co(iii)cbl species , is consistent with the unique abs spectrum obtained experimentally for nocbl . from nocbl to nocbi , td - dft predicts the most intense feature in the visible region of the abs spectrum to blue shift by 3500 cm , in qualitative agreement with the 1000 cm shift of the / bands observed experimentally ( figure 6 ) . however , additional features are present in the near - ir region of the computed abs spectrum that have no discernible counterparts in the experimental spectrum . as revealed by their eddms ( supporting information , figure s15 ) , the transitions associated with the first two features in regions 0 and ia cause a large electron redistribution within the co no moiety ( see table 4 ) . the fact that analogous features are not observed experimentally indicates that tddft incorrectly predicts the intensities and/or energies of these charge transfer ( ct)-type transitions of nocbi ; similar cases where tddft fails to properly describe ct excited states are well - documented in the literature . the remaining changes in the computed abs spectra from nocbl to nocbi pertain to a significant intensity redistribution among the dominant features in the region , which is again in good qualitative agreement with our experimental results . because our tddft computations reproduce the key differences between the abs spectra of nocbl and nocbi observed experimentally quite well , the computed mo diagrams can be used as the basis for exploring the electronic structural origin of these differences . as described above , the dmb h2o ligand substitution causes a stabilization of mos with significant co 3dz orbital contributions relative to the other mos . of particular importance with respect to the / band transition is the stabilization of the corrin 7-based donor mo relative to the corrin 8-based acceptor mo from nocbl ( mos # 163 and # 166 ) to nocbi ( mos # 128 and # 132 ) , which readily explains the blue shift of the / bands observed experimentally ( figures 2 , 3 , and 6 ) . interestingly , in the case of alkylcobalamins that also possess a strong -donor ligand in the upper axial position , the blue shift of the / bands in response to dmb h2o ligand substitution is considerably larger than it is in the case of nocbl ; for example , 2500 cm for mecbl versus 1000 cm for nocbl . this difference can be understood in terms of the larger -donation from the upper ligand in nocbl than in alkylcobalamins , which leads to a weaker co n(dmb ) bond and , thus , to less pronounced changes in the electronic structure upon lower - ligand substitution in the former species . in fact , our computed mo description for nocbl bears some intriguing similarities to that developed previously for co(ii)cbl , especially with regard to the energies of the co 3d - based mos relative to the corrin /*-based mos . this observation indicates that the high degree of -donation from the occupied no * orbital into the formally unoccupied co 3dz orbital leads to a significant decrease in the effective nuclear charge experienced by the co 3d orbitals in nocbl . however , the no co charge donation is partially compensated for by the strong -backbonding interaction involving the doubly occupied co 3dyz orbital and formally empty no * orbital . a more detailed analysis of the salient co no bonding interactions within the nbo formalism is presented next . while a relatively straightforward correlation can be established between the dft / tddft - predicted and experimentally observed properties of nocbl and nocbi , a direct evaluation of the key co no bonding interactions is complicated by the partial delocalization of the relevant mos over the corrin ring . to overcome this challenge , we resorted to the nbo formalism within which the calculated electron density is partitioned into chemically intuitive bonding mos and electron pairs so as to generate a bonding description closely adhering to the lewis structure formalism . n(o ) bonding interactions in nocbl and nocbi are essentially identical as judged on the basis of our experimental rr data and dft results ( vide supra ) , the following analysis will focus on the electronic structure of nocbi . the relevant nbos , labeled by their primary orbital contributors and classified either as bonding ( bd ) or antibonding ( bd * ) with respect to the co no bond , are shown in figure 10 ( left ) . as expected on the basis of the dft results described above , the co 3dz bd - nbo is characterized by a highly covalent co no -bonding interaction . however , shortcomings of the lewis structure - like description for nocbi are evident from the relatively large occupancies of several formally empty orbitals , most importantly the antibonding co 3dxy bd*-nbo and co 4s bd*-nbo , as well as the occupancies of considerably less than 2 of some of the formally doubly occupied orbitals . these observations indicate that deviations from a single lewis structure , treated within the nbo framework as donor acceptor hyperconjugation interactions , are important for properly describing the bonding interactions in nocbi . these interactions can be fully accounted for by a transformation of the canonical mos to a set of natural localized mos ( nlmos ) , which are constructed to retain a large amount of nbo character . these nlmos possess integer occupancies , like the canonical mos , and thus provide a useful connection between the canonical mos and the nbos . isosurface plots of the metal - based nbos ( in blue / gray ) and corresponding nlmos ( in orange / gray ) of nocbi . the composition of the nbos and their percent contributions to the nlmos are provided in table 4 the relevant nlmos for nocbi are shown in figure 10 ( right ) , and their compositions are provided in table 5 . inspection of the co 3dz bd - nlmo discloses a very covalent -bonding interaction between the co ion and no ( 58% co 3dz and 42% n(o ) 2pz orbital contributions , see table 5 ) , as anticipated on the basis of the canonical mo and nbo compositions . intriguingly , while the co 3dz bd - nlmo as well as the nonbonding co 3dx y and 3dxz nlmos are very similar to their corresponding nbos , the 3dyz bd - nlmo shows distinct differences . the composition of this nlmo reveals that it only retains 83.1% of pure co 3dyz nbo character ( in comparison to > 95% for the other co - based nlmos ) due to a sizable -backbonding interaction between the co 3dyz orbital and the no * orbital ( figure 10 , center columns ) . since the co 3dyz orbital only mixes with the no * orbital ( figure 10 ) , the amount of charge backdonation from co to no can be estimated to be 0.32 electrons . because of the presence of this -backbonding interaction , the co ion does not retain a co(ii)-like electronic structure as might be anticipated on the basis of the large donation from the no * orbital to the co 3dz orbital . lastly , the unoccupied co 3dz bd*-nlmo and co 3dxy bd*-nlmo are -antibonding with respect to the corrin ring and the axial ligand orbitals , as expected , and retain > 95% of nbo character ( see table 5 and figure 10 , outside columns ) . na indicates values not availalable from nlmo composition analysis , but estimated to be close to 100% . the nbo / nlmo results can also be used to estimate the number of electrons in the co 3d atomic orbitals ( aos ) and thus an effective oxidation state of the co ion . as the number of d electrons is sensitive to the covalency of the metal ligand bonding interactions as well as the delocalization of the 3d orbitals , the following approach was used to estimate this value ( see supporting information for additional details ) . first , the % nbo composition of each nlmo ( see table 5 ) was used to calculate the fractional occupancy of the co 3d - based nbo(s ) contributing to this particular nlmo . this electron count was then partitioned into co 3d - based and ligand - based contributions , estimated based on the % co 3d contributions to each nbo . finally , the fractional co 3d electron counts for all nlmos obtained using this procedure were added up to estimate the total number of co 3d electrons . using this approach , an effective number of co 3d electrons of 6.27 was obtained for nocbi , larger than a value of 6 expected for a co(iii ) species . this result corroborates the conclusion drawn from the energies and compositions of the canonical mos regarding the relatively high co(ii ) character in the electronic structures of nocbl and nocbi . thus , while the large amount of electron donation from no to co in the co 3dz bd - nlmo is partially offset by the strong -backbonding interaction in the co 3dyz bd - nlmo , our nbo / nlmo analysis clearly shows that a co(iii)no description for nocbi , and by analogy nocbl , is inaccurate . rather , these species are better described as resonance hybrids with both co(iii)no and co(ii)no limiting structures being major contributors . given the roles of no as a blood - pressure regulator , neurotransmitter , and second messenger , as well as a cytotoxic agent in immunological response , the ability of co(ii)cbl to scavenge this molecule in vivo to form nocbl is of considerable interest . additionally , nocbl has been shown to be potentially useful for targeted no delivery as a chemotherapeutic or vasodilating agent , since its co no bond can be broken under physiological conditions for controlled no release . thus , elucidating the nature of the bonding interaction between the no ligand and the co center in nocbl represents an important step toward the development of an improved understanding of the chemical and biological properties of this molecule . previous reports have highlighted the unique structural properties of nocbl relative to other biologically relevant cobalamins , most notably the presence of an unprecedentedly long co however , because nocbl exhibits similar spectroscopic signatures as the well - characterized alkyl - co(iii)cbls , such as mecbl and adocbl , it has generally been described as a co(iii)no species . to test this assumption , we developed experimentally validated electronic structure descriptions for nocbl in its base - on and base - off states . while our abs spectrum of nocbl in fluid solution at 300 k is similar to those reported in the literature , we discovered a previously unobserved thermochromism for this species ( supporting information , figures s3 ) . from a comparison to the abs data obtained for nocbi , a model of base - off nocbl , we conclude that an increase in temperature from 4.5 to 300 k causes a shift in the base - on to base - off equilibrium for nocbl , favoring dissociation of the n(dmb ) ligand at high temperatures . this change in co coordination environment has , however , negligible effects on the co no bonding interaction , as evidenced by our rr data , tddft results , and nbo analysis . the origin and implications of these findings are discussed below . collectively , our spectroscopic and computational data reveal that nocbl is inadequately described as a co(iii)no species , because significant charge donation from the no ligand notably alters the effective nuclear charge of the metal center . compared to the methyl group of mecbl , the no ligand of nocbl is an even stronger -donor , therefore inducing additional co 3dz orbital character into the homo and further enhancing the co our dft - computed mo description indicates that the formally unoccupied co 3dz orbital contributes as much as 29% to the homo of nocbl , as compared to 7% in mecbl . n(dmb ) bond from 2.16 in mecbl to 2.35 in nocbl as determined by x - ray crystallography . the high degree of electron donation from no is particularly evident from an analysis of the nlmos and nbos ( figure 10 ) derived from the canonical mos obtained with dft . on the basis of this analysis , the doubly occupied 3dz bd - nlmo contains 58% co 3d orbital character ( table 5 ) , another unique feature of the co no bond in nocbl and nocbi compared to the axial bonds in other co(iii ) corrinoid species is the presence of a sizable -backbonding interaction involving the co 3dyz and no * orbitals . inspection of the co 3dyz bd - nlmo reveals that the no ligand contribution to this orbital is 16% , leading to an estimate for the extent of donation of 0.32 electrons . this backbonding partially compensates for the large amount of donation from the no ligand into the co 3dz orbital , and represents an unprecedented mechanism by which the electronic properties of the co(iii ) ion can be modulated by the axial ligands . the high degree of no co donation in nocbl weakens the co n(dmb ) bond to the point that at high temperature , the base - on to base - off equilibrium favors a unique pentacoordinate species in aqueous solution . in the case of nocbi , an effectively five - coordinate species is present at all temperatures , suggesting that the strong trans influence exerted by the no ligand precludes the binding of a weakly donating water molecule even at 4.5 k. nonetheless , during the geometry optimization of a complete nocbi model , the lower water ligand did not fully dissociate . a comparison of this structure and that obtained for the truncated model reveals major differences in terms of the orientation of the water molecule , indicating that the propionamide side chains of the corrin ligand , which were absent in the truncated model , may play an important role in modulating the lower axial bonding interaction in cobalamins . further experimental support for our bonding description for nocbl is provided by published nmr data , which indicate that the n - resonance of the no ligand undergoes a 40 ppm upfield shift upon protonation and dissociation of the dmb ligand in the lower axial position . although the observed change in electron shielding experienced by the n nucleus was originally attributed to an increase in the co o bond angle from base - on to base - off nocbl , our rr and dft results provide compelling evidence that the co no core structures of these two species are in fact virtually identical . instead , our computational data suggest that the enhanced shielding of the n nucleus in base - off nocbl reflects the weaker -antibonding interaction between co and the lower axial ligand in that species ( cf . mos # 164 of nocbl and # 130 of nocbi in figures 8 and 9 , respectively ) , with the consequent increase in n(o ) natural charge by 4% ( see table 2 ) . previous crystallographic and epr studies have indicated that co(ii)cbl binds molecular oxygen reversibly to yield superoxocobalamin ( o2cbl ) as the first step in the autoxidation process that eventually yields h2ocbl . o2cbl may also be of physiological importance as free cobalamin is present predominantly in the co(ii)cbl state in vivo . given the similar frontier orbitals of no and o2 , which has been widely exploited to mimic the binding of dioxygen to metal centers in proteins by using nitric oxide , it is interesting to compare the electronic structures of nocbl and o2cbl . while crystallographic data for these species have revealed that both the no and o2 molecules ligate to the co center in a bent end - on fashion , the axial co n(dmb ) bond lengths differ by 0.29 between nocbl ( 2.35 ) and o2cbl ( 2.06 ) . n(dmb ) bond length in o2cbl is also considerably shorter than it is in mecbl ( 2.16 ) but very similar to that reported for cncbl ( 2.041 ) , which is considered to possess a moderately strong donor in the upper axial position based on computational analyses and the fact that this species exhibits a typical the nlmos for o2cbl are shown in figure 11 , and the compositions of the relevant nbos and nlmos are listed in table 6 ( note that our electronic structure description for o2cbl is supported by a previous single - crystal epr characterization of this species , given the good agreement between the computed and experimental g values and hyperfine coupling constants , see table 7 ) . because of the spin - unrestricted formalism used in these calculations , the nlmos have occupancies of 1.00 and are divided into a set of spin - up ( , majority spin . figure 11 , left ) and spin - down orbitals ( , minority spin . inspection of the nlmos of o2cbl reveals that these orbitals are very similar in composition to the nlmos of nocbl , although with some important differences . for both sets of spin orbitals , the co 3dxz and co 3dx y nlmos of o2cbl are nonbonding with respect to the ligand framework , in an analogous manner as the nlmos of nocbl . additionally , the compositions of the nlmos of o2cbl ( table 6 , right ) reflect the presence of a relatively covalent co o2 bond , with a co 3d orbital contribution to the co 3dz(bd ) nlmo of 41% , as compared to 58% predicted for nocbl ( see table 5 ) . however , the co contribution to the spin - up counterpart ( i.e. , the co 3dz(bd ) nlmo ) is merely 24% . thus , the net donation to the co(iii ) ion from o2 in o2cbl is smaller than it is from no in nocbl , which is consistent with the greater oxidizing power of o2 ( e = 0.16 v vs nhe ) versus no ( e = 0.8 v nhe ) . likewise , the effective number of co 3d electrons of o2cbl , computed as described above for nocbl , is 6.08 , consistent with the classification of this species as a typical co(iii)cbl species based on its short co thus , our computational results indicate that o2cbl is adequately described as a co(iii)o species . the orbitals ( majority spin , on left ) are shown in orange / gray , while orbitals ( minority spin , on right ) are shown in yellow / gray . nlmos are labeled according to the co - based nbo providing the largest contribution . the compositions of the nlmos in terms of the corresponding nbos are provided in table 5 na indicates values not availalable from nlmo composition analysis , but estimated to be close to 100% . biochemical studies have shown that no can inhibit the two b12-dependent enzymes found in humans , namely , meth and mmcm , which could provide an additional pathway for the regulation of these enzymes . specifically , it was shown that inhibition of mmcm in the presence of no donors resulted in the formation of nocbl in the active site of the enzyme . for this and related enzymes , the formation of nocbl in the active site and consequent inhibition of catalytic activity would require a repair mechanism involving no dissociation or replacement of nocbl by adocbl . given the strong co no bond in nocbl as revealed by the present investigation , nocbl removal from the active site of these enzymes is expected to be the main pathway for reactivation . this process could be facilitated by the strong trans influence exerted by the no ligand . conformation , and on the basis of the results obtained in this study , it can be speculated that no coordination to the transiently formed co(ii)cbl species in the enzyme active site will considerably weaken the co similarly , for b12-dependent enzymes that bind adocbl in the base - on conformation , the co n(dmb ) bond elongation and additional geometric changes in response to no coordination to the co(ii)cbl intermediate will likely facilitate removal of the nocbl product from the active site . although more experimental evidence is needed to support these hypotheses , the results obtained from this study offer unprecedented insights into the spectral and electronic properties of nocbl and provide useful spectroscopic markers for probing the interaction of this species with b12-dependent enzymes .

nitrosylcobalamin ( nocbl ) is readily formed when co(ii)balamin reacts with nitric oxide ( no ) gas . nocbl has been implicated in the inhibition of various b12-dependent enzymes , as well as in the modulation of blood pressure and of the immunological response . previous studies revealed that among the known biologically relevant cobalamin species , nocbl possesses the longest bond between the co ion and the axially bound 5,6-dimethylbenzimidazole base , which was postulated to result from a strong trans influence exerted by the no ligand . in this study , various spectroscopic ( electronic absorption , circular dichroism , magnetic circular dichroism , and resonance raman ) and computational ( density functional theory ( dft ) and time - dependent dft ) techniques were used to generate experimentally validated electronic structure descriptions for the base - on and base - off forms of nocbl . further insights into the principal co ligand bonding interactions were obtained by carrying out natural bond orbital analyses . collectively , our results indicate that the formally unoccupied co 3dz2 orbital engages in a highly covalent bonding interaction with the filled no * orbital and that the co no bond is strengthened further by sizable -backbonding interactions that are not present in any other co(iii)cbl characterized to date . because of the substantial no to co(iii ) charge donation , nocbl is best described as a hybrid of co(iii)no and co(ii)no resonance structures . in contrast , our analogous computational characterization of a related species , superoxocobalamin , reveals that in this case a co(iii)o2 description is adequate due to the larger oxidizing power of o2 versus no . the implications of our results with respect to the unusual structural features and thermochromism of nocbl and the proposed inhibition mechanisms of b12-dependent enzymes by nocbl are discussed .

Introduction Methods Results Discussion

the biologically active forms of cobalamin differ with respect to the identity of the variable upper axial ligand , with the best characterized forms being methylcobalamin ( mecbl ) and adenosylcobalamin ( adocbl ) . in support of this assumption , in vitro studies have indicated that no reacts with co(ii)cbl on a microsecond time scale to yield nitrosylcobalamin ( nocbl ) , while in vivo studies in animals revealed that hydroxycobalamin supplementation can inhibit the physiological response to no , due to efficient no scavenging by the resulting co(ii)cbl formed in the cell . from a comparison to { m no } metalloporphyrin species , the co no fragment of nocbl is expected to adopt a bent geometry , with the no ligand exerting a strong trans influence . however , detailed ph titration and nmr studies by van eldik and co - workers led to the identification of a pka value of 5.1 for the protonation and consequent dissociation of the dmb nitrogen , indicating that base - on nocbl is favored at neutral ph . led to the suggestion that at neutral ph , 33% of nocbl is present in the base - off form , with the remaining 67% being in the base - on form . to improve the current understanding of the electronic structures of nocbl in its base - on and base - off forms , we carried out abs , circular dichroism ( cd ) , magnetic cd ( mcd ) , and resonance raman ( rr ) spectroscopic studies of nocbl and nitrosylcobinamide ( nocbi ) . the spectroscopic data were analyzed within the framework of density functional theory ( dft ) and time - dependent - dft ( tddft ) calculations , employed previously with great success in computational studies of other co(iii)cbl and co(iii)cbi species . to identify the principal co ligand bonding interactions collectively , our results provide significant new insights into the spectral and electronic properties of nocbl and nocbi . the room - temperature abs spectra of this low - ph nocbl species and nocbi were found to be superimposable , indicating that the former species was cleanly converted to its base - off form , as expected on the basis of the pka value of 5.1 reported for the ligating dmb nitrogen of nocbl . as in those latter spectra , the pair of features at 19 000 and 20 000 cm in the abs spectrum of nocbl also appear to correspond to the origin and first member of a vibrational progression in a totally symmetric breathing mode of the corrin ring . previous studies of other co(iii ) corrinoids have revealed that the spectral changes in the / region that occur in response to a dmb h2o lower ligand substitution reflect a stabilization of the homo relative to the lumo , the extent of which depends on the -donating strength of the upper axial ligand . however , since the / bands of nocbl occur at higher energies than those of mecbl and all other alkylcobalamins , their small blue shifts from nocbl to nocbi could also be due to the fact that the dmb moiety is only weakly interacting with the co ion in the former species , as suggested by the unusually long co abs ( top ) , cd ( center ) , and 7 t mcd ( bottom ) spectra at various temperatures of nocbi , a spectroscopic model of base - off nocbl . however , consistent with published nmr results , a sizable fraction of nocbl must remain in the base - on state even under ambient conditions , since the 300 k abs spectra of nocbl and nocbi are not superimposable ( see supporting information , figure s1 ) . yet , while the nmr data were interpreted to indicate that at room temperature 67% of the nocbl molecules are in the base - on state , the traces obtained by adding the 4.5 k abs or cd spectra of nocbl ( scaled by 0.67 ) and nocbi ( scaled by 0.33 ) differ from the 300 k abs and cd spectra of nocbl . an analogous isotope - sensitive feature is present in the rr spectrum of base - off nocbl ( our model of nocbi , figure 4 , bottom traces ) , though in this case a much better - resolved difference spectrum is obtained . indeed , a gaussian deconvolution of the rr spectra in figure 4 reveals that the vibrational modes of base - on and base - off nocbl at 515 and 532 cm shift to 500 and 521 cm , respectively , upon no no substitution ( see supporting information , figure s9 ) . additionally , considering that mode coupling typically leads to lower isotope shifts than expected , the reported isotope shift of 18 cm , which largely exceeds the 14 cm decrease in vibrational frequency calculated using a harmonic oscillator model for a localized co low - energy region of rr spectra of nocbl and its no - enriched isotopomer in the base - on ( top ) and base - off ( bottom ) conformations , obtained at 77 k with 488 nm ( 20 491 cm ) laser excitation . high - energy region of rr spectra of nocbl and its no - enriched isotopomer in the base - on and base - off conformations , obtained at 77 k with 488 nm ( 20 491 cm ) laser excitation . the high similarity between the low - energy regions of the rr spectra of base - on and base - off nocbl ( figure 4 ) indicates that the co no bonding interaction is largely unperturbed by the dmb h2o lower - ligand substitution . consistent with this conclusion , the rr spectra of fluid solution samples of base - on and base - off nocbl are very similar to each other as well as to the corresponding 77 k spectra ( see supporting information , figures s7 and s8 ) , despite the thermochromism exhibited by nocbl . as shown in table 1 , the (la ) values derived from the crystal structure and dft - optimized complete model of nocbl vary quite substantially , as expected from the sizable variation in the corresponding co in contrast , the (sa ) values are nearly identical , suggesting that the corrin ring conformation observed experimentally is minimally affected by crystal - packing effects and/or intermolecular h - bonding interactions . although this distance shortens by 0.60 upon removal of the propionamide side chains , the lone pairs on oxygen in our truncated model are not actually oriented properly to engage in a bonding interaction with the co ion . finally , as expected in light of the strong co no bonding interaction in nocbl , the co no unit is largely unaffected by the dmb h2o lower - ligand substitution ( table 2 , see nbo analysis for more details ) , with the most notable change being a small ( 0.02 ) decrease in the co n(dmb ) bonds in nocbl and mecbl , the base does play a small role in modulating the upper axial bonding interactions in these species . this observation , as well as the lack of mechanical coupling between the n o and corrin ring stretches and the weak electronic coupling between the no moiety and corrin system ( see below ) predicted computationally are consistent with the absence of a feature attributable to the n o stretch in our experimental rr spectra of nocbl and nocbi . among these orbitals ( see figure 8) , the co 3dx y - based mo ( # 158 in the case of nocbl ) is essentially nonbonding and thus lowest in energy , whereas the co 3dxy - based mo ( distributed over mos # 167 and # 168 due to mixing with another , energetically proximate mo ) is strongly -antibonding with respect to the co n(corrin ) bonds and therefore highest in energy ( note that the x and y axes are rotated by 45 about the z axis from their usual orientations due to the co intriguingly , the homo and lumo of nocbl derive mostly from co 3d and no *orbitals , in contrast to the case of alkylcobalamins ( e.g. specifically , the homo of nocbl contains 28% co 3dz and 40% no * orbital contributions ( to differentiate between the two no * orbitals , the one with its lobes roughly parallel to the co n(o ) bond axis will be denoted as * and the other as * ) , making it strongly -bonding with respect to the co n(o ) bond ( see figure 8 for mo plots and supporting information , table s1 for compositions ) . alternatively , the lumo corresponds to the -antibonding combination of the co 3dyz and the no * orbitals ( 15% and 78% , respectively ) , with the bonding counterpart being considerably lower in energy ( mo # 162 ) . upon replacement of the axial dmb ligand of nocbl with a more weakly -donating water molecule in nocbi , mos with large co 3dz orbital contributions ( mos # 163 , # 164 , # 167 , and # 168 of nocbl , corresponding to mos # 128 , # 130 , # 133 , and # 134 of nocbi , see figure 9 ) are stabilized by 0.1 ev relative to the co 3dx y - based mo , which was chosen as the reference point because its composition remains essentially unchanged from nocbl to nocbi ( see supporting information , tables s1 and s2 ) . this observation indicates that the high degree of -donation from the occupied no * orbital into the formally unoccupied co 3dz orbital leads to a significant decrease in the effective nuclear charge experienced by the co 3d orbitals in nocbl . while a relatively straightforward correlation can be established between the dft / tddft - predicted and experimentally observed properties of nocbl and nocbi , a direct evaluation of the key co no bonding interactions is complicated by the partial delocalization of the relevant mos over the corrin ring . the composition of this nlmo reveals that it only retains 83.1% of pure co 3dyz nbo character ( in comparison to > 95% for the other co - based nlmos ) due to a sizable -backbonding interaction between the co 3dyz orbital and the no * orbital ( figure 10 , center columns ) . because of the presence of this -backbonding interaction , the co ion does not retain a co(ii)-like electronic structure as might be anticipated on the basis of the large donation from the no * orbital to the co 3dz orbital . lastly , the unoccupied co 3dz bd*-nlmo and co 3dxy bd*-nlmo are -antibonding with respect to the corrin ring and the axial ligand orbitals , as expected , and retain > 95% of nbo character ( see table 5 and figure 10 , outside columns ) . as the number of d electrons is sensitive to the covalency of the metal ligand bonding interactions as well as the delocalization of the 3d orbitals , the following approach was used to estimate this value ( see supporting information for additional details ) . given the roles of no as a blood - pressure regulator , neurotransmitter , and second messenger , as well as a cytotoxic agent in immunological response , the ability of co(ii)cbl to scavenge this molecule in vivo to form nocbl is of considerable interest . thus , elucidating the nature of the bonding interaction between the no ligand and the co center in nocbl represents an important step toward the development of an improved understanding of the chemical and biological properties of this molecule . to test this assumption , we developed experimentally validated electronic structure descriptions for nocbl in its base - on and base - off states . from a comparison to the abs data obtained for nocbi , a model of base - off nocbl , we conclude that an increase in temperature from 4.5 to 300 k causes a shift in the base - on to base - off equilibrium for nocbl , favoring dissociation of the n(dmb ) ligand at high temperatures . collectively , our spectroscopic and computational data reveal that nocbl is inadequately described as a co(iii)no species , because significant charge donation from the no ligand notably alters the effective nuclear charge of the metal center . compared to the methyl group of mecbl , the no ligand of nocbl is an even stronger -donor , therefore inducing additional co 3dz orbital character into the homo and further enhancing the co our dft - computed mo description indicates that the formally unoccupied co 3dz orbital contributes as much as 29% to the homo of nocbl , as compared to 7% in mecbl . on the basis of this analysis , the doubly occupied 3dz bd - nlmo contains 58% co 3d orbital character ( table 5 ) , another unique feature of the co no bond in nocbl and nocbi compared to the axial bonds in other co(iii ) corrinoid species is the presence of a sizable -backbonding interaction involving the co 3dyz and no * orbitals . inspection of the co 3dyz bd - nlmo reveals that the no ligand contribution to this orbital is 16% , leading to an estimate for the extent of donation of 0.32 electrons . this backbonding partially compensates for the large amount of donation from the no ligand into the co 3dz orbital , and represents an unprecedented mechanism by which the electronic properties of the co(iii ) ion can be modulated by the axial ligands . in the case of nocbi , an effectively five - coordinate species is present at all temperatures , suggesting that the strong trans influence exerted by the no ligand precludes the binding of a weakly donating water molecule even at 4.5 k. nonetheless , during the geometry optimization of a complete nocbi model , the lower water ligand did not fully dissociate . a comparison of this structure and that obtained for the truncated model reveals major differences in terms of the orientation of the water molecule , indicating that the propionamide side chains of the corrin ligand , which were absent in the truncated model , may play an important role in modulating the lower axial bonding interaction in cobalamins . further experimental support for our bonding description for nocbl is provided by published nmr data , which indicate that the n - resonance of the no ligand undergoes a 40 ppm upfield shift upon protonation and dissociation of the dmb ligand in the lower axial position . although the observed change in electron shielding experienced by the n nucleus was originally attributed to an increase in the co o bond angle from base - on to base - off nocbl , our rr and dft results provide compelling evidence that the co no core structures of these two species are in fact virtually identical . instead , our computational data suggest that the enhanced shielding of the n nucleus in base - off nocbl reflects the weaker -antibonding interaction between co and the lower axial ligand in that species ( cf . while crystallographic data for these species have revealed that both the no and o2 molecules ligate to the co center in a bent end - on fashion , the axial co n(dmb ) bond lengths differ by 0.29 between nocbl ( 2.35 ) and o2cbl ( 2.06 ) . n(dmb ) bond length in o2cbl is also considerably shorter than it is in mecbl ( 2.16 ) but very similar to that reported for cncbl ( 2.041 ) , which is considered to possess a moderately strong donor in the upper axial position based on computational analyses and the fact that this species exhibits a typical the nlmos for o2cbl are shown in figure 11 , and the compositions of the relevant nbos and nlmos are listed in table 6 ( note that our electronic structure description for o2cbl is supported by a previous single - crystal epr characterization of this species , given the good agreement between the computed and experimental g values and hyperfine coupling constants , see table 7 ) . thus , the net donation to the co(iii ) ion from o2 in o2cbl is smaller than it is from no in nocbl , which is consistent with the greater oxidizing power of o2 ( e = 0.16 v vs nhe ) versus no ( e = 0.8 v nhe ) . this process could be facilitated by the strong trans influence exerted by the no ligand . conformation , and on the basis of the results obtained in this study , it can be speculated that no coordination to the transiently formed co(ii)cbl species in the enzyme active site will considerably weaken the co similarly , for b12-dependent enzymes that bind adocbl in the base - on conformation , the co n(dmb ) bond elongation and additional geometric changes in response to no coordination to the co(ii)cbl intermediate will likely facilitate removal of the nocbl product from the active site .

non - small cell lung cancer ( nsclc ) is the leading cause of cancer mortality in the united states and the world , with a 5-year survival rate of only 15% for all stages combined , despite some recent advances in chemotherapeutic agents.1 , 2 the size and distribution of nsclc makes cytoreductive surgery ineffective . consequently , chemotherapy and/or radiation have been the treatments of choice . conventional chemotherapy regimens have had limited efficacy , mainly because of resistance of these cancer cells to chemotherapeutic agents.4 , 5 small - molecule inhibitors such as erlotinib and afatinib that target the tyrosine kinase domain of epidermal growth factor receptor ( egfr ) produce responses in approximately 10% of patients with nsclc.6 , 7 , 8 in patients with mutant egfr , responses to egfr - tyrosine kinase inhibitors ( egfr - tkis ) can be dramatic and may last longer than a year . in contrast , k - ras gene mutation , which occurs in approximately 30% of nsclc , has been associated with a poor response to egfr - tkis.9 , 10 , 11 , 12 , 13 it has been demonstrated previously that somatic mutations in the tyrosine kinase domain of egfr are associated with sensitivity to egfr - tkis , whereas mutations in k - ras , which encodes a guanosine triphosphotase ( gtpase ) downstream of egfr , are associated with primary resistance . however , there is an unmet need in developing the most appropriate therapy for mutant k - ras nsclc . several strategies , such as farnesyltransferase inhibitors , have been explored as possible inhibitors of mutant k - ras in lung adenocarcinomas . to date , none of these strategies have proved to be successful , mainly because of lack of specificity . small interfering rna ( sirna ) inhibition is a very viable alternative given the specificity of this technology . here we aim to harness the advantages of sirna technology as a therapeutic modality for mutant k - ras in nsclc . sirna as a therapeutic molecule is very effective at knocking down molecular pathways that are pathogenic . however , its application in the clinic is often limited by its susceptibility to enzymatic degradation in blood , non - specific uptake by cells , and the difficulty involved in the transfection of sirna to cells because of its relatively large size and polarity.16 , 17 . clearance by the mononuclear phagocyte system ( mps ) is another limiting factor affecting the possible therapeutic application of sirna.18 , 19 , 20 recently , our group developed a novel hybrid nanoparticle delivery system composed of human immunoglobulin g ( igg ) and poloxamer-188 ( a polyoxyethylene - polyoxypropylene block copolymer ) for stable and efficient sirna delivery to lung adenocarcinoma cells.21 , 22 , 23 we hypothesized that our hybrid nanoparticles would efficiently deliver loaded anti - mutant k - ras sirna into the cytosol of lung adenocarcinoma cells , downregulate g12s - k - ras in a549 cells , and inhibit cancer cell proliferation . further , in view of the fact that human igg is the main immunoglobulin that protects the body against infection , we hypothesized that these hybrid nanoparticles would not produce the well documented immunogenic / inflammatory reaction experienced with most nanoparticle formulations . poloxamer- 188 , a nonionic triblock copolymer , is added to the surface of the nanoparticles to help circumvent the mps during systemic circulation . data obtained in vitro in cell culture supported these hypotheses and demonstrated the effectiveness of these hybrid nanoparticles in accomplishing the set objectives.21 , 23 an anti - proliferation effect of anti - mutant k - ras sirna - loaded hybrid nanoparticles ( akslhns ) in a549 cells was also demonstrated.21 , 22 further , our findings demonstrated that the double - layer protection provided by these nanoparticles helps to protect enclosed sirna from serum nucleases for up to a minimum of 48 hr . the main objective of the present study was to validate the results obtained from in vitro studies under in vivo conditions using a metastatic orthotopic murine model of nsclc . to achieve this , we hypothesize that akslhns will inhibit the progression of metastatic lung cancer by efficiently downregulating mutant - k - ras in mouse models without eliciting an immune / inflammatory reaction . further , we hypothesize that akslhns will not cause unnecessary toxicity to normal tissues because of their preferential accumulation in tumors compared with normal tissues . this is based on the fact that tumors are naturally more permeable to particles between 100800 nm because of an enhanced permeation and retention ( epr ) effect . to test these hypotheses , metastatic orthotopic murine models of nsclc were created by injecting a549-luc cells into the tail veins of female severe combined immunodeficiency ( scid ) beige mice . the efficacy of intraperitoneally injected akslhns at inhibiting or downregulating mutant k - ras and slowing down tumor progression was compared with several control formulations . akslhns composed of human igg in the inner layer and poloxamer-188 in the outer layer were prepared using our previously reported nanoprecipitation process.21 , 22 , 23 figure 1 shows a schematic of akslhn development . akslhns were spontaneously produced at ph 7.0 , which is the isoelectric point of human igg . photon correlation spectroscopy ( pcs ) demonstrated that these particles were 135.4 5.4 nm in diameter , with a polydispersity index ( pi ) of 0.07 0.03 . these particles demonstrated a zeta potential of + 16.7 0.2 and encapsulation efficiency and loading efficiency of 60% 0.4% and 2.04% 0.06% , respectively . the maximum tolerated dose ( mtd ) of sig12s in akslhns was determined for in vivo experiments by administering different doses of akslhns ( 0.1 , 0.3 , 0.6 , 0.9 , and 1.2 mg / kg body weight equivalent of sig12s ) progressively by intraperitoneal injection to female nu / nu nude mice . 24 hr after the last dose was injected , the animals were euthanized , and their kidneys , lungs , and livers were harvested . the results demonstrated that the akslhns had no effect on animal mortality , behavior , or body weight up to 0.9 mg / kg body weight . apoptosis evaluation demonstrated that the nanoparticle formulation did not cause any significant damage to the liver , lung , and kidney compared with the untreated control up to 0.9 mg / kg ( figure 2 ) . mice treated with akslhns showed regression in tumor burden over the 4-week experimental period . in contrast , pbs - treated control , erlotinib - treated , and combination - treated ( akslhns and erlotinib ) groups showed lack of sensitivity to their respective treatment , as demonstrated by tumor progression ( figure 3 ) . the tumor burden was quantified by measuring the photon counts for each tumor , which was then plotted against time ( weeks ) to further demonstrate the antitumor effect of injected akslhns , as shown in figure 4a . the tumor burden in pbs - treated mice continued to progress until metastasis in the colorectal area was observed after 2 weeks of treatment . metastasis of tumors to both the left and right lumber lymph nodes and the colorectal region could also be conspicuously observed after 3 weeks of monitoring . we also observed metastasis to the right or left popliteal lymph nodes in some of the mice ( figure s1 ) . to confirm metastasis to other organs , the lungs , heart , liver , and kidneys of pbs - treated mice were harvested after the mice were euthanized and imaged ex vivo using xenogen ivis bioluminescence . metastasis to the liver and heart was demonstrated in these mice , as shown in figure s2 . similarly , the erlotinib - treated and combination - treated groups demonstrated evidence of metastasis to the colorectal region of the mice . interestingly , tumor metastasis to the brain was observed in one of the five mice in the erlotinib - treated group after 3 weeks of treatment ( figure s2 ) . apoptosis determination by cell death elisa and tunel showed that akslhns enhanced apoptosis induction in lungs with tumors ( figures 4b and 4c ) . in contrast , erlotinib- , combination- , and pbs - treated lungs with tumors only demonstrated limited evidence of apoptosis . expression of mutant k - ras g12s mrna was studied in the tumors using taqman qrt - pcr and western blot analysis ( figures 5a and 5b ) . the western blot data in figure 5a demonstrate that akslhns downregulated mutant k - ras compared with pbs , combination , and erlotinib . qrt - pcr showed that akslhns significantly downregulated mutant k - ras mrna by approximately 60% compared with pbs . further , combination and erlotinib treatments did not show any effect on mutant k - ras expression , as shown in figure 5b . adenocarcinoma of mice treated with akslhns showed smaller tumors with thin fibrovascular stroma and rare mitotic figures ( figure 6a ) . mice that received a combination of erlotinib and akslhns showed few mitotic figures on h&e , as shown in figure 6b . adenocarcinomas from mice treated with erlotinib had numerous mitotic figures ( figure 6c ) . mice from the control group ( pbs ) had multinucleated tumor cells with eosinophilic cytoplasm , as shown in figure 6d . toxicity to healthy tissues was evaluated using cell death detection elisa to detect the induction of apoptosis . figure 7a demonstrates that akslhns did not lead to apoptosis in the lung , kidney , and liver of treated animals after 4 weeks of treatment . similarly , the combination - treated group did not show any apoptosis in any of these critical organs . however , the erlotinib - treated group showed signs of apoptosis in the liver , as demonstrated in figure 7a . cytokines interleukin-6 ( il-6 ) and tumor necrosis factor ( tnf- ) levels were measured in both cultured splenocytes isolated from spleens harvested from treated mice and serum using the respective elisa kits . figure 7b shows that akslhns did not significantly increase il-6 expression in the spleens of treated mice compared with pbs except for day 2 post - treatment . similarly , tnf- did not significantly increase in the splenocytes of treated mice throughout the 8-day study period . expression of both il-6 and tnf- in the sera of treated mice was also evaluated . figure s3 shows that there was no significant increase in the levels of both il-6 and tnf- throughout the period of study . akslhns were specially designed to facilitate the delivery of anti - mutant k - ras sirna to mutant k - ras - expressing metastatic lung cancer in an orthotopic mouse model . it was hypothesized that akslhns will inhibit the progression of metastatic lung cancer by efficiently downregulating mutant - k - ras in mouse models without eliciting immune / inflammatory reactions . further , we hypothesized that akslhns will not cause unnecessary toxicity to normal tissues because of their preferential accumulation in tumors compared with normal tissues . it has been reported that an unmet need still exists in finding an appropriate therapy for mutant k - ras - expressing lung cancer because various treatment modalities , including farnesyltransferase inhibitors , have yet to yield positive results . however , sirnas are known to have the potential to silence critical molecular pathways responsible for pathogenic conditions . the use of sirna as a therapeutic tool is , however , being compromised by a lack of safe and efficient delivery systems , limiting the efficacy of these molecules in the clinic . in this study , akslhns are being evaluated as a potential treatment modality in mutant k - ras - expressing metastatic lung cancer . akslhns were prepared based on the fact that proteins have minimum solubility but maximum precipitation at the isoelectric point.21 , 22 akslhns were very effective in regressing lung tumors in mice , as shown in figure 3 , after 4 weeks of treatment . a combination of akslhns and erlotinib was evaluated under the hypothesis that the downregulation of mutant k - ras by akslhns would improve the sensitivity of mutant k - ras - expressing lung cancer to egfr - tkis because mutant k - ras has been implicated in the lack of efficacy of egfr - tkis in lung cancer with mutant k - ras expression.9 , 14 , 26 , 27 surprisingly , this combination treatment produced an inferior antitumor effect compared with akslhns alone . we previously reported that our hybrid nanoparticles are able to escape endocytic recycling and are successfully delivered to the cytosol of target cells because of the buffering capacity of the hybrid nanoparticles in the endosome . the solubility of igg and poloxamer-188 at acidic ph levels makes it possible for the dissolved igg in the endosome to activate the proton pump that raises osmotic pressure in the endosome , leading to the swelling and subsequent escape of sirna from endosomes into the cytosol.21 , 27 , 28 however , the presence of erlotinib , a very basic drug , in the endosome at the same time may increase the ph level of the endosome , making it impossible for the nanoparticles to dissolve and release the sirna as it would normally do . the lack of release of sirna because of the presence of erlotinib is assumed to subsequently lead to an inferior antitumor effect of the combination therapy compared with akslhns alone . the interaction between a ph - sensitive nucleic acid - loaded nanoparticle system and a concurrently administered basic drug needs to be investigated further for a clear understanding of this phenomenon . nevertheless , metastasis of the tumor to the brain , colorectal region , right and left lumber lymph nodes , and right popliteal lymph nodes was observed in the other groups of animals , except the akslhn - treated group . the effective antitumor effect of akslhns was further validated by induction of apoptosis in the tumor - bearing lungs of the akslhn - treated group using both tunel and cell death detection elisa . further , both mutant k - ras protein and mrna were significantly downregulated in the lungs of akslhn - treated mice compared with the other mouse groups , further validating the effectiveness of this therapy . previously , we reported the superior and stable downregulation of mutant k - ras by akslhns at the cellular level ( using a549 cells ) over both scramble sirna and lipofectamine - delivered anti - k - ras sirna.21 , 29 toxicity and inflammatory reaction remain the limiting factors preventing the translational application of most nanomedicines.22 , 23 , 30 for any nanoparticle - based therapeutic agent to have a chance of progressing to the clinic , it is important that it is not toxic to healthy tissues and does not elicit a significant immunogenic / inflammatory reaction . on that note , we evaluated the effect of akslhns , erlotinib , and a combination of both on the induction of apoptosis in healthy tissues . induction of apoptosis in healthy tissues has been reported previously to represent toxicity in such tissues . akslhns and the combination treatment did not lead to induction of apoptosis in the lung , kidney , and liver of treated mice . this is not surprising because erlotinib has been implicated previously in acute hepatotoxicity in nsclc patients . it is , however , interesting to note that the combination therapy involving both akslhns and erlotinib did not lead to hepatotoxicity . this further corroborates our hypothesis that an interaction occurs between akslhns and erlotinib at the cellular level . to evaluate the ability of akslhns to elicit an immune / inflammatory response , naive balb / c mice were used . scid beige mice used for development of the metastatic orthotopic murine nsclc model are immunocompromised and would not be suitable for the immunogenic / inflammatory reaction experiment . scid mice are characterized by the complete inability of the adaptive immune system to mount , coordinate , and sustain an appropriate immune response , usually because of absent or atypical t and b lymphocytes . il-6 and tnf- expression in isolated splenocytes and sera of akslhn - treated mice was similar to that of pbs - treated ( control ) mice , except for a certain time point where insignificant elevated expression of these cytokines was observed . this could be attributed to two factors : the presence of poloxamer-188 on the surface of the nanoparticles or the fact that the nanoparticles were fabricated with human igg and tested in mice . this interspecies variation could contribute to the slight elevation in these cytokines at these time points . future study will involve the use of mouse igg in the fabrication of the nanoparticles to eliminate interspecies variation as a contributory factor to this observation . in conclusion , akslhns are a promising prospect as a treatment modality for mutant k - ras - expressing nsclc as a single therapy . a further understanding of the cellular - level interaction between akslhns and egfr - tkis during concurrent administration would help in developing an optimal dosage regimen to further harness this treatment modality in nsclc patients . poloxamer-188 , rnase - free water , and fetal bovine serum were obtained from fisher scientific . sirna against mutated k - ras g12s was designed by and purchased from thermo scientific ( formerly dharmacon ) . the sig12s sense and antisense sequences were guuggagcuaguggcguagdtdt and cuacgccacuagcuccaacdtdt , respectively . monoclonal antibodies specific for mutant g12s k - ras proteins was obtained from neweast biosciences . the tnf- elisa kit was obtained from thermo scientific , and the il-6 elisa kit was obtained from becton dickinson . human adenocarcinoma cell line a549-luciferase , expressing a k - ras mutation at g12s , was obtained from perkinelmer . these cells were maintained in 10% fetal bovine serum and 1% antibiotics - supplemented f12k . female scid beige mice , 78 weeks old and weighing approximately 28 g , and female balb / c mice were supplied by taconic and maintained in the american association for the accreditation of laboratory animal care ( aaalac)-accredited facility at thomas jefferson university . all animal studies were approved by the institutional animal care and use committee ( iacuc ) of thomas jefferson university . 50 mg of excipient - free human igg was dissolved in 0.01 n hcl containing 20 mg of poloxamer-188 . 187 g of sirna was then added to make a 10-ml total solution in a 50-ml beaker . the final concentration of human igg in the solution amounted to 5 mg / ml . this solution was then slowly titrated with 0.01 n naoh to bring the ph of the mixture to 7 , which is the isoelectric point ( pi ) of human igg as determined in our laboratory using isoelectric focusing . the nanoparticles were continuously stirred on a magnetic stirrer for 10 min . to isolate the nanoparticles from the precipitation medium , the suspension was centrifuged with a microcentrifuge ( eppendorf centrifuge 5418 ) at 2,000 rpm for 5 min . nanoparticles were then rinsed with double - distilled deionized water before being redispersed in water and snap - frozen using liquid nitrogen . this was then loaded into a freeze dryer ( labconco freezezone 4.6 ) , and lyophilization was performed for 48 hr . lung cancer models of nsclc were created by injecting 5 10 a549-luciferase cells in sterile pbs into the tail veins of female scid beige mice . cancer development and progression were monitored using the xenogen ivis ( perkinelmer ) bioluminescence imaging system . 100 l of 30 mg / ml xenolight rediject d - luciferin ( perkinelmer ) was injected intraperitoneally into the mice approximately 10 min before the animals were imaged . following the establishment of detectable lung tumors ( 2 weeks from the time of induction ) , mice were divided into four groups of five animals each . the first group was treated with 20 mg / kg erlotinib daily for 4 weeks by oral gavage ( 100 l ) . the second group was treated with akslhns twice a week for 4 weeks at a dose of 0.3 mg / kg by intraperitoneal injection ( 100 l ) . a third group was treated with a combination of akslhns and erlotinib , whereas the fourth group was treated with pbs as a control twice a week by intraperitoneal injection ( 100 l ) . one part of the tissue was fixed in 10% phosphate - buffered formalin ( fisher scientific ) for paraffin embedding , histology , and immunohistochemistry ( ihc ) analysis , and other three were snap - frozen for real - time pcr , western blot , and cell death detection elisa . five mice were treated with a single dose of 0.9 mg / kg akslhns by intraperitoneal injection , whereas the remaining four served as controls and were injected with pbs ( 100 l per mouse ) . blood was collected by retro - orbital puncture under isoflurane anesthesia into edta - covered vacutainers ( becton dickinson ) on days 1 , 2 , 6 , and 8 . blood was then immediately centrifuged at 1,000 g for 10 min at 4c , and serum was collected and frozen at 80c until ready to be used . splenocytes were isolated from mice injected with functionalized nanoparticles and pbs on days 1 , 2 , 6 , and 8 . conditioned medium was collected after 24 hr and frozen at 80c until ready to be used . the levels of il-6 and tnf- were measured using the respective elisa kits according to the manufacturers instructions . spleens of mice injected with akslhns and pbs were collected , placed into a tube with rpmi medium , and kept on ice . the tube was then transferred to the hood , and the spleen was homogenized by two microscope glass slides . the spleen slurry in 10 ml of rpmi medium was transferred to a 70-m cell strainer ( falcon ) and filtered from clumps . the cell suspension the cell pellet was resuspended in 3 ml of ammonium - chloride - potassium ( ack ) lysis buffer ( gibco ) to get rid of red blood cells . the hemolysis was carried out at room temperature with periodic shaking for 5 min , after which the suspension was reconstituted with 10 ml of fresh rpmi medium and centrifuged at 400 g for 5 min at 20c . the cell pellet was washed with 10 ml of rpmi medium and spun down at 400 g for 5 min . the resulting cell pellet was dissolved in complete rpmi medium ( rpmi 1640 medium , 10% fbs , 1 penicillin / streptomycin [ pen / strep ] , 10 mm hepes ) , 500,000 cells were seeded in triplicate into wells of a 96-well plate . the cells were incubated for 24 hr , and the conditioned medium was collected and frozen until ready to be used for determination of the proinflammatory cytokines tnf and il-6 . tumor - bearing lungs were collected from mice , snap - frozen in liquid nitrogen , and kept at 80c until ready to be used . tissue lysates were prepared in pierce radioimmunoprecipitation ( ripa ) buffer ( thermo scientific ) with addition of pierce protease inhibitor mini tablets ( thermo scientific ) . the protein concentration was determined with the coomassie plus ( bradford ) assay kit ( thermo scientific ) . 80 g of total protein was separated on nupage 4%12% bis - tris gels ( life technologies ) with nupage mes sds running buffer ( life technologies ) and subsequently transferred onto a nitrocellulose membrane of 0.45-m pore size ( life technologies ) . the membrane with proteins was blocked according to the manufacturer s instructions ( invitrogen ) for 1 hr at room temperature and probed with primary antibodies kras g12s monoclonal antibody ( 1:500 , new east biosciences ) and -actin ( 1:5,000 , sigma - aldrich)overnight at 4c . the membranes were washed three times for 5 min in wash buffer according to the manufacturer s instructions ( invitrogen ) and incubated with secondary goat anti - mouse antibodies conjugated with horseradish peroxidase ( molecular probes ) at a dilution of 1:1,000 . immune complexes were detected with chemoluminescent substrate , pierce ecl western blotting substrate ( thermo scientific ) in a dark room on a tabletop processor , srx-101a ( konica minolta ) . total rna from snap - frozen lung tissues was isolated with the qiagen rnaeasy kit , and cdna was made with the verzo cdna kit ( thermo scientific ) using polya primers . real - time pcr was carried out in 20-l reaction mixtures containing 25 ng of cdna , 10 l of 2 taqman gene expression master mix ( applied biosystems ) , and 1 l of specific taqman gene expression assay ( 20 , fam dye - labeled ) . gene expression assays ( obtained from thermo scientific ) contained mutant kras at g12s and -actin primers ( -actin served as a normalizing gene ) . apoptosis in samples collected from mice with nsclc was detected by tunel assay with the tacs 2tdt - fluor in situ apoptosis detection kit ( trevigen ) according to the manufacturer s instructions . briefly , lung tissue section slides were deparaffinized in xylene and 100% , 95% , 70% ethanol , followed by two changes of pbs . next , the samples were digested with 50 l of cytonin solution for 30 min and washed with water and terminal deoxynucleotidyl transferase ( tdt ) labeling buffer . next , the slides were covered with 50 l of labeling reaction mix ( tdt deoxynucleoside triphosphate [ dntp ] , co , tdt enzyme and tdt labeling buffer ) and incubated for 60 min at 37c in a humidity chamber . at this step , positive control was made before the labeling step by incubating the slide with tacs nuclease ( generating dna breaks in every cell ) at room temperature for 40 min . the labeling reaction was halted by stop buffer , and samples were washed in pbs and covered with 50 l of strep - fluorescein solution for 20 min . next , the slides were washed with pbs , mounted , and viewed under a fluorescence microscope with a 495-nm filter . cell death detection elisa was carried out according to the manufacturer s instructions ( roche ) . sandwich elisa was carried out to determine the amount of mono- and oligonucleosomes in the cytoplasmic fraction of tissue lysates . 2% homogenate of lung tissue was prepared in incubation buffer and centrifuged for 10 min at 15,000 g. the supernatant was collected and kept at 80c until ready to use . plastic wells were incubated with coating solution ( containing anti - histone antibodies ) overnight at 4c . the next day , it was substituted for incubation buffer for 30 min and washed three times with washing solution . 100 l of homogenate , diluted 29 times in incubation buffer , was placed into the wells and kept for 90 min at room temperature with mild shaking . the plate was washed three times and incubated with conjugate solution containing anti - dna - peroxidase antibodies for 90 min . the wells were washed three times and exposed to abts substrate for 30 min until the color developed . absorption values were read on a biotek instruments epoch microplate spectrophotometer with gen5 1.10 software . heat - mediated antigen retrieval was performed in citrate buffer ( ph 6.0 ) at 98c for 20 min . 9664 , cell signaling technology ) , was diluted 1:300 and incubated at room temperature for 30 min ; human tonsil was used as a positive control . the ihc procedure was done with the dako autostainer plus platform using the vectastain elite abc kit ( standard , catalog no . statistically significant differences between two groups were determined by two - tailed student s t test . m.p . carried out most of the experiments and was also involved in data interpretation . were involved in taking pictures of the histology slides and the interpretation of histopathology data . were involved in the development and monitoring of the orthotopic metastatic mouse lung cancer models . s.a.s . initiated the study , planned the experiments , interpreted the data , and wrote the manuscript .

there is an unmet need in the development of an effective therapy for mutant k - ras - expressing non - small - cell lung cancer ( nsclc ) . although various small molecules have been evaluated , an effective therapy remains a dream . sirnas have the potential to downregulate mutant k - ras both at the protein and mrna levels . however , a safe and effective delivery of sirnas to tumors remains a limitation to their translational application in the treatment of this highly debilitating disease . here we developed a novel hybrid nanoparticle carrier for effective delivery of anti - mutant k - ras to nsclc ( akslhn ) . the ability of this treatment modality to regress lung tumors in mouse models was evaluated as a monotherapy or as a combination treatment with erlotinib . further , the toxicity of this treatment modality to healthy tissues was evaluated , along with its ability to elicit immune / inflammatory reactions . the results suggest that this treatment modality is a promising prospect for the treatment of mutant k - ras - expressing nsclc without any accompanying toxicity . however , further understanding of the cellular - level interaction between ahslhn and erlotinib needs to be attained before this promising treatment modality can be brought to the bedside .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

non - small cell lung cancer ( nsclc ) is the leading cause of cancer mortality in the united states and the world , with a 5-year survival rate of only 15% for all stages combined , despite some recent advances in chemotherapeutic agents.1 , 2 the size and distribution of nsclc makes cytoreductive surgery ineffective . consequently , chemotherapy and/or radiation have been the treatments of choice . conventional chemotherapy regimens have had limited efficacy , mainly because of resistance of these cancer cells to chemotherapeutic agents.4 , 5 small - molecule inhibitors such as erlotinib and afatinib that target the tyrosine kinase domain of epidermal growth factor receptor ( egfr ) produce responses in approximately 10% of patients with nsclc.6 , 7 , 8 in patients with mutant egfr , responses to egfr - tyrosine kinase inhibitors ( egfr - tkis ) can be dramatic and may last longer than a year . in contrast , k - ras gene mutation , which occurs in approximately 30% of nsclc , has been associated with a poor response to egfr - tkis.9 , 10 , 11 , 12 , 13 it has been demonstrated previously that somatic mutations in the tyrosine kinase domain of egfr are associated with sensitivity to egfr - tkis , whereas mutations in k - ras , which encodes a guanosine triphosphotase ( gtpase ) downstream of egfr , are associated with primary resistance . however , there is an unmet need in developing the most appropriate therapy for mutant k - ras nsclc . several strategies , such as farnesyltransferase inhibitors , have been explored as possible inhibitors of mutant k - ras in lung adenocarcinomas . to date , none of these strategies have proved to be successful , mainly because of lack of specificity . small interfering rna ( sirna ) inhibition is a very viable alternative given the specificity of this technology . here we aim to harness the advantages of sirna technology as a therapeutic modality for mutant k - ras in nsclc . sirna as a therapeutic molecule is very effective at knocking down molecular pathways that are pathogenic . however , its application in the clinic is often limited by its susceptibility to enzymatic degradation in blood , non - specific uptake by cells , and the difficulty involved in the transfection of sirna to cells because of its relatively large size and polarity.16 , 17 . clearance by the mononuclear phagocyte system ( mps ) is another limiting factor affecting the possible therapeutic application of sirna.18 , 19 , 20 recently , our group developed a novel hybrid nanoparticle delivery system composed of human immunoglobulin g ( igg ) and poloxamer-188 ( a polyoxyethylene - polyoxypropylene block copolymer ) for stable and efficient sirna delivery to lung adenocarcinoma cells.21 , 22 , 23 we hypothesized that our hybrid nanoparticles would efficiently deliver loaded anti - mutant k - ras sirna into the cytosol of lung adenocarcinoma cells , downregulate g12s - k - ras in a549 cells , and inhibit cancer cell proliferation . further , in view of the fact that human igg is the main immunoglobulin that protects the body against infection , we hypothesized that these hybrid nanoparticles would not produce the well documented immunogenic / inflammatory reaction experienced with most nanoparticle formulations . poloxamer- 188 , a nonionic triblock copolymer , is added to the surface of the nanoparticles to help circumvent the mps during systemic circulation . data obtained in vitro in cell culture supported these hypotheses and demonstrated the effectiveness of these hybrid nanoparticles in accomplishing the set objectives.21 , 23 an anti - proliferation effect of anti - mutant k - ras sirna - loaded hybrid nanoparticles ( akslhns ) in a549 cells was also demonstrated.21 , 22 further , our findings demonstrated that the double - layer protection provided by these nanoparticles helps to protect enclosed sirna from serum nucleases for up to a minimum of 48 hr . the main objective of the present study was to validate the results obtained from in vitro studies under in vivo conditions using a metastatic orthotopic murine model of nsclc . to achieve this , we hypothesize that akslhns will inhibit the progression of metastatic lung cancer by efficiently downregulating mutant - k - ras in mouse models without eliciting an immune / inflammatory reaction . the efficacy of intraperitoneally injected akslhns at inhibiting or downregulating mutant k - ras and slowing down tumor progression was compared with several control formulations . akslhns composed of human igg in the inner layer and poloxamer-188 in the outer layer were prepared using our previously reported nanoprecipitation process.21 , 22 , 23 figure 1 shows a schematic of akslhn development . 24 hr after the last dose was injected , the animals were euthanized , and their kidneys , lungs , and livers were harvested . in contrast , pbs - treated control , erlotinib - treated , and combination - treated ( akslhns and erlotinib ) groups showed lack of sensitivity to their respective treatment , as demonstrated by tumor progression ( figure 3 ) . the tumor burden in pbs - treated mice continued to progress until metastasis in the colorectal area was observed after 2 weeks of treatment . we also observed metastasis to the right or left popliteal lymph nodes in some of the mice ( figure s1 ) . to confirm metastasis to other organs , the lungs , heart , liver , and kidneys of pbs - treated mice were harvested after the mice were euthanized and imaged ex vivo using xenogen ivis bioluminescence . metastasis to the liver and heart was demonstrated in these mice , as shown in figure s2 . similarly , the erlotinib - treated and combination - treated groups demonstrated evidence of metastasis to the colorectal region of the mice . interestingly , tumor metastasis to the brain was observed in one of the five mice in the erlotinib - treated group after 3 weeks of treatment ( figure s2 ) . expression of mutant k - ras g12s mrna was studied in the tumors using taqman qrt - pcr and western blot analysis ( figures 5a and 5b ) . the western blot data in figure 5a demonstrate that akslhns downregulated mutant k - ras compared with pbs , combination , and erlotinib . qrt - pcr showed that akslhns significantly downregulated mutant k - ras mrna by approximately 60% compared with pbs . further , combination and erlotinib treatments did not show any effect on mutant k - ras expression , as shown in figure 5b . mice that received a combination of erlotinib and akslhns showed few mitotic figures on h&e , as shown in figure 6b . adenocarcinomas from mice treated with erlotinib had numerous mitotic figures ( figure 6c ) . toxicity to healthy tissues was evaluated using cell death detection elisa to detect the induction of apoptosis . figure 7a demonstrates that akslhns did not lead to apoptosis in the lung , kidney , and liver of treated animals after 4 weeks of treatment . similarly , the combination - treated group did not show any apoptosis in any of these critical organs . however , the erlotinib - treated group showed signs of apoptosis in the liver , as demonstrated in figure 7a . figure 7b shows that akslhns did not significantly increase il-6 expression in the spleens of treated mice compared with pbs except for day 2 post - treatment . similarly , tnf- did not significantly increase in the splenocytes of treated mice throughout the 8-day study period . expression of both il-6 and tnf- in the sera of treated mice was also evaluated . figure s3 shows that there was no significant increase in the levels of both il-6 and tnf- throughout the period of study . akslhns were specially designed to facilitate the delivery of anti - mutant k - ras sirna to mutant k - ras - expressing metastatic lung cancer in an orthotopic mouse model . it was hypothesized that akslhns will inhibit the progression of metastatic lung cancer by efficiently downregulating mutant - k - ras in mouse models without eliciting immune / inflammatory reactions . further , we hypothesized that akslhns will not cause unnecessary toxicity to normal tissues because of their preferential accumulation in tumors compared with normal tissues . it has been reported that an unmet need still exists in finding an appropriate therapy for mutant k - ras - expressing lung cancer because various treatment modalities , including farnesyltransferase inhibitors , have yet to yield positive results . however , sirnas are known to have the potential to silence critical molecular pathways responsible for pathogenic conditions . the use of sirna as a therapeutic tool is , however , being compromised by a lack of safe and efficient delivery systems , limiting the efficacy of these molecules in the clinic . in this study , akslhns are being evaluated as a potential treatment modality in mutant k - ras - expressing metastatic lung cancer . akslhns were prepared based on the fact that proteins have minimum solubility but maximum precipitation at the isoelectric point.21 , 22 akslhns were very effective in regressing lung tumors in mice , as shown in figure 3 , after 4 weeks of treatment . a combination of akslhns and erlotinib was evaluated under the hypothesis that the downregulation of mutant k - ras by akslhns would improve the sensitivity of mutant k - ras - expressing lung cancer to egfr - tkis because mutant k - ras has been implicated in the lack of efficacy of egfr - tkis in lung cancer with mutant k - ras expression.9 , 14 , 26 , 27 surprisingly , this combination treatment produced an inferior antitumor effect compared with akslhns alone . we previously reported that our hybrid nanoparticles are able to escape endocytic recycling and are successfully delivered to the cytosol of target cells because of the buffering capacity of the hybrid nanoparticles in the endosome . the solubility of igg and poloxamer-188 at acidic ph levels makes it possible for the dissolved igg in the endosome to activate the proton pump that raises osmotic pressure in the endosome , leading to the swelling and subsequent escape of sirna from endosomes into the cytosol.21 , 27 , 28 however , the presence of erlotinib , a very basic drug , in the endosome at the same time may increase the ph level of the endosome , making it impossible for the nanoparticles to dissolve and release the sirna as it would normally do . the lack of release of sirna because of the presence of erlotinib is assumed to subsequently lead to an inferior antitumor effect of the combination therapy compared with akslhns alone . the interaction between a ph - sensitive nucleic acid - loaded nanoparticle system and a concurrently administered basic drug needs to be investigated further for a clear understanding of this phenomenon . nevertheless , metastasis of the tumor to the brain , colorectal region , right and left lumber lymph nodes , and right popliteal lymph nodes was observed in the other groups of animals , except the akslhn - treated group . the effective antitumor effect of akslhns was further validated by induction of apoptosis in the tumor - bearing lungs of the akslhn - treated group using both tunel and cell death detection elisa . further , both mutant k - ras protein and mrna were significantly downregulated in the lungs of akslhn - treated mice compared with the other mouse groups , further validating the effectiveness of this therapy . previously , we reported the superior and stable downregulation of mutant k - ras by akslhns at the cellular level ( using a549 cells ) over both scramble sirna and lipofectamine - delivered anti - k - ras sirna.21 , 29 toxicity and inflammatory reaction remain the limiting factors preventing the translational application of most nanomedicines.22 , 23 , 30 for any nanoparticle - based therapeutic agent to have a chance of progressing to the clinic , it is important that it is not toxic to healthy tissues and does not elicit a significant immunogenic / inflammatory reaction . on that note , we evaluated the effect of akslhns , erlotinib , and a combination of both on the induction of apoptosis in healthy tissues . induction of apoptosis in healthy tissues has been reported previously to represent toxicity in such tissues . akslhns and the combination treatment did not lead to induction of apoptosis in the lung , kidney , and liver of treated mice . it is , however , interesting to note that the combination therapy involving both akslhns and erlotinib did not lead to hepatotoxicity . this further corroborates our hypothesis that an interaction occurs between akslhns and erlotinib at the cellular level . to evaluate the ability of akslhns to elicit an immune / inflammatory response , naive balb / c mice were used . scid beige mice used for development of the metastatic orthotopic murine nsclc model are immunocompromised and would not be suitable for the immunogenic / inflammatory reaction experiment . scid mice are characterized by the complete inability of the adaptive immune system to mount , coordinate , and sustain an appropriate immune response , usually because of absent or atypical t and b lymphocytes . this could be attributed to two factors : the presence of poloxamer-188 on the surface of the nanoparticles or the fact that the nanoparticles were fabricated with human igg and tested in mice . future study will involve the use of mouse igg in the fabrication of the nanoparticles to eliminate interspecies variation as a contributory factor to this observation . in conclusion , akslhns are a promising prospect as a treatment modality for mutant k - ras - expressing nsclc as a single therapy . a further understanding of the cellular - level interaction between akslhns and egfr - tkis during concurrent administration would help in developing an optimal dosage regimen to further harness this treatment modality in nsclc patients . sirna against mutated k - ras g12s was designed by and purchased from thermo scientific ( formerly dharmacon ) . monoclonal antibodies specific for mutant g12s k - ras proteins was obtained from neweast biosciences . human adenocarcinoma cell line a549-luciferase , expressing a k - ras mutation at g12s , was obtained from perkinelmer . female scid beige mice , 78 weeks old and weighing approximately 28 g , and female balb / c mice were supplied by taconic and maintained in the american association for the accreditation of laboratory animal care ( aaalac)-accredited facility at thomas jefferson university . this solution was then slowly titrated with 0.01 n naoh to bring the ph of the mixture to 7 , which is the isoelectric point ( pi ) of human igg as determined in our laboratory using isoelectric focusing . lung cancer models of nsclc were created by injecting 5 10 a549-luciferase cells in sterile pbs into the tail veins of female scid beige mice . following the establishment of detectable lung tumors ( 2 weeks from the time of induction ) , mice were divided into four groups of five animals each . a third group was treated with a combination of akslhns and erlotinib , whereas the fourth group was treated with pbs as a control twice a week by intraperitoneal injection ( 100 l ) . one part of the tissue was fixed in 10% phosphate - buffered formalin ( fisher scientific ) for paraffin embedding , histology , and immunohistochemistry ( ihc ) analysis , and other three were snap - frozen for real - time pcr , western blot , and cell death detection elisa . conditioned medium was collected after 24 hr and frozen at 80c until ready to be used . the levels of il-6 and tnf- were measured using the respective elisa kits according to the manufacturers instructions . the tube was then transferred to the hood , and the spleen was homogenized by two microscope glass slides . the cells were incubated for 24 hr , and the conditioned medium was collected and frozen until ready to be used for determination of the proinflammatory cytokines tnf and il-6 . the protein concentration was determined with the coomassie plus ( bradford ) assay kit ( thermo scientific ) . the membrane with proteins was blocked according to the manufacturer s instructions ( invitrogen ) for 1 hr at room temperature and probed with primary antibodies kras g12s monoclonal antibody ( 1:500 , new east biosciences ) and -actin ( 1:5,000 , sigma - aldrich)overnight at 4c . the membranes were washed three times for 5 min in wash buffer according to the manufacturer s instructions ( invitrogen ) and incubated with secondary goat anti - mouse antibodies conjugated with horseradish peroxidase ( molecular probes ) at a dilution of 1:1,000 . total rna from snap - frozen lung tissues was isolated with the qiagen rnaeasy kit , and cdna was made with the verzo cdna kit ( thermo scientific ) using polya primers . gene expression assays ( obtained from thermo scientific ) contained mutant kras at g12s and -actin primers ( -actin served as a normalizing gene ) . apoptosis in samples collected from mice with nsclc was detected by tunel assay with the tacs 2tdt - fluor in situ apoptosis detection kit ( trevigen ) according to the manufacturer s instructions . next , the samples were digested with 50 l of cytonin solution for 30 min and washed with water and terminal deoxynucleotidyl transferase ( tdt ) labeling buffer . next , the slides were covered with 50 l of labeling reaction mix ( tdt deoxynucleoside triphosphate [ dntp ] , co , tdt enzyme and tdt labeling buffer ) and incubated for 60 min at 37c in a humidity chamber . next , the slides were washed with pbs , mounted , and viewed under a fluorescence microscope with a 495-nm filter . cell death detection elisa was carried out according to the manufacturer s instructions ( roche ) . sandwich elisa was carried out to determine the amount of mono- and oligonucleosomes in the cytoplasmic fraction of tissue lysates . plastic wells were incubated with coating solution ( containing anti - histone antibodies ) overnight at 4c . the plate was washed three times and incubated with conjugate solution containing anti - dna - peroxidase antibodies for 90 min . 9664 , cell signaling technology ) , was diluted 1:300 and incubated at room temperature for 30 min ; human tonsil was used as a positive control . were involved in the development and monitoring of the orthotopic metastatic mouse lung cancer models .

the most important one is carbondioxide gas resulted from the combustion of fossil fuels and deforestation . thus , it is necessary to search for the sources of the renewable energy such as solar , wind , hydraulic , geothermal , and tide energy . on the other hand , the biofuels from biomass can also represent the promising type of energy source . bioethanol is now considered interesting biofuel and has been attracting attention since it can be directly used in place of benzene or diesel . it can be applied in the form of the mixture with benzene called gasohol or blended with diesel called diesohol . bioethanol can be produced from biomass such as starch , sugar , and lignocellulosic materials . previous studies revealed that the cheaper and suitable one for the production of ethanol can be derived from corncobs , grasses , wooden spills , and bagasses . however , the use of starch and sugar from cassava , corn , and sugarcane which are basically human - food might possibly lead to the problem of food crisis . therefore , lignocellulosic biomass which is abundant and low on production cost obtained from nonfood sources should be considered as an appropriate feedstock for ethanol production . the energy crops are the types of plants expected to be cultivated as the raw materials for the production of the biofuels such as ethanol . therefore , the plants with the fast growing rate such as grasses or shrubs should represent the appropriate energy crops for the future . these plants are perennial crops considered suitable as feedstock for lignocellulosic ethanol production because of high yields , low costs , fit for infertile land , and less environmental impacts . grasses are targeted as potential energy crops because of high productivity per hectare , abundancy , availability , and utilization of the whole plants . fibres and storage carbohydrates within some species of grass can be used as substrates to produce ethanol whereas the species of grasses that contain high amounts of proteins can be used as nitrogenous waste for biorefineries . most of the researches in this area have been conducted on bioethanol production from either cellulose or hemicellulose hydrolysis to glucose or xylose for ethanol fermentation . in this research , both cellulose and hemicellulose from grasses were aimed to be used as the plant biomass as much as possible . the reason is from the fact that grasses contain very high percentages of total cellulose and hemicellulose . in general , the average compositions of grass biomass are 2540% of cellulose , 2550% of hemicellulose , and 1030% of lignin [ 2 , 5 ] . the scientific researches on ethanol production from grasses have been mostly performed in switch grass [ 6 , 7 ] . moreover , there were studies carried out in timothy grass , reed canary grass , bermuda grass ( cynodon dactylon ) , and silver grass ( miscanthus floridulus ) , and so forth . these grasses are fast to grow likewise require less nutrient , thus suitable to be used as an energy crops for bioethanol production . in particular , vetiver grass also has a potential for biofuel production by using a raw material in biomass power plant as well as ethanol production . ethanol production can be achieved from cellulose and hemicellulose hydrolysis to sugar by acid or enzyme catalysts . the use of acid is disadvantageous from the high temperature required , vigorous and nonspecific reactions resulting in the unwanted products . therefore , the application of enzymes appears more interesting from the high specificity , neutral reaction without the unwanted by products , and therefore cost reduction in the waste management . obviously , the production of ethanol from agricultural wastes can only be applied for the short - term purpose since the amount of such waste wo n't be sufficient in the future . therefore , in order to maintain long - term and successfully sustainable production of the alternative energy , the cultivation of energy crops together with the utilization of agricultural wastes and the search for the potential plants which can produce ethanol should be implemented . grasses are considered one of the most suitable energy crops from their longevity , regeneration after the cutoff , and effective capability to withstand the drought . hence , the objective of this research was to select the types of grasses in thailand suitable as the feedstocks for cellulosic ethanol production by biological process . the fungus , trichoderma reesei , has been widely accepted as the organism capable of producing the enzymes , cellulase , and hemicellulase in considerable quantities , appropriate for lignocellulosic biomass digestion . although the fermentation can be achieved from various processes , the application of simultaneous saccharification and cofermentation ( sscf ) this method allows the raw materials to be hydrolyzed to hexose and pentose sugars for the subsequent fermentation to obtain ethanol in the single step . the obtained glucose gradually released from the hydrolysis will be continuously utilized in the fermentation . this low concentration of the glucose will result in the improved fermentation of xylose sugar . similarly , the yeasts , saccharomyces cerevisiae and pichia stipites , are widely known organisms that can utilize glucose and xylose for ethanol fermentation , respectively . although , p. stipitis can ferment both glucose and xylose , but ethanol production rate from glucose is at least five times less than obtained with s. cerevisiae . 18 samples of grasses were collected from chiang mai , lampang , ratchaburi , and petchburi provinces . all samples were dried at 60c for 3 days , ground to small particles and later filtered through 0.4 mm mesh . biomass contents were analyzed according to the method of ruminant nutrition laboratory , university of nebraska . neutral detergent fiber ( ndf ) , acid detergent fiber ( adf ) , and permanganate lignin ( pml ) were determined . hemicellulose content of biomass was determined as ndf minus adf , lignin content of biomass as adf minus pml , and cellulose content of biomass as pml minus residue after ash . t. reesei tistr 3081 ( t. reesei qm9414 ) was obtained from thailand institute of scientific and technological research ( tistr ) , thailand and was later cultured in potato dextrose agar at 30c for 8 days . a cork borer ( 7 mm diameter ) was used to cut agar plugs from a culture on pda . five agar plugs with the filaments of fungi were inoculated into 250 ml flask containing 100 ml of mandels medium for cellulase production in the presence of 10 g / l -cellulose as a carbon source . the induction of xylanase production was carried out in xylan medium containing 10 g / l birchwood xylan as a carbon source , 5 g / l corn steep liquor , 5 g / l polypeptone , 1 g / l yeast extract , 4 g / l k2hpo4 , 0.2 g / l kcl , 1 g / l mg2so4 7h2o , and 0.02 g after incubation at 30c and shaken at 150 rpm for 7 days , the mycelia were removed by filtration through whatman no.1 filter paper . crude enzyme was collected and determined for the activities of cellulase and xylanase using whatman no.1 filter paper and 1% birchwood xylan as the substrates , respectively , according to ghose . one unit of enzyme was referred as the amount of enzyme which could convert substrates to 1 mole of reducing sugar in 1 minute . protein was determined by micro lowry 's assay to obtain the specific activity of enzyme ( u / mg protein ) . the milled grasses were suspended in 7.5% ( v / v ) h2o2 and naoh was then added to adjust the ph to 11.5 . the pretreated samples were then incubated at 35c , shaken at 250 rpm for 24 hours . finally , conc . the liquid phase with solubilized hemicellulose and the solid phase with cellulose of the samples were subsequently hydrolyzed by the enzymes . cellulase ( 60 u / g substrate ) , xylanase ( 1200 u / g substrate ) , and 5 ml of 0.05 m sodium citrate buffer ( ph 4.8 ) were added to each substrate ( 0.6 g of milled grass ) and positive control ( 0.3 g of -cellulose mixed with 0.3 g of xylan ) . after the addition of the enzymes , the samples were incubated at 50c and shaken at 150 rpm for 72 hours . total reducing sugar was analyzed by dns method and subsequently calculated for the percentages of conversion to cellulose and hemicellulose according to the equations below . ( 1)% conversion of cellulose and hemicellulose to sugars = ba100 . total reducing sugar after the hydrolysis ( ton / ha / year ) was considered for selection of suitable grasses as the substrates for ethanol production by sscf process . ( 2)total reducing sugar after hydrolysis= bc1000 , where a is total cellulose and hemicellulose before hydrolysis ( mg / g dry weight of substrate ) . b is total reducing sugar after hydrolysis ( mg / g dry weight of substrate ) . c is dry matter yield of grass ( ton / ha / year ) . p. stipitis cbs 5773 was obtained from the centraalbureau voor schimmelcultures ( cbs ) , the netherlands . yeast strains were maintained at 4c on ym agar containing 3 g / l yeast extract , 3 g / l malt extract , 5 g / l peptone , 10 g / l glucose , and 20 g / l agar . s. cerevisiae and p. stipitis were precultured as inocula for fermentation in erlenmeyer flasks containing 50 ml of 3 g / l yeast extract , 3 g / l malt extract , 5 g / l ( nh4)2so4 , and 20 g / l glucose for s. cerevisiae or 20 g / l xylose for p. stipitis . flasks were incubated at 30c and shaken at 150 rpm for 9 h ( s. cerevisiae ) and 10 h ( p. stipitis ) . 1.2 g of pretreated substrates was supplemented in fermentation medium containing 0.45 g yeast extract , 0.45 g malt extract , and 0.75 g ( nh4)2so4 in 7.5 ml of 0.05 m sodium citrate buffer ( ph 5.0 ) and sterilized by autoclaving at 121c for 15 min . cellulase and xylanase were filtered and sterilized through a 0.2 m filters and the enzymes were added with the activities of 72 u and 1,440 u in each flask , respectively . then , 7.5 ml ( 5% v / v ) of each yeast strain was added last . the samples were incubated with shaking at 35c , 150 rpm for 7 days , and ethanol production was determined by gas chromatography ( shimadzu , japan ) collected data ( 3 replicates ) were analyzed by one - way anova at 95% confidence level . comparison was performed by duncan 's multiple range test ( dmrt ) using spss for window version 15.0 . the first group was composed of 8 types of the forage grasses ( napier leaves , dwarf napier leaves , king napier leaves , bana leaves , purple guinea , ruzi , pangola , and atratum ) and the second one contained 10 ecotypes of vetiver grasses ( kamphaeng phet 2 , songkhla 3 , surat thani , sri lanka , roi et , loei , nakhon sawan , prachuap khiri khan , ratchaburi , and kamphaeng phet 1 ) as shown in figure 1 . since the height of all the grasses in this study is more than 1 metre , and the harvest can be carried out several times per year , considerably huge amount of dry biomass can be obtained from the conversion . both leaves and stems from the purple guinea grass , ruzi grass , pangola grass , atratum grass , and vetiver grasses were used since their stems are rather small . however , only the leaves were employed from napier grass , dwarf napier grass , king napier grass , and bana grass from their strong , hard stems together with the limitation of the specific types of facilities required for the chopping and grinding . as the compositions of biomass in each type of grass diverse immensely , even the same type possibly differs depending on the environment or the plant ages . the factors that can affect growth , production and composition of grasses can be genetics , soil properties , maintenance , amount , and distribution of rain . moreover , the cuttings of the stems and leaves of plants for various purposes result in the decrease of photosynthetic activities thereby reducing the accumulation of carbohydrates . on the contrary , the proteins will be increased . from the analysis of the composition of the biomass in 18 types of grasses studied , it was found that all types contained the average quantities of cellulose in the range of 31.8538.51% , 31.1342.61% of hemicellulose and 3.105.64% of lignin ( table 1 ) . the low level of lignin present in the samples of grasses used in this study appeared more advantageous than the other types , for example , 6.4% in coastal bermuda grass , 12% in switch grass , 7.3% in s32 rye grass ( seed setting ) , 4.7% in orchard grass [ 2 , 5 ] , and 10.7% in miscanthus grass . moreover , since the free sugars were not detectable from the samples , the hydrolysis was necessary in order to obtain the sugar prior to the further fermentation process . the contents of plant biomass can be used to initially indicate the potentiality of grasses whether they are suitable for the application as the energy crops . all the studied 18 types of plants contained high contents of the total cellulose and hemicellulose of approximately 6080% . therefore , the determination of cellulose and hemicellulose can be applied to quantify the theoretical production of ethanol . the enzymes produced from the fungus t. reesei tistr 3081 were assayed for the activities of cellulase using -cellulose and xylanase using xylan as the carbon sources , respectively . the results revealed that cellulase and xylanase showed the activities of 0.948 0.05 and 92.13 6.86 u / ml , respectively , whereas the obtained specific activities were 1.09 0.09 and 65.32 1.59/mg protein , respectively . the various strains of fungus , trichoderma , especially t. reesei including the mutants , have been popularly applied from the high capacity of the cellulase production suitable for the hydrolysis . the enzymes have been shown to be tolerant against many inhibitors and show stability at 50c comparatively higher than other types of fungi . the advantages of the application of t. reesei are the fungal capability to produce the mixture of cellulases containing activities of at least 2 types of cellobiohydrolases , 5 types of endoglucanases together with -glucosidases . in addition , hemicellulases can also be produced which was reported by zhang and lynd . the studies on the production of cellulases and hemicellulases from the fungus , t. reesei rut c30 in the presence of 7.5 g / l of solka floc as the carbon source were also conducted by juhsz et al . . the results revealed that the obtained specific activities of cellulase and xylanase by the fpu assay were approximately 0.58 and 119 u / mg protein , respectively . it can be clearly seen that the fungus , t. reesei can produce both cellulases and hemicellulases . hence , the organism appears suitable for the production of the hydrolytic enzymes for lignocellulosic material degradation . as a result , the number of the types of enzymes required for the process can be reduced thereby reducing the costs . in this research , only the glucose and xylose produced from the hydrolysis of cellulose and xylan by cellulase and xylanase were studied , respectively , since the main composition of hemicellulose is generally xylan . the comparison of the efficiency for the hydrolysis of each type of grass was evaluated from the percentages of the conversion of the total reducing sugar since each type contained different quantities of cellulose and hemicellulose . the pretreatment of the raw materials was conducted to disaggregate lignocellulose into the various compositions , namely , cellulose , hemicellulose , and lignin . the objective was to remove lignin , reduce cellulose crystallinity , increase the porosity of the raw materials resulting in cellulose accessibility to hydrolysis for conversion to fuels [ 2 , 25 , 28 ] . the use of alkaline hydrogen peroxide was an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production . in addition , it has been widely applied with various substrates such as wheat straw [ 29 , 30 ] , rice hulls , sugarcane bagasse , barley straw , and miscanthus grass . the advantages of this pretreatment are the use of reagents with low environmental impact and avoidance of special reaction chambers . as a consequence , no reducing sugars were obtained without the raw material pretreatment before the enzymatic hydrolysis ( data not shown ) . hence , the necessity of the pretreatment is very imperative . from the hydrolysis of 18 types of grasses by chemical pretreatment with alkaline peroxide followed by hydrolysis with cellulase and xylanase produced from the fungus t. reesei tistr 3081 , the total reducing sugar obtained from the hydrolysis most of the grass samples ' total reducing sugar showed the values in the range of 500600 mg / g of raw materials equivalent to the concentration of 45 these chemicals are always detected after the pretreatment and the hydrolysis by the use of acid and act as the inhibitors for the growth of yeasts used for the ethanol fermentation . rice hulls and wheat straw pretreated with alkaline peroxide could be converted to fermentable sugars with an excellent yield ( 9697% ) by enzymatic saccharification [ 19 , 29 ] . the values were high since the commercial enzymes , namely , celluclast 1.5 l ( cellulase ) , novozyme 188 ( -glucosidase ) , and viscostar 150 l ( xylanase ) were applied in their work . these commercial enzymes must absolutely give much higher activities than the enzyme produced from the fungus t. reesei in our research . moreover , the presence of -glucosidase facilitated the hydrolysis of cellobiose to glucose resulting in the higher percentages of sugar conversion . since the sugar content and the value of sugar conversion from each grass obtained by the enzymatic hydrolysis were rather indifferent , the decisive selection of the suitable grass for the subsequent fermentation was not accurately possible . therefore , the dry mass values of the grass samples were included in the calculations of the obtained reducing sugar contents from the enzymatic hydrolysis and the theoretical production of ethanol could be later calculated from the reducing sugar , assuming that the theoretical ethanol yield for fermenting is 0.511 g per g of hexose or pentose . from the results tabulated in table 2 , 11 types of grassses with the total reducing sugars over 3.9 ton / ha / year or the theoretical values for the ethanol production exceeding 2,550 l / ha / year were selected for the subsequent fermentation in descending order from the maximum , namely , pangola , purple guinea , dwarf napier leaves , atratum , ruzi , prachuap khiri khan , ratchaburi , bana leaves , napier leaves , king napier leaves and sri lanka , respectively . hydrolysis of the main composition , cellulose and hemicellulose , into glucose and xylose as the substrates of fermentation is necessarily required for the production of ethanol . in this research , the mixture of 2 types of yeasts was used , s. cerevisiae and p. stipites , since the former organism can use only glucose while the latter can catalyze both glucose and xylose as the substrates . there are various methods to achieve the fermentation but sscf has been shown to be more efficient than the others from the fact that the raw materials can be hydrolyzed to hexose and pentose for further ethanol fermentation in single step . the gradual release of glucose from the hydrolysis can be continuously used for the fermentation resulting in the low concentration of glucose which yields better fermentation for xylose . when the initially selected 11 types of grasses were fermented by sscf by the addition of 2 types of yeasts ; s. cerevisiae and p. stipitis together with 2 enzymes produced from t. reesei tistr 3081 ; cellulase and xylanase at 35c for 7 days , the ethanol production from various raw materials was tabulated in table 3 . the highest yield of ethanol , 1.14 g / l or 0.14 g / g substrate equivalent to 32.72% of the theoretical values , was obtained from sri lanka ecotype vetiver grass . for purple guinea grass and pangola grass , it was found that the lowest yield of ethanol was obtained at 6.08 and 6.8% , respectively , compared to the theoretical values . this result conflicted with the preliminary results of enzymatic hydrolysis that purple guinea grass and pangola grass gave the highest conversion of sugar of 85.56 3.44 and 76.03 0.84% respectively . this indicated that these 2 types of grasses may contain certain chemicals such as tannin or silica in the quantities that could possibly affect the activities of the enzyme and the growth of the yeasts resulting in the reduction of the ethanol production obtained from fermentation . moreover , the presence of other substances toxic to the microorganisms in the system , such as the derivatives of lignin which are the phenolic compounds , may reduce the efficiency of the fermentation . studied the ssf fermentation in switchgrass by aqueous ammonia pretreatment and found that lignin could be eliminated by 4050% . the yield of ethanol at 72% of the theoretical values was obtained from the application of the fermentation mixture between commercial cellulase ( spezyme cp ) with the activity of 77 fpu / ml together with s. cerevisiae d5a for the period of 10 days . the differences could be from the fact that hemicellulose and lignin which can act as the inhibitors were preseparated . only cellulose was used in the ethanol production by cellulase hydrolysis and single microorganism in ssf fermentation resulting in higher amount of ethanol . in the analysis to determine the potential suitable grasses for the cultivation as the energy crops , the dry mass production of each type of grass ( table 1 ) was included in the calculation . the quantities of ethanol production were calculated in 3 cases as tabulated in table 4 . for the first case , the theoretical ethanol production was calculated from the determination of cellulose and hemicellulose from each type of grass . the complete hydrolysis of cellulose and hemicellulose and the amount of sugar totally converted to ethanol were used for the calculations . the overall theoretical yield for conversion was 0.581 g of ethanol per g of xylan and 0.568 g of ethanol per g of cellulose , assuming that xylan represents the major sugar source in all the biomass hemicellulose . the theoretical ethanol yield for fermentation is 0.511 g per g of hexose or pentose . the results showed that the highest yield of ethanol was obtained from the pangola grass at 18,706.69 l / ha / year from the highest value of dry biomass at 37.5 ton / ha / year . as a result , it might have been suitable to be applied as the energy crops for the production of ethanol . nevertheless , there are still many necessary factors required for the analysis such as the plantation areas for the cultivation , maintenance , and the composition in various grasses which may contain inhibitors affecting the activities of certain enzymes and the growth of the microorganisms in the fermentation process . apparently , the obtained values of the sugar contents from the hydrolysis and the obtained ethanol from the fermentation were inconsistent with the theory . hence , further studies on the hydrolysis of the biomass composition and the ethanol production should be conducted to determine the real quantity of the ethanol production . when the analysis has been performed in more details , other appropriate types of grasses may also have been discovered . for the second case , when the theoretical values of ethanol production were calculated from the total reducing sugars obtained after the enzymatic hydrolysis , it was found that pangola grass still produced the highest theoretical values of ethanol production of 12,654.31 l / ha / yr despite the rather similar values of reducing sugars obtained from each type of grass after the enzymatic hydrolysis . therefore , the highest yield of dry mass obtained from pangola grass consistently resulted in the highest value of theoretical ethanol production . for the final third case , it was found that dwarf napier grass leaves showed the highest yield of 2,720.56 l / ha / year . therefore , this grass should have potential to be developed as the energy crop for the production of ethanol by this sscf fermentation . further studies can be carried out on the optimal conditions of fermentation to increase the production of ethanol approximately closed to the theoretical values . in this research , since the samples of rather aged grasses with the very strong and hard stems were collected , only the leaves were selectively applied for the experiments . nevertheless , if the dwarf napier grass leaves were to be utilized for the application in the future , they should be harvested at the earlier stage so that both the stems and the leaves can be used . when the theoretical ethanol production from pangola grass obtained from the second case was comparatively considered with the values obtained from the process of ssfc , it can be seen that the theoretical ethanol production from pangola grass was 5-folds higher than what obtained from dwarf napier grass from the process of ssfc . on the contrary , the ethanol production from pangola grass obtained from ssfc was much lower than the theoretical values since the concentration of ethanol obtained was considerably low when compared with the others . this result suggested that the fermentation by the method in this study might not have been suitable for this grass . pangola grass may not be appropriate for the ethanol production by sscf fermentation . however , pangola grass yielded the highest quantities of ethanol when the analysis was derived from both theoretical calculations . more importantly , it seems appropriately applicable as the hay from the tremendously high contents of dry mass in this grass . hence , pangola grass may also present potentiality as the energy crop in thailand . in case this type of grass would be further developed for the production of ethanol that would yield the closest values to the theory , the fermentation process should be further investigated with some alterations . in addition , the fermentation by shf can represent the good alternative process since the high level of sugar can be produced from the enzymatic hydrolysis of this type of grass . recently , bank of thailand , in january 2012 reported the consumption of ethanol in thailand at approximately 1.3 million litres per day or 457 million litres per year . in case that the dwarf napier grass is to be applied for the production of ethanol by the process of ssfc , at least 0.17 million hectares of the plantation area are necessarily required . when the production of ethanol from other types of energy crops was comparatively studied , it has been found that the yield from sugar cane was equal to 4,900 kg / ha / year or 6,210 l / ha / year , whereas from cassava was 6,000 kg / ha / year or 7,604 l / ha / year . although the production of ethanol from the sscf process derived from dwarf napier grass was found to be 2,720.56 l / ha / year or 2.3 and 2.8-folds less than what obtained from the sugar cane and cassava , respectively , both of the mentioned energy crops are mostly utilized for human food consumption . hence , the search for the alternative nonfood energy crops for the production of ethanol appears undeniably and continuously imperative in order to sustain the availability of the food energy crops for the prevention of food shortage problem in the future . after the process , the total raw materials did not appear to be completely hydrolyzed . this might be from the fact that the optimal temperature for the activity of the enzymes should be in the range of 4550c . however , the temperature applied in this process was at 35c . for the fermentation by yeast , furthermore , the other conditions for the fermentation by these 2 microorganisms might not be optimal resulting in less ethanol production than it should have been . therefore , the investigation on the optimal conditions about the various factors such as ph , temperature , oxygen content , and the medium ingredients affecting the fermentation is considerably imperative . this will enhance the efficiency of the sugar cofermentation from both s. cerevisiae and p. stipitis . the method to produce high concentration of ethanol can be accomplished by producing the enzyme with high hydrolytic activities to increase the efficiency of the hydrolysis . moreover since the enzyme applied in this research was totally isolated from the fungi in our lab , the activities were therefore considerably low compared to the commercials . as a consequence , the large quantities of the enzyme were necessarily applied for the process . the improvement of the production media such as the sources of carbon and the growth conditions for the culture should greatly enhance the production of the enzyme with higher activities . in addition , the development of the better strain by genetic manipulation also represents the interesting method to increase the fungal capacity for the production of the better enzyme with higher activities . this can be achieved through the higher efficiency of the fermentation in various aspects , for example , the strains that can possibly use various types of sugars as the substrates , the capability of the organisms to tolerate the high temperature , and high concentration of ethanol . in particular , in case that cellulosic ethanol will be further produced for the industries in the future , the development of the strains with the capability of simultaneous hydrolysis and fermentation of lignocellulose should be more advantageous . lignocellulosic biomass , like grasses , could be successfully used in cellulosic ethanol production by biological process . pangola grass has potential for saccharification before fermentation process , whereas dwarf napier grass has potential for fermentation by sscf process . therefore , they are potential alternative energy crops to serve as feedstocks for cellulosic ethanol production in the future for thailand .

the grasses in thailand were analyzed for the potentiality as the alternative energy crops for cellulosic ethanol production by biological process . the average percentage composition of cellulose , hemicellulose , and lignin in the samples of 18 types of grasses from various provinces was determined as 31.8538.51 , 31.1342.61 , and 3.105.64 , respectively . the samples were initially pretreated with alkaline peroxide followed by enzymatic hydrolysis to investigate the enzymatic saccharification . the total reducing sugars in most grasses ranging from 500600 mg / g grasses ( 7080% yield ) were obtained . subsequently , 11 types of grasses were selected as feedstocks for the ethanol production by simultaneous saccharification and cofermentation ( sscf ) . the enzymes , cellulase and xylanase , were utilized for hydrolysis and the yeasts , saccharomyces cerevisiae and pichia stipitis , were applied for cofermentation at 35c for 7 days . from the results , the highest yield of ethanol , 1.14 g / l or 0.14 g / g substrate equivalent to 32.72% of the theoretical values was obtained from sri lanka ecotype vetiver grass . when the yields of dry matter were included in the calculations , sri lanka ecotype vetiver grass gave the yield of ethanol at 1,091.84 l / ha / year , whereas the leaves of dwarf napier grass showed the maximum yield of 2,720.55 l / ha / year ( 0.98 g / l or 0.12 g / g substrate equivalent to 30.60% of the theoretical values ) .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

the energy crops are the types of plants expected to be cultivated as the raw materials for the production of the biofuels such as ethanol . therefore , the plants with the fast growing rate such as grasses or shrubs should represent the appropriate energy crops for the future . grasses are targeted as potential energy crops because of high productivity per hectare , abundancy , availability , and utilization of the whole plants . in general , the average compositions of grass biomass are 2540% of cellulose , 2550% of hemicellulose , and 1030% of lignin [ 2 , 5 ] . therefore , the application of enzymes appears more interesting from the high specificity , neutral reaction without the unwanted by products , and therefore cost reduction in the waste management . obviously , the production of ethanol from agricultural wastes can only be applied for the short - term purpose since the amount of such waste wo n't be sufficient in the future . therefore , in order to maintain long - term and successfully sustainable production of the alternative energy , the cultivation of energy crops together with the utilization of agricultural wastes and the search for the potential plants which can produce ethanol should be implemented . hence , the objective of this research was to select the types of grasses in thailand suitable as the feedstocks for cellulosic ethanol production by biological process . the fungus , trichoderma reesei , has been widely accepted as the organism capable of producing the enzymes , cellulase , and hemicellulase in considerable quantities , appropriate for lignocellulosic biomass digestion . although the fermentation can be achieved from various processes , the application of simultaneous saccharification and cofermentation ( sscf ) this method allows the raw materials to be hydrolyzed to hexose and pentose sugars for the subsequent fermentation to obtain ethanol in the single step . similarly , the yeasts , saccharomyces cerevisiae and pichia stipites , are widely known organisms that can utilize glucose and xylose for ethanol fermentation , respectively . 18 samples of grasses were collected from chiang mai , lampang , ratchaburi , and petchburi provinces . the induction of xylanase production was carried out in xylan medium containing 10 g / l birchwood xylan as a carbon source , 5 g / l corn steep liquor , 5 g / l polypeptone , 1 g / l yeast extract , 4 g / l k2hpo4 , 0.2 g / l kcl , 1 g / l mg2so4 7h2o , and 0.02 g after incubation at 30c and shaken at 150 rpm for 7 days , the mycelia were removed by filtration through whatman no.1 filter paper . crude enzyme was collected and determined for the activities of cellulase and xylanase using whatman no.1 filter paper and 1% birchwood xylan as the substrates , respectively , according to ghose . the liquid phase with solubilized hemicellulose and the solid phase with cellulose of the samples were subsequently hydrolyzed by the enzymes . cellulase ( 60 u / g substrate ) , xylanase ( 1200 u / g substrate ) , and 5 ml of 0.05 m sodium citrate buffer ( ph 4.8 ) were added to each substrate ( 0.6 g of milled grass ) and positive control ( 0.3 g of -cellulose mixed with 0.3 g of xylan ) . after the addition of the enzymes , the samples were incubated at 50c and shaken at 150 rpm for 72 hours . total reducing sugar after the hydrolysis ( ton / ha / year ) was considered for selection of suitable grasses as the substrates for ethanol production by sscf process . c is dry matter yield of grass ( ton / ha / year ) . p. stipitis cbs 5773 was obtained from the centraalbureau voor schimmelcultures ( cbs ) , the netherlands . s. cerevisiae and p. stipitis were precultured as inocula for fermentation in erlenmeyer flasks containing 50 ml of 3 g / l yeast extract , 3 g / l malt extract , 5 g / l ( nh4)2so4 , and 20 g / l glucose for s. cerevisiae or 20 g / l xylose for p. stipitis . cellulase and xylanase were filtered and sterilized through a 0.2 m filters and the enzymes were added with the activities of 72 u and 1,440 u in each flask , respectively . the samples were incubated with shaking at 35c , 150 rpm for 7 days , and ethanol production was determined by gas chromatography ( shimadzu , japan ) collected data ( 3 replicates ) were analyzed by one - way anova at 95% confidence level . the first group was composed of 8 types of the forage grasses ( napier leaves , dwarf napier leaves , king napier leaves , bana leaves , purple guinea , ruzi , pangola , and atratum ) and the second one contained 10 ecotypes of vetiver grasses ( kamphaeng phet 2 , songkhla 3 , surat thani , sri lanka , roi et , loei , nakhon sawan , prachuap khiri khan , ratchaburi , and kamphaeng phet 1 ) as shown in figure 1 . since the height of all the grasses in this study is more than 1 metre , and the harvest can be carried out several times per year , considerably huge amount of dry biomass can be obtained from the conversion . both leaves and stems from the purple guinea grass , ruzi grass , pangola grass , atratum grass , and vetiver grasses were used since their stems are rather small . however , only the leaves were employed from napier grass , dwarf napier grass , king napier grass , and bana grass from their strong , hard stems together with the limitation of the specific types of facilities required for the chopping and grinding . moreover , the cuttings of the stems and leaves of plants for various purposes result in the decrease of photosynthetic activities thereby reducing the accumulation of carbohydrates . from the analysis of the composition of the biomass in 18 types of grasses studied , it was found that all types contained the average quantities of cellulose in the range of 31.8538.51% , 31.1342.61% of hemicellulose and 3.105.64% of lignin ( table 1 ) . the low level of lignin present in the samples of grasses used in this study appeared more advantageous than the other types , for example , 6.4% in coastal bermuda grass , 12% in switch grass , 7.3% in s32 rye grass ( seed setting ) , 4.7% in orchard grass [ 2 , 5 ] , and 10.7% in miscanthus grass . the contents of plant biomass can be used to initially indicate the potentiality of grasses whether they are suitable for the application as the energy crops . all the studied 18 types of plants contained high contents of the total cellulose and hemicellulose of approximately 6080% . therefore , the determination of cellulose and hemicellulose can be applied to quantify the theoretical production of ethanol . the enzymes produced from the fungus t. reesei tistr 3081 were assayed for the activities of cellulase using -cellulose and xylanase using xylan as the carbon sources , respectively . the results revealed that cellulase and xylanase showed the activities of 0.948 0.05 and 92.13 6.86 u / ml , respectively , whereas the obtained specific activities were 1.09 0.09 and 65.32 1.59/mg protein , respectively . the studies on the production of cellulases and hemicellulases from the fungus , t. reesei rut c30 in the presence of 7.5 g / l of solka floc as the carbon source were also conducted by juhsz et al . the results revealed that the obtained specific activities of cellulase and xylanase by the fpu assay were approximately 0.58 and 119 u / mg protein , respectively . as a result , the number of the types of enzymes required for the process can be reduced thereby reducing the costs . in this research , only the glucose and xylose produced from the hydrolysis of cellulose and xylan by cellulase and xylanase were studied , respectively , since the main composition of hemicellulose is generally xylan . the comparison of the efficiency for the hydrolysis of each type of grass was evaluated from the percentages of the conversion of the total reducing sugar since each type contained different quantities of cellulose and hemicellulose . the pretreatment of the raw materials was conducted to disaggregate lignocellulose into the various compositions , namely , cellulose , hemicellulose , and lignin . as a consequence , no reducing sugars were obtained without the raw material pretreatment before the enzymatic hydrolysis ( data not shown ) . from the hydrolysis of 18 types of grasses by chemical pretreatment with alkaline peroxide followed by hydrolysis with cellulase and xylanase produced from the fungus t. reesei tistr 3081 , the total reducing sugar obtained from the hydrolysis most of the grass samples ' total reducing sugar showed the values in the range of 500600 mg / g of raw materials equivalent to the concentration of 45 these chemicals are always detected after the pretreatment and the hydrolysis by the use of acid and act as the inhibitors for the growth of yeasts used for the ethanol fermentation . rice hulls and wheat straw pretreated with alkaline peroxide could be converted to fermentable sugars with an excellent yield ( 9697% ) by enzymatic saccharification [ 19 , 29 ] . the values were high since the commercial enzymes , namely , celluclast 1.5 l ( cellulase ) , novozyme 188 ( -glucosidase ) , and viscostar 150 l ( xylanase ) were applied in their work . since the sugar content and the value of sugar conversion from each grass obtained by the enzymatic hydrolysis were rather indifferent , the decisive selection of the suitable grass for the subsequent fermentation was not accurately possible . therefore , the dry mass values of the grass samples were included in the calculations of the obtained reducing sugar contents from the enzymatic hydrolysis and the theoretical production of ethanol could be later calculated from the reducing sugar , assuming that the theoretical ethanol yield for fermenting is 0.511 g per g of hexose or pentose . from the results tabulated in table 2 , 11 types of grassses with the total reducing sugars over 3.9 ton / ha / year or the theoretical values for the ethanol production exceeding 2,550 l / ha / year were selected for the subsequent fermentation in descending order from the maximum , namely , pangola , purple guinea , dwarf napier leaves , atratum , ruzi , prachuap khiri khan , ratchaburi , bana leaves , napier leaves , king napier leaves and sri lanka , respectively . hydrolysis of the main composition , cellulose and hemicellulose , into glucose and xylose as the substrates of fermentation is necessarily required for the production of ethanol . in this research , the mixture of 2 types of yeasts was used , s. cerevisiae and p. stipites , since the former organism can use only glucose while the latter can catalyze both glucose and xylose as the substrates . when the initially selected 11 types of grasses were fermented by sscf by the addition of 2 types of yeasts ; s. cerevisiae and p. stipitis together with 2 enzymes produced from t. reesei tistr 3081 ; cellulase and xylanase at 35c for 7 days , the ethanol production from various raw materials was tabulated in table 3 . the highest yield of ethanol , 1.14 g / l or 0.14 g / g substrate equivalent to 32.72% of the theoretical values , was obtained from sri lanka ecotype vetiver grass . for purple guinea grass and pangola grass , it was found that the lowest yield of ethanol was obtained at 6.08 and 6.8% , respectively , compared to the theoretical values . this result conflicted with the preliminary results of enzymatic hydrolysis that purple guinea grass and pangola grass gave the highest conversion of sugar of 85.56 3.44 and 76.03 0.84% respectively . this indicated that these 2 types of grasses may contain certain chemicals such as tannin or silica in the quantities that could possibly affect the activities of the enzyme and the growth of the yeasts resulting in the reduction of the ethanol production obtained from fermentation . moreover , the presence of other substances toxic to the microorganisms in the system , such as the derivatives of lignin which are the phenolic compounds , may reduce the efficiency of the fermentation . the yield of ethanol at 72% of the theoretical values was obtained from the application of the fermentation mixture between commercial cellulase ( spezyme cp ) with the activity of 77 fpu / ml together with s. cerevisiae d5a for the period of 10 days . only cellulose was used in the ethanol production by cellulase hydrolysis and single microorganism in ssf fermentation resulting in higher amount of ethanol . in the analysis to determine the potential suitable grasses for the cultivation as the energy crops , the dry mass production of each type of grass ( table 1 ) was included in the calculation . for the first case , the theoretical ethanol production was calculated from the determination of cellulose and hemicellulose from each type of grass . the complete hydrolysis of cellulose and hemicellulose and the amount of sugar totally converted to ethanol were used for the calculations . the results showed that the highest yield of ethanol was obtained from the pangola grass at 18,706.69 l / ha / year from the highest value of dry biomass at 37.5 ton / ha / year . as a result , it might have been suitable to be applied as the energy crops for the production of ethanol . nevertheless , there are still many necessary factors required for the analysis such as the plantation areas for the cultivation , maintenance , and the composition in various grasses which may contain inhibitors affecting the activities of certain enzymes and the growth of the microorganisms in the fermentation process . apparently , the obtained values of the sugar contents from the hydrolysis and the obtained ethanol from the fermentation were inconsistent with the theory . hence , further studies on the hydrolysis of the biomass composition and the ethanol production should be conducted to determine the real quantity of the ethanol production . when the analysis has been performed in more details , other appropriate types of grasses may also have been discovered . for the second case , when the theoretical values of ethanol production were calculated from the total reducing sugars obtained after the enzymatic hydrolysis , it was found that pangola grass still produced the highest theoretical values of ethanol production of 12,654.31 l / ha / yr despite the rather similar values of reducing sugars obtained from each type of grass after the enzymatic hydrolysis . therefore , the highest yield of dry mass obtained from pangola grass consistently resulted in the highest value of theoretical ethanol production . for the final third case , it was found that dwarf napier grass leaves showed the highest yield of 2,720.56 l / ha / year . in this research , since the samples of rather aged grasses with the very strong and hard stems were collected , only the leaves were selectively applied for the experiments . nevertheless , if the dwarf napier grass leaves were to be utilized for the application in the future , they should be harvested at the earlier stage so that both the stems and the leaves can be used . when the theoretical ethanol production from pangola grass obtained from the second case was comparatively considered with the values obtained from the process of ssfc , it can be seen that the theoretical ethanol production from pangola grass was 5-folds higher than what obtained from dwarf napier grass from the process of ssfc . on the contrary , the ethanol production from pangola grass obtained from ssfc was much lower than the theoretical values since the concentration of ethanol obtained was considerably low when compared with the others . pangola grass may not be appropriate for the ethanol production by sscf fermentation . hence , pangola grass may also present potentiality as the energy crop in thailand . in addition , the fermentation by shf can represent the good alternative process since the high level of sugar can be produced from the enzymatic hydrolysis of this type of grass . in case that the dwarf napier grass is to be applied for the production of ethanol by the process of ssfc , at least 0.17 million hectares of the plantation area are necessarily required . when the production of ethanol from other types of energy crops was comparatively studied , it has been found that the yield from sugar cane was equal to 4,900 kg / ha / year or 6,210 l / ha / year , whereas from cassava was 6,000 kg / ha / year or 7,604 l / ha / year . although the production of ethanol from the sscf process derived from dwarf napier grass was found to be 2,720.56 l / ha / year or 2.3 and 2.8-folds less than what obtained from the sugar cane and cassava , respectively , both of the mentioned energy crops are mostly utilized for human food consumption . hence , the search for the alternative nonfood energy crops for the production of ethanol appears undeniably and continuously imperative in order to sustain the availability of the food energy crops for the prevention of food shortage problem in the future . this might be from the fact that the optimal temperature for the activity of the enzymes should be in the range of 4550c . for the fermentation by yeast , furthermore , the other conditions for the fermentation by these 2 microorganisms might not be optimal resulting in less ethanol production than it should have been . therefore , the investigation on the optimal conditions about the various factors such as ph , temperature , oxygen content , and the medium ingredients affecting the fermentation is considerably imperative . as a consequence , the large quantities of the enzyme were necessarily applied for the process . the improvement of the production media such as the sources of carbon and the growth conditions for the culture should greatly enhance the production of the enzyme with higher activities . this can be achieved through the higher efficiency of the fermentation in various aspects , for example , the strains that can possibly use various types of sugars as the substrates , the capability of the organisms to tolerate the high temperature , and high concentration of ethanol . in particular , in case that cellulosic ethanol will be further produced for the industries in the future , the development of the strains with the capability of simultaneous hydrolysis and fermentation of lignocellulose should be more advantageous . lignocellulosic biomass , like grasses , could be successfully used in cellulosic ethanol production by biological process . therefore , they are potential alternative energy crops to serve as feedstocks for cellulosic ethanol production in the future for thailand .

a disintegrin and metalloproteinase with thrombospondin motifs ( adamts ) family consists of a precisely ordered modular organization that includes at least one thrombospondin type i repeat . this family is thought to be comprised of a zinc - dependent group of proteases , which play vital roles in a variety of normal and pathological conditions [ 27 ] . the adamts family has grown to include 19 members , which are involved in a plethora of diseases ranging from arthritis to coagulation disorders [ 1 , 919 ] . the adamts family is divided into four subgroups based on their structural and functional similarities [ 8 , 2022 ] . adamts-17 and -19 , adamts-16 and -18 , adamts-7 and -12 , and adamts-6 and -10 compose the structurally related adamts pairs division . it has been well established that the adamts-12 gene is expressed in the musculoskeletal system ( skeletal muscles , cartilage , and tendons ) and in the fetal lung [ 1 , 23 , 24 ] for instance , adamts-12 has been proven to be associated with the pathogenesis of arthritis [ 816 ] , intervertebral disc degeneration [ 25 , 26 ] , inflammation [ 27 , 28 ] , and invasion and metastasis of tumor [ 2931 ] . although adamts-7 and -12 share similar structure and domain organization , adamts-7 and -12 may have different pathological effects or be responsible for different phases in a disease [ 26 , 32 ] . for instance , adamts-7 mrna is found to be significantly increased in the cartilage of patients with rheumatoid arthritis ( ra ) and only slightly increased in the cartilage of patients with osteoarthritis ( oa ) , whereas adamts-12 mrna is significantly increased in both oa and ra cartilage [ 10 , 32 ] . moreover , in intervertebral disc degenerative ( ivd ) rat model , the level of adamts-7 was significantly increased in the early phase while the level of adamts-12 was increased in the latter phase . in this review , we will discuss the structure and perspective of adamts-12 and focus on its roles and the underlying mechanisms in arthritis ( especially osteoarthritis ) and other diseases , including inflammation , tumorigenesis and antiangiogenesis , intervertebral disc degenerative disease , schizophrenia , gonad differentiation , trophoblast invasion , and pediatric stroke ( figure 1 ) . in addition , we used adamts12 and adamts-12 as keywords to search articles through pubmed , google scholar , and web of science . the structure of adamts-12 includes a signal peptide , a prodomain , a catalytic domain , a disintegrin - like domain , a central thrombospondin i ( ts ) repeat , a cysteine - rich domain , two spacer regions , and c - terminal tsp - like repeat region ( figure 2 ) [ 33 , 34 ] . it was believed that the c - terminal tail of adamts-12 might be important for the ability of adamts-12 binding to the components of extracellular matrix ( ecm ) . indeed , it was found that the four tsp - like repeat regions of adamts-12 directly bound to the egf - like domain of cartilage oligomeric matrix protein ( comp ) . it was also reported that the c - terminal four tsp - like repeat inhibited chondrocyte differentiation . to determine the effects of each functional domain of adamts-12 on chondrocyte differentiation , numerous deleted mutants were generated , such as the removal of c - terminal four thrombospondin motifs known to bind comp , the removal of a spacer-2 domain , the deletion of three thrombospondin motifs , and the deletion of a spacer-1 domain . all of the above mentioned deletions did not change the cell surface localization of the protein in chondrocytes . however , the deletion of the cysteine - rich domain released the enzyme from cell surface into the medium . these data indicated that the cysteine - rich domain of adamts-12 was required for its binding to ecm and cell surface appearance in chondrocytes . in addition , the antiangiogenesis properties of adamts-12 did not rely on the catalytic domain , but , more likely , on its tsp-1 domains . in contrast , the catalytic activity was implicated in the protective effect against allergen - induced inflammation . arthritis is the leading cause of physical disability , health care usage , and impaired quality of life [ 3638 ] . the impact of arthritic conditions is expected to grow as the elderly population increases in the coming decades [ 39 , 40 ] . osteoarthritis ( oa ) and rheumatoid arthritis ( ra ) are the most common categories of arthritis [ 8 , 4143 ] . oa is associated with age - related loss of homeostatic balance between degradation and repair mechanisms in articular cartilage , which is characterized by synovitis , cartilage degeneration , and osteophyte formation [ 4447 ] . oa is a multifactorial disorder which can be affected by genetic alternation , inflammation , mechanical stress , obesity , and aging as well as biochemical , molecular , and enzymatic factors [ 4851 ] . ra is a chronic systemic inflammatory disease that is characterized by the inflammatory proliferation of synovial tissues , leading to cartilage and bone damage and eventual disability [ 5254 ] . both genetic and environmental factors , as well as aberrant immune responses , play a key role in the pathogenesis of ra [ 55 , 56 ] . the dysregulation in both oa and ra induces senescence , differentiation , proliferation , and cell death in joint cells through gene and/or protein expression networks that switch from anabolic to catabolic outcomes . recent evidence has demonstrated the significance of proteases , in the pathological processes of arthritis [ 5759 ] . the destruction of the extracellular matrix of articular cartilage and bone in arthritic joints is thought to be mediated by excessive proteolytic activity . in accordance with this concept , recent studies show that adamts-12 also plays a critical role in the pathological process of arthritis [ 8 , 32 , 35 , 43 , 61 ] . osteoarthritis is an age - related or posttraumatic degenerative disease of the joint that is characterized by loss of articular cartilage , chondrocyte proliferation and hypertrophic differentiation , subchondral bone remodeling , inflammation , and finally osteophyte formation [ 62 , 63 ] . there is a strong relevance between cartilage degradation and osteoarthritis [ 6466 ] . in that case , anything that can affect cartilage may play a role in the initiation and progression of osteoarthritis . the finding that adamts-12 was isolated as a binding metalloproteinase of comp , an extracellular matrix highly expressed in cartilage [ 8 , 32 , 6771 ] , promoted us to determine the role of adamts-12 in cartilage development and degeneration . it was reported that adamts-12 , functioning as a downstream molecule of parathyroid hormone related peptide ( pthrp ) signaling , negatively regulated chondrogenesis . adamts-12 was prominently expressed in proliferating and prehypertrophic chondrocytes within the wild - type embryonic growth plate , whereas adamts-12 was hardly detected in pthrp(/ ) embryos . in addition , within the adamts-12 overexpressing cells , the expressions of both early ( collagen ii and sox9 ) and late ( collagen x ) marker genes for chondrogenesis were all repressed . furthermore , pthrp signaling was nearly abolished when adamts-12 was repressed , and pthrp action was largely restored when adamts-12 was reexpressed . notably , the negative regulation of chondrocyte differentiation by adamts-12 depended on its enzymatic activity since its point mutant lacking enzymatic activity completely lost this activity . the expression of adamts-12 in the course of chondrogenesis was regulated by c - maf transcription factor . c - maf , the cellular homologue of the avian viral oncogene c - maf , was a member of the basic leucine zipper ( bzip ) superfamily [ 7375 ] . c - maf proteins bind to a specific dna sequences termed the maf recognition element ( mare ) through the basic dna recognition domain . in addition , c - maf was overexpressed during embryonic development and postnatal growth in hypertrophic chondrocytes as well as in the osteoarthritic chondrocytes , which was similar to the expression pattern of that of adamts-12 [ 7375 ] . furthermore , c - maf transcriptional factor directly binds to the proximal mare sequences of adamts-12 at position -61 in vitro . interestingly , adamts-12 was found to function as the downstream target of parathyroid hormone - like hormone / parathyroid hormone related peptide ( pthlh / pthrp ) in chondrocytes , and both pthlh and adamts-12 were all downregulated along with impaired chondrogenic differentiation within brachydactyly type e ( bde ) . bde is an autosomal - dominant disease characterized by bilateral manifestation of shortened metacarpals , metatarsals , and/or phalanges [ 7880 ] . additionally , pthlh and both of its targets , adamts-7 and -12 , were downregulated in the fibroblasts of bde patients . furthermore , the expressions of chondrogenic markers ( aggrecan , collagen ii , collagen x , and indian hedgehog ( ihh ) ) in chondrogenically differentiated bde were all significantly altered . collectively , adamts-12 , whose expression was regulated by c - maf transcription factor , acted as a downstream molecule of pthrp signaling , negatively regulated chondrocyte differentiation , and hypertrophy ( figure 3 ) . in addition , proper expression of adamts-12 is required for normal cartilage development and its dysregulation may lead to pathologies with defects in the musculoskeletal system , such as bde . abnormal level and/or function of adamts-12 may induce dysregulation of cartilage , which finally leads to arthritis . adamts-12 's involvements in osteoarthritis is most probably due to its association and degradation of cartilage oligomeric matrix protein ( comp ) [ 1 , 8 , 32 , 6771 ] . comp , a prominent noncollagenous component of cartilage , accounts for approximately 1% of the wet weight of the tissue . the function of comp may be involved in stabilizing cartilage ecm via specific interactions with matrix components such as collagen types ii and ix , aggrecan , and fibronectin [ 8183 ] . comp also has a role in mediating chondrocyte attachment via an integrin receptor [ 8487 ] . it was reported that mutations in the type iii or c - terminal globular domain of the human comp gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia , which were autosomal - dominant forms of short - limb dwarfism [ 88 , 89 ] . comp fragments have been detected in the diseased cartilage , synovial fluid , and serum of patients with knee injuries . the level of adamts-12 was significantly increased in the cartilage and synovium of patients with ra or oa [ 9 , 10 ] . both the in vitro glutathione s - transferase ( gst ) pull - down assay and coimmunoprecipitation assay demonstrated that adamts-12 could bind to comp directly . importantly , the size of comp fragments generated by adamts-12 was similar to those of comp - degraded fragments seen in arthritic patients . in addition , antibodies against adamts-12 dramatically inhibited tumor necrosis factor - induced ( tnf - induced ) and interleukin-1-induced ( il-1-induced ) comp degradation in cartilage explants . suppression of adamts-12 expression using the small interfering rna silencing approach in human chondrocytes also markedly prevented comp degradation . in addition , adamts-7 and -12 were colocalized with comp both in the cytoplasm and on the surface of human chondrocytes [ 25 , 35 ] . while adamts-7 was unable to digest aggrecan , adamts-12 , with a similar structure , did have the ability to digest aggrecan . it was reported that alpha-2-macroglobulin ( 2 m ) acted as a substrate for both adamts-7 and adamts-12 and efficiently protected comp degradation by these enzymes by in vitro digestion assays . in addition , granulin - epithelin precursor ( gep , a newly identified chondrogenic growth factor , which was also known as proepithelin , acrogranin , progranulin ( pgrn ) , and gp88/pc cell - derived growth factor ) inhibited the action of adamts-12 preventing comp degradation via two distinct mechanisms [ 61 , 9297 ] . in detail , gep inhibits the induction of adamts-12 by inflammatory cytokines such as tnf- and gep is able to disrupt the association between adamts-12 and comp via a direct protein - to - protein interaction [ 8 , 61 ] . it was approved that adamts-12 , comp , aggrecan , gep , 2 m , and tnf- constitute an interplay and interaction network in mediating cartilage degradation in arthritis ( figure 4 ) . tnf- upregulates adamts-12 leading to the degradation of comp [ 43 , 98 , 99 ] . in addition , tgf- was reported to significantly induce the adamts-12 in human fetal fibroblasts . this in vitro data provide insight into the critical role of adamts-12 in the initiation and progression of osteoarthritis which needs to be further confirmed using in vivo genetically modified animal models . interestingly , such in vivo models for adamts-7 , which shares similar domain organization and structure , have been generated , and the critical role of adamts-7 in the pathogenesis of oa has been elucidated . it was reported that adamts-7 formed a positive feedback loop with tnf- in the pathogenesis of osteoarthritis . targeted overexpression of adamts-7 in chondrocytes led to chondrodysplasia characterized by short - limbed dwarfism and a delay in endochondral ossification in young mice and a spontaneous oa - like phenotype in aged mice . in addition , overexpression of adamts-7 led to exaggerated breakdown of cartilage and accelerated oa progression , while knockdown of adamts-7 attenuated degradation of cartilage matrix and protected against oa development , in surgically induced destabilization of medial meniscus oa models [ 58 , 100 ] . adamts-7 upregulated tnf- and metalloproteinases associated with oa ; in addition , tnf- induced adamts-7 through nf-b signaling . these data indicated that adamts-7 and tnf- form a positive feedback loop in the regulation of cartilage degradation and oa progression [ 58 , 101 ] . aside from the fact that adamts-12 shares a similar structure with adamts-7 , adamts-12 may play a critical role in the oa development in vivo [ 10 , 23 , 58 ] . rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease , characterized by inflammation of the synovial membrane and progressive destruction of the articular cartilage and bone [ 102 , 103 ] . as we have previously stated , adamts-12 is an inflammation - related protein , which can digest comp ; it was reported that adamts-12 might play a role in ra [ 104 , 105 ] . three single nucleotide polymorphisms ( snps ) ( rs1364044 , intron c / t ; rs10461703 , intron c / t ; rs25754 , missense thr1495ile ) of adamts12 were genotyped by using a direct sequencing method in 303 ra patients and 495 control subjects . multiple logistic regression models , snpstats and snpanalyzer pro programs , and bonferroni 's correction ( pc ) were used and found that the genotype frequency of rs10461703 was associated with the ra development . however , no significant correlation was observed between the three tested snps and ra patients with regard to their clinical features . adamts-12 was reported to associate with sex - specific disparities in ra resulting in cartilage degradation . the involvement of sex chromosomes in ra and the relationship between animal models of ra and gender have been reported by several laboratories [ 108114 ] . in addition , it was suggested that collagen antibody - induced arthritis ( caia ) in congenic mice can be used to identify homologous ra associated molecular pathways . it was reported that balb / c.dba/2-pgia8- ( c.d2-pgia8- ) congenic mice carry a sex - affected arthritis - suppressive genomic interval which was associated with pgia . it was revealed that caia severity in pgia8-congenic females was 20% higher than in balb / c females . however , the inflammation severity of the pgia8-congenic males was 3045% lower than wild - type balb / c males , indicating that the genetic anti - inflammatory effect was specific towards male . criteria were set as a 1.5-fold change threshold in the caia severity change and three genes were found including collagen triple - helix repeat - containing 1 ( cthrc1 ) , c1q and tnf - related protein 3 ( c1qtnf3 ) , and adamts-12 , which were all associated with both gender and arthritis . in addition , these three genes were highly involved in the severity of arthritis with r = 0.91 , 0.89 , and 0.87 , respectively ; thus there is a strong correlation among these 3 genes . it was also demonstrated that the cthrc1 protein positively regulated the pcp - wnt pathway altering chondrocyte maturation and cartilage formation [ 115117 ] . collectively , these genetic and in vitro studies indicate that adamts-12 plays an important role in the pathogenesis of arthritis and may be a potential target for developing new treatments for arthritis . adamts-12-deficient mice model with arthritis are critical for verifying its role in the course of both osteoarthritis and rheumatoid arthritis in vivo . adamts-12 has also been reported to be a critical mediator in inflammation , especially allergic inflammation [ 24 , 27 , 28 ] . adamts-12 has been identified as one asthma - associated gene in the human genome screening program . asthma is characterized by a specific pattern of inflammation and airway / bronchial hyperresponsiveness ( ahr / bhr ) [ 118 , 119 ] . fine mapping and positional candidate studies were performed in the hutterites ( south dakota ) and an outbred case - control sample from germany . genotyping on chromosome 5p showed that snps in adamts-12 were primarily associated with bronchial hyperresponsiveness ( bhr ) in the hutterites . in addition , furthermore , there was a significant difference between asthma cases and controls in the frequencies of long range haplotypes composed of snps at adamts-12 . recently , asthma ova and hdm model were generated in the adamts-12-deficient mice to determine the role of adamts-12 in this disease . it was found that adamts-12 deficiency could exacerbate an allergen - induced inflammatory and airway response by inducing th2 inflammation . adamts-12-deficient mice models displayed a more severe phenotype of allergen - induced ahr when compared to their corresponding wt littermates by histological analysis . in addition , the markers of th2-skewed inflammatory response , such as ag - specific ige and ag - specific igg1 , were all significantly increased in allergen - induced mice . and there appeared to be no difference between the groups when evaluating the th1-associated marker , igg2a . the cytokine levels of il-4 and il-13 ( typical th2 cytokines ) were highly increased in adamts-12-deficient mice . on the other hand the levels of il-10 and ifn- ( th1 cytokines ) did not exhibit significant changes . furthermore , the levels of rantes ( ccl5 ) ( a major eosinophil chemoattractant ) , il-5 ( known to regulate eosinophil differentiation and survival ) , and il-33 ( known to further contribute to exacerbated eosinophil [ 122 , 123 ] ) were all increased and coordinate with the increased amount of eosinophilia . the levels of both the mast cells and st2 receptor were higher within the lungs of allergen - induced adamts-12-deficient mice [ 124 , 125 ] . in addition to its involvements in allergic inflammation , adamts-12 was found to be required in normal inflammation . to elucidate the role of adamts-12 in normal inflammation , several in vivo murine models adamts-12-deficient mice were used to create the animal models for colitis , endotoxic sepsis , and pancreatitis . they also experienced a delay in the recovery process from these challenges by comparison with their wild - type littermates . the adamts-12-deficient tissues showed a significant increase in several inflammatory markers in both rna and protein levels after analysis . in addition , all adamts-12-deficient mice models exhibited severe inflammatory symptoms accompanied by neutrophilia in the affected tissues . furthermore , hemopexin and cytokines such as granulocyte - colony stimulating factor ( gcsf ) and il-6 were increased in the adamts-12-deficient mice . notably , hemopexin and gcsf had the ability to promote duration and inhibit the apoptosis of neutrophils [ 126128 ] . above all , adamts-12 has a genetic linkage with asthma and a deficiency in adamts-12 leads to defects in both the normal and hyperresponsive inflammatory response . it may act as an important mediator maintaining the immune balance in both biological and pathological process . clearly , the molecular events and mechanisms underlying adamts-12-mediated regulation of inflammation warrant further investigations . within intervertebral disc ( ivd ) this process may negatively influence both the diffusion and nutrition of the ivd , which can lead to a more sever phenotype . it was reported that the expressions of adamts-7 and adamts-12 in the endplate cells isolated from patients with degenerative disc disease were significantly increased . a rat caudal intervertebral disc model was used to examine the expression profiling of adamts-7 and -12 in the course of the degeneration of intervertebral disc . adamts-7 and adamts-12 were detected in the nps and their expressions were slightly changed within the first 18 hours after loading . after that window of time both adamts-7 and -12 increased significantly and coordinated with the dramatic increase of comp . within a recent load - induced ivd degenerative rat model , the expression of adamts-7 peaked on the first day and adamts-12 expression reached peak level on the 7th day . however , on 7th day , the level of adamts-12 was much higher than that of adamts-7 . this data suggests that adamts-7 and adamts-12 may contribute to the early and late degeneration stage of ivd , respectively . adamts-12 was found to be expressed in the fibroblasts adjacent to the tumor cells in the colorectal cancer specimens from human patients . in addition , the expression of adamts-12 was not associated with the tumor origin , nor gender or age , but depended on the differentiation of the cancer cells . well - differentiated cells exhibited a higher staining score while poorly differentiated cells displayed negative staining . moreover , the adamts-12 expression in cancer stroma of patients with lymph node metastasis was greater than that within the patients without lymphatic metastasis the adamts-12 gene promoter was hypermethylated in colorectal carcinomas and colon cancer cell lines resulting in the silence of adamts-12 expression . in addition , it was reported that adamts-12 was expressed in fibroblastic - like cells surrounding malignant cells , and it was confirmed by histological staining from clinic . moreover , well and moderately differentiated carcinomas showed a strong immunoreactivity for adamts-12 whereas poorly differentiated cells showed weak staining . the expression of adamts-12 was doubled when fibroblasts coculture with colon cancer cells compared with fibroblasts cultures alone . it has been documented that the tumor cell growth rate was significantly decreased when fibroblasts coculture with colon cancer cells , as compared to colon cancer cell cultures alone . furthermore , the apoptotic cancer cell population of cocultured cells with colon fibroblasts was much higher than that of tumor cell cultures alone . it was reported that fibroblasts - expressed adamts-12 acted as a compensatory strategy for the epigenetic silencing of this gene within malignant cells . adamts-12 was reported to exact an tumorigenesis and angiogenesis - inhibitory effect [ 5 , 2931 , 131 ] . adamts-12 was found to play a protective role in angiogenesis and cancer progression via using adamts-12-deficient - mice models . malignant keratinocyte ( pdva cell line ) transplantation demonstrated that tumor vascularization and invasion were increased in adamts-12-deficient - mice . the aortic explants assay and basic fibroblast growth factor ( bfgf ) injection indicated that adamts-12 was a negative regulator of angiogenesis . additionally , the overexpression of adamts-12 in breast carcinoma ( mcf7 cell line ) resulting in reduced new vessel formation exhibited an inhibitory effect in angiogenesis as well . adamts-12 was also found to abolish vascular endothelial growth factor- ( vegf- ) induced tubule formation in the bovine aortic endothelial ( bae ) cells . furthermore adamts-12 inhibited tumor growth in vivo as well , since injection of a549 cells overexpressing adamts-12 into the immunodeficient - scid - mice showed a reduced tumor growth . adamts-12 was reported to play its tumorigenesis role by modulating the ras - dependent erk signaling pathway . there was a notable lower level of phosphorylated erk in the madin - darby canine kidney ( mdck ) cells expressing adamts-12 than those lacking adamts-12 after being stimulated by hepatocyte growth factor ( hgf ) . in addition , adamts-12 may function as a tumor suppressor through limiting the proliferation of tumor cells . interestingly , the catalytic activity of adamts-12 appeared to be dispensable for its angiogenic - inhibitory function , since the mutations in the catalytic domain inactivated its enzymatic activity but did not impair the antiangiogenic effect . new findings have illustrated that adamts-12 could elicit tumorigenesis effects when it interacts with fibulin-2 in breast cancer . fibulin-2 belongs to fibulins , which are components of basement membranes and elastic matrix fibers in connective tissue and also play a role in tumorigenesis [ 134136 ] . both in vitro and in vivo data have shown that adamts-12 itself could exhibit protumor effect while interaction of adamts-12 and fibulin-2 could exhibit tumorigenesis effect . furthermore , clinically detection of both proteins in breast cancer patients predicted a good prognosis . taken together , both in vivo and in vitro experiments demonstrate that adamts-12 expression associates with the differentiation of the cancer cells and has tumorigenesis and antiangiogenic effects . thus , adamts-12 may provide a new molecular target for treating various types of cancer in addition to the potential of being employed as a prognostic indicator in tumor malignant grade and metastasis . members of adamts family have been implicated in neurodegenerative and neuroinflammatory diseases [ 137139 ] . in addition , some metalloproteinases , including mmp-9 and adamtsl3 , were found to be implicated in schizophrenia [ 140 , 141 ] . the adamts-12 gene had been shown to be a functional and positional candidate in the susceptibility of schizophrenia by mutation analysis in puerto rican patients of spanish descent . moreover , the intronic variant rs256792 and the two - snp haplotype rs256603rs256792 were closely associated with the disorder , although the function of single nucleotide polymorphisms remains largely unknown . , 110 genes were selected for evaluation of their expression during chicken gonad differentiation , and adamts-12 showed higher level of expression within the testis and was increased during gonadal development . furthermore , whole mount in situ hybridization assay reveled that adamts-12 was expressed in the testis on day 7 , the time of histological differentiation of the gonads . a previous study reported that adamts-12 acted as an inhibitor of sox9 expression during in vitro chondrocyte differentiation and the reverse expression profiling between adamts-12 and sox9 in the course of chicken gonad differentiation indicating that adamts12 may be a novel regulator of gonad differentiation through modulating sox9 transcription factor . in addition , it was reported that adamts-12 might be an early marker of male gonad differentiation . in addition to its potential role in male gonad differentiation , adamts-12 was also found to be involved in the trophoblastic cell invasion [ 144 , 145 ] . adamts-12 was found to be detectable in samples of human placental tissue from the first trimester . adamts-12 altered the cell - extracellular matrix ( ecm ) interactions leading to the reduction of cell adhesion capability and promotion of cell invasion by measuring the first trimester trophoblastic cells . expression of adamts-12 was significantly higher in cultures of invasive extravillous cytotrophoblast ( evt ) than in poorly invasive jeg-3 choriocarcinoma cells . in addition , it was reported that gonadotropin - releasing hormone- ( gnrh- ) i and ii increased adamts-12 's expression in evt within a time- and concentration - dependent manner and transforming growth factor-1 ( tgf-1 ) promoted cytotrophoblast invasion in vitro while interleukin-1 ( il-1 ) restrained cytotrophoblast invasion in vitro . in addition , adamts-12 reduced cell - emc adhesion by regulating the v3 integrin heterodimer . furthermore , the ability of adamts-12 to promote invasion largely depended on the disintegrin - like domain and ancillary domains . it was reported that there was a relationship between the adamts family and pediatric stroke . although snps residing in adamts12 were strongly associated with pediatric stroke , the mechanisms through which the genetic variants in adamts genes lead to pediatric stroke still remain unknown . the dysregulation of adamts-12 is associated with musculoskeletal degenerative diseases , including arthritis and ivd degeneration , which probably resulted from its degradation of comp , aggrecan , and other unidentified matrix proteins and its role in inflammation . in addition , adamts-12 functions as a protective mediator in both normal and hyperresponsive inflammation by inducing neutrophil apoptosis and interfering with the t response , respectively . furthermore , adamts-12 is found to exhibit tumorigenesis and angiogenic - inhibitory effect in certain cancer types and adamts-12 has the potential to be used as an indicator of cancer prognosis . adamts-12 also promotes the invasive ability of trophoblastic cells and acts as an early marker of male gonad differentiation in chicken . although growing evidence indicates that adamts-12 is a metalloproteinase with multiple functions , the exact expression profiling , regulation , and function of adamts-12 in various pathophysiological processes , especially the signaling pathways and molecular events involved , remain to be delineated . moreover , the function of each domain should be taken into account since it has been reported that thrombospondin type 1 repeats interact with mmp-2 , whose role in osteoarthritis is currently emerging . by determining the functional domain of adamts-12 in osteoarthritis , we can develop a more efficient inhibitor to rescue this disease . in addition , the protein partner of adamts-12 in the progression of osteoarthritis is also a big challenge to us . although global knockout mice of adamts-12 are viable , timely controlled ( i.e. , inducible ) , tissue - specific knockout and/or transgenic mice are needed for precisely dissecting the role of adamts-12 in the development of various tissues . in addition , these mouse strains are especially useful for generating various kinds of diseases models . for instance , in order to figure out the potential effect of adamts-12 in cartilage development and the initiation and/or progression of oa , cartilage specific deletion of adamts-12 in the adult mice is required for generating surgically induced oa models .

adamts-12 is a member of a disintegrin and metalloproteinase with thrombospondin motifs ( adamts ) family of proteases , which were known to play important roles in various biological and pathological processes , such as development , angiogenesis , inflammation , cancer , arthritis , and atherosclerosis . in this review , we briefly summarize the structural organization of adamts-12 ; concentrate on the emerging role of adamts-12 in several pathophysiological conditions , including intervertebral disc degeneration , tumorigenesis and angioinhibitory effects , pediatric stroke , gonad differentiation , trophoblast invasion , and genetic linkage to schizophrenia and asthma , with special focus on its role in arthritis and inflammation ; and end with the perspective research of adamts-12 and its potential as a promising diagnostic and therapeutic target in various kinds of diseases and conditions .

1. Introduction 2. Structural Organization of ADAMTS-12 3. Biological Functions of ADAMTS-12 4. Other Conditions and Diseases 5. Summary and Perspective

a disintegrin and metalloproteinase with thrombospondin motifs ( adamts ) family consists of a precisely ordered modular organization that includes at least one thrombospondin type i repeat . this family is thought to be comprised of a zinc - dependent group of proteases , which play vital roles in a variety of normal and pathological conditions [ 27 ] . the adamts family has grown to include 19 members , which are involved in a plethora of diseases ranging from arthritis to coagulation disorders [ 1 , 919 ] . adamts-17 and -19 , adamts-16 and -18 , adamts-7 and -12 , and adamts-6 and -10 compose the structurally related adamts pairs division . it has been well established that the adamts-12 gene is expressed in the musculoskeletal system ( skeletal muscles , cartilage , and tendons ) and in the fetal lung [ 1 , 23 , 24 ] for instance , adamts-12 has been proven to be associated with the pathogenesis of arthritis [ 816 ] , intervertebral disc degeneration [ 25 , 26 ] , inflammation [ 27 , 28 ] , and invasion and metastasis of tumor [ 2931 ] . moreover , in intervertebral disc degenerative ( ivd ) rat model , the level of adamts-7 was significantly increased in the early phase while the level of adamts-12 was increased in the latter phase . in this review , we will discuss the structure and perspective of adamts-12 and focus on its roles and the underlying mechanisms in arthritis ( especially osteoarthritis ) and other diseases , including inflammation , tumorigenesis and antiangiogenesis , intervertebral disc degenerative disease , schizophrenia , gonad differentiation , trophoblast invasion , and pediatric stroke ( figure 1 ) . in addition , we used adamts12 and adamts-12 as keywords to search articles through pubmed , google scholar , and web of science . the structure of adamts-12 includes a signal peptide , a prodomain , a catalytic domain , a disintegrin - like domain , a central thrombospondin i ( ts ) repeat , a cysteine - rich domain , two spacer regions , and c - terminal tsp - like repeat region ( figure 2 ) [ 33 , 34 ] . to determine the effects of each functional domain of adamts-12 on chondrocyte differentiation , numerous deleted mutants were generated , such as the removal of c - terminal four thrombospondin motifs known to bind comp , the removal of a spacer-2 domain , the deletion of three thrombospondin motifs , and the deletion of a spacer-1 domain . these data indicated that the cysteine - rich domain of adamts-12 was required for its binding to ecm and cell surface appearance in chondrocytes . in addition , the antiangiogenesis properties of adamts-12 did not rely on the catalytic domain , but , more likely , on its tsp-1 domains . arthritis is the leading cause of physical disability , health care usage , and impaired quality of life [ 3638 ] . oa is associated with age - related loss of homeostatic balance between degradation and repair mechanisms in articular cartilage , which is characterized by synovitis , cartilage degeneration , and osteophyte formation [ 4447 ] . oa is a multifactorial disorder which can be affected by genetic alternation , inflammation , mechanical stress , obesity , and aging as well as biochemical , molecular , and enzymatic factors [ 4851 ] . ra is a chronic systemic inflammatory disease that is characterized by the inflammatory proliferation of synovial tissues , leading to cartilage and bone damage and eventual disability [ 5254 ] . the dysregulation in both oa and ra induces senescence , differentiation , proliferation , and cell death in joint cells through gene and/or protein expression networks that switch from anabolic to catabolic outcomes . recent evidence has demonstrated the significance of proteases , in the pathological processes of arthritis [ 5759 ] . in accordance with this concept , recent studies show that adamts-12 also plays a critical role in the pathological process of arthritis [ 8 , 32 , 35 , 43 , 61 ] . osteoarthritis is an age - related or posttraumatic degenerative disease of the joint that is characterized by loss of articular cartilage , chondrocyte proliferation and hypertrophic differentiation , subchondral bone remodeling , inflammation , and finally osteophyte formation [ 62 , 63 ] . in that case , anything that can affect cartilage may play a role in the initiation and progression of osteoarthritis . the finding that adamts-12 was isolated as a binding metalloproteinase of comp , an extracellular matrix highly expressed in cartilage [ 8 , 32 , 6771 ] , promoted us to determine the role of adamts-12 in cartilage development and degeneration . it was reported that adamts-12 , functioning as a downstream molecule of parathyroid hormone related peptide ( pthrp ) signaling , negatively regulated chondrogenesis . furthermore , pthrp signaling was nearly abolished when adamts-12 was repressed , and pthrp action was largely restored when adamts-12 was reexpressed . notably , the negative regulation of chondrocyte differentiation by adamts-12 depended on its enzymatic activity since its point mutant lacking enzymatic activity completely lost this activity . the expression of adamts-12 in the course of chondrogenesis was regulated by c - maf transcription factor . c - maf , the cellular homologue of the avian viral oncogene c - maf , was a member of the basic leucine zipper ( bzip ) superfamily [ 7375 ] . in addition , c - maf was overexpressed during embryonic development and postnatal growth in hypertrophic chondrocytes as well as in the osteoarthritic chondrocytes , which was similar to the expression pattern of that of adamts-12 [ 7375 ] . furthermore , c - maf transcriptional factor directly binds to the proximal mare sequences of adamts-12 at position -61 in vitro . collectively , adamts-12 , whose expression was regulated by c - maf transcription factor , acted as a downstream molecule of pthrp signaling , negatively regulated chondrocyte differentiation , and hypertrophy ( figure 3 ) . in addition , proper expression of adamts-12 is required for normal cartilage development and its dysregulation may lead to pathologies with defects in the musculoskeletal system , such as bde . abnormal level and/or function of adamts-12 may induce dysregulation of cartilage , which finally leads to arthritis . the function of comp may be involved in stabilizing cartilage ecm via specific interactions with matrix components such as collagen types ii and ix , aggrecan , and fibronectin [ 8183 ] . it was reported that mutations in the type iii or c - terminal globular domain of the human comp gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia , which were autosomal - dominant forms of short - limb dwarfism [ 88 , 89 ] . comp fragments have been detected in the diseased cartilage , synovial fluid , and serum of patients with knee injuries . the level of adamts-12 was significantly increased in the cartilage and synovium of patients with ra or oa [ 9 , 10 ] . in addition , adamts-7 and -12 were colocalized with comp both in the cytoplasm and on the surface of human chondrocytes [ 25 , 35 ] . while adamts-7 was unable to digest aggrecan , adamts-12 , with a similar structure , did have the ability to digest aggrecan . it was reported that alpha-2-macroglobulin ( 2 m ) acted as a substrate for both adamts-7 and adamts-12 and efficiently protected comp degradation by these enzymes by in vitro digestion assays . in addition , granulin - epithelin precursor ( gep , a newly identified chondrogenic growth factor , which was also known as proepithelin , acrogranin , progranulin ( pgrn ) , and gp88/pc cell - derived growth factor ) inhibited the action of adamts-12 preventing comp degradation via two distinct mechanisms [ 61 , 9297 ] . in detail , gep inhibits the induction of adamts-12 by inflammatory cytokines such as tnf- and gep is able to disrupt the association between adamts-12 and comp via a direct protein - to - protein interaction [ 8 , 61 ] . it was approved that adamts-12 , comp , aggrecan , gep , 2 m , and tnf- constitute an interplay and interaction network in mediating cartilage degradation in arthritis ( figure 4 ) . in addition , tgf- was reported to significantly induce the adamts-12 in human fetal fibroblasts . this in vitro data provide insight into the critical role of adamts-12 in the initiation and progression of osteoarthritis which needs to be further confirmed using in vivo genetically modified animal models . interestingly , such in vivo models for adamts-7 , which shares similar domain organization and structure , have been generated , and the critical role of adamts-7 in the pathogenesis of oa has been elucidated . aside from the fact that adamts-12 shares a similar structure with adamts-7 , adamts-12 may play a critical role in the oa development in vivo [ 10 , 23 , 58 ] . rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease , characterized by inflammation of the synovial membrane and progressive destruction of the articular cartilage and bone [ 102 , 103 ] . as we have previously stated , adamts-12 is an inflammation - related protein , which can digest comp ; it was reported that adamts-12 might play a role in ra [ 104 , 105 ] . multiple logistic regression models , snpstats and snpanalyzer pro programs , and bonferroni 's correction ( pc ) were used and found that the genotype frequency of rs10461703 was associated with the ra development . criteria were set as a 1.5-fold change threshold in the caia severity change and three genes were found including collagen triple - helix repeat - containing 1 ( cthrc1 ) , c1q and tnf - related protein 3 ( c1qtnf3 ) , and adamts-12 , which were all associated with both gender and arthritis . in addition , these three genes were highly involved in the severity of arthritis with r = 0.91 , 0.89 , and 0.87 , respectively ; thus there is a strong correlation among these 3 genes . collectively , these genetic and in vitro studies indicate that adamts-12 plays an important role in the pathogenesis of arthritis and may be a potential target for developing new treatments for arthritis . adamts-12-deficient mice model with arthritis are critical for verifying its role in the course of both osteoarthritis and rheumatoid arthritis in vivo . adamts-12 has also been reported to be a critical mediator in inflammation , especially allergic inflammation [ 24 , 27 , 28 ] . recently , asthma ova and hdm model were generated in the adamts-12-deficient mice to determine the role of adamts-12 in this disease . in addition , the markers of th2-skewed inflammatory response , such as ag - specific ige and ag - specific igg1 , were all significantly increased in allergen - induced mice . on the other hand the levels of il-10 and ifn- ( th1 cytokines ) did not exhibit significant changes . furthermore , the levels of rantes ( ccl5 ) ( a major eosinophil chemoattractant ) , il-5 ( known to regulate eosinophil differentiation and survival ) , and il-33 ( known to further contribute to exacerbated eosinophil [ 122 , 123 ] ) were all increased and coordinate with the increased amount of eosinophilia . in addition to its involvements in allergic inflammation , adamts-12 was found to be required in normal inflammation . to elucidate the role of adamts-12 in normal inflammation , several in vivo murine models adamts-12-deficient mice were used to create the animal models for colitis , endotoxic sepsis , and pancreatitis . the adamts-12-deficient tissues showed a significant increase in several inflammatory markers in both rna and protein levels after analysis . above all , adamts-12 has a genetic linkage with asthma and a deficiency in adamts-12 leads to defects in both the normal and hyperresponsive inflammatory response . it may act as an important mediator maintaining the immune balance in both biological and pathological process . within intervertebral disc ( ivd ) this process may negatively influence both the diffusion and nutrition of the ivd , which can lead to a more sever phenotype . it was reported that the expressions of adamts-7 and adamts-12 in the endplate cells isolated from patients with degenerative disc disease were significantly increased . after that window of time both adamts-7 and -12 increased significantly and coordinated with the dramatic increase of comp . however , on 7th day , the level of adamts-12 was much higher than that of adamts-7 . in addition , the expression of adamts-12 was not associated with the tumor origin , nor gender or age , but depended on the differentiation of the cancer cells . moreover , the adamts-12 expression in cancer stroma of patients with lymph node metastasis was greater than that within the patients without lymphatic metastasis the adamts-12 gene promoter was hypermethylated in colorectal carcinomas and colon cancer cell lines resulting in the silence of adamts-12 expression . in addition , it was reported that adamts-12 was expressed in fibroblastic - like cells surrounding malignant cells , and it was confirmed by histological staining from clinic . the expression of adamts-12 was doubled when fibroblasts coculture with colon cancer cells compared with fibroblasts cultures alone . it was reported that fibroblasts - expressed adamts-12 acted as a compensatory strategy for the epigenetic silencing of this gene within malignant cells . adamts-12 was found to play a protective role in angiogenesis and cancer progression via using adamts-12-deficient - mice models . additionally , the overexpression of adamts-12 in breast carcinoma ( mcf7 cell line ) resulting in reduced new vessel formation exhibited an inhibitory effect in angiogenesis as well . adamts-12 was reported to play its tumorigenesis role by modulating the ras - dependent erk signaling pathway . in addition , adamts-12 may function as a tumor suppressor through limiting the proliferation of tumor cells . interestingly , the catalytic activity of adamts-12 appeared to be dispensable for its angiogenic - inhibitory function , since the mutations in the catalytic domain inactivated its enzymatic activity but did not impair the antiangiogenic effect . fibulin-2 belongs to fibulins , which are components of basement membranes and elastic matrix fibers in connective tissue and also play a role in tumorigenesis [ 134136 ] . both in vitro and in vivo data have shown that adamts-12 itself could exhibit protumor effect while interaction of adamts-12 and fibulin-2 could exhibit tumorigenesis effect . taken together , both in vivo and in vitro experiments demonstrate that adamts-12 expression associates with the differentiation of the cancer cells and has tumorigenesis and antiangiogenic effects . thus , adamts-12 may provide a new molecular target for treating various types of cancer in addition to the potential of being employed as a prognostic indicator in tumor malignant grade and metastasis . in addition , some metalloproteinases , including mmp-9 and adamtsl3 , were found to be implicated in schizophrenia [ 140 , 141 ] . moreover , the intronic variant rs256792 and the two - snp haplotype rs256603rs256792 were closely associated with the disorder , although the function of single nucleotide polymorphisms remains largely unknown . , 110 genes were selected for evaluation of their expression during chicken gonad differentiation , and adamts-12 showed higher level of expression within the testis and was increased during gonadal development . a previous study reported that adamts-12 acted as an inhibitor of sox9 expression during in vitro chondrocyte differentiation and the reverse expression profiling between adamts-12 and sox9 in the course of chicken gonad differentiation indicating that adamts12 may be a novel regulator of gonad differentiation through modulating sox9 transcription factor . in addition , it was reported that adamts-12 might be an early marker of male gonad differentiation . in addition to its potential role in male gonad differentiation , adamts-12 was also found to be involved in the trophoblastic cell invasion [ 144 , 145 ] . expression of adamts-12 was significantly higher in cultures of invasive extravillous cytotrophoblast ( evt ) than in poorly invasive jeg-3 choriocarcinoma cells . furthermore , the ability of adamts-12 to promote invasion largely depended on the disintegrin - like domain and ancillary domains . although snps residing in adamts12 were strongly associated with pediatric stroke , the mechanisms through which the genetic variants in adamts genes lead to pediatric stroke still remain unknown . the dysregulation of adamts-12 is associated with musculoskeletal degenerative diseases , including arthritis and ivd degeneration , which probably resulted from its degradation of comp , aggrecan , and other unidentified matrix proteins and its role in inflammation . in addition , adamts-12 functions as a protective mediator in both normal and hyperresponsive inflammation by inducing neutrophil apoptosis and interfering with the t response , respectively . furthermore , adamts-12 is found to exhibit tumorigenesis and angiogenic - inhibitory effect in certain cancer types and adamts-12 has the potential to be used as an indicator of cancer prognosis . although growing evidence indicates that adamts-12 is a metalloproteinase with multiple functions , the exact expression profiling , regulation , and function of adamts-12 in various pathophysiological processes , especially the signaling pathways and molecular events involved , remain to be delineated . by determining the functional domain of adamts-12 in osteoarthritis , we can develop a more efficient inhibitor to rescue this disease . in addition , the protein partner of adamts-12 in the progression of osteoarthritis is also a big challenge to us . , inducible ) , tissue - specific knockout and/or transgenic mice are needed for precisely dissecting the role of adamts-12 in the development of various tissues . in addition , these mouse strains are especially useful for generating various kinds of diseases models . for instance , in order to figure out the potential effect of adamts-12 in cartilage development and the initiation and/or progression of oa , cartilage specific deletion of adamts-12 in the adult mice is required for generating surgically induced oa models .

chilli peppers ( capsicum annuum l. ) are used in a multitude of food preparations and are marketed in different regions . the greatest genetic variety of c. annuum l. can be found in mexico , where there are chilli peppers of many different shapes , sizes , and colours , and they are among the most frequently consumed products . the genus capsicum is classified into the family of solanaceae , which is constituted by 25 wild and 5 domesticated species ( c. annuum l. , c. frutescens l. , c. chinense jacq , c. baccatum jacq , and c. pubescens l. ) , including more than 200 varieties [ 2 , 3 ] . chilli peppers have been object of study mainly due to containing capsaicin , which produces different pungency levels , as well as carotenoids and phenolic compounds , which are used as natural pigments and antioxidant agents . chilli peppers may have different content and profiles of such compounds , depending on the genotype and variety , maturity of the fruit , and the environmental conditions of the cultures . in order to preserve , manage , and improve the different chilli species , the evaluation of the extent of genetic variation within species , as this has now become a fundamental tool in biology and agriculture . among the main criteria used for that purpose the rapd ( random amplified polymorphic dna ) method is a widely used technique for molecular marking and is based on the amplification of genomic dna fragments by using primers of arbitrary nucleotide sequences ; which in turn detects polymorphisms that can be employed as genetic markers without previous genetic sequences . in addition to being simple , fast , and low - cost , this method does not require radioactive markers and consumes minimum amounts of dna [ 911 ] . however , one of the inconveniences reported about the rapd technique , since it was published for the first time , is the low reproducibility found mainly in low - intensity bands . the most important factor affecting reproducibility in rapd analysis is the preparation of the dna template . thereby , the differences in the concentration of dna template between samples are observed as the gain or loss of some bands . recently , to improve the results , phytochemical markers have been combined with molecular techniques . silva et al . correlated the flavonoid content of medicinal plants from brazil with molecular markers obtained by rapd , whereas ercisli et al . ( 20072008 ) [ 13 , 14 ] obtained rapid results for evaluation of genotypic diversity and distances by using methyl esters of fatty acids as markers . previously , the content and composition of fatty acids were tested as taxonomic markers in hippophae rhamnoides l. , showing that these substances , such as linoleic acid , can be used as biochemical markers for this species . on the other hand , fller et al . analyzed the variability of morphological characteristics as well as the content of phenolic compounds and essential oils in plants from southern brazil . described the state of preservation of a meadow by comparing the content of phenolic compounds and the rapds . another essential technique for determining the biological nature of agriculture products is fourier transform - infrared spectroscopy ( ftir ) , which is a technique that has been widely employed for food characterization because of several important advantages , such as its nondestructive nature and its capacity to yield structural information that constitutes a molecular fingerprint of the sample . a combination of ftir and chemometric techniques like principal component analysis ( pca ) has been employed as a direct and rapid way to discriminate properties in foods , including geographic origin , adulteration , and quality control . the aim of the present study is to contribute to management and improvement of the different chilli species by determining the relationship between molecular and biochemical markers , such as the content of carotenoids and phenolic compounds among different commercial cultivars of chilli peppers from diverse geographic origins . the samples were collected from central and north regions of mexico ( table 1 ) . the chilli peppers were considered fresh when the samples presented bright green colour , and smooth , firm texture . the mature specimens were derived from the green samples , by exposure to sunlight and moisture - free storage in the laboratory for 25 to 35 days . the samples were considered mature when the chilli peppers had a bright red , yellow , or orange colour and less firmness and texture . length , width , and weight of the fruits were determined in accordance to the mexican official standard . briefly , the length was taken from the base to the apex of the fruit without considering the peduncle ; the width was measured at the widest part of the chilli pepper ; for the weight , an analytical balance was used . the extraction of carotenoids from the chilli samples was carried out with 2 g portions of pulp and peel , which were mixed with diatomaceous earth and acetone at 10c . afterwards , the extract was filtered at vacuum through whatman grade 2-filter paper and transferred to 10 ml of petroleum ether . the oil phase was extensively washed with distilled water to remove the residue of acetone , while the residual water was removed by shaking with anhydrous sodium sulphate . a 1 ml aliquot was used to determine the content of carotenoids using the molar extinction coefficient for -carotene in hexane at 450 nm . the extracts of carotenoids were reconstituted in 500 l of hplc - grade ethyl acetate and filtered through nylon membranes ( 0.45 m , pore size ) and then introduced into the c18 column ( 25 cm 4.6 mm ; 5 m ) of the hplc equipment ( perkin elmer binary lc pump 250 ; series 200 , uv / vis detector ) . an isocratic elution system was established with acetonitrile - methanol - ethyl acetate ( 73 : 20 : 7 ) working at 0.6 ml / min for 70 min , and the absorbance was measured at 450 nm . a total of 2 g of pulp and peel chilli peppers were mixed with 8 ml of 80% ethanol , and shaken for 10 min at 200 rpm . the sediment was reconstituted into 8 ml of 80% ethanol , and the procedure was repeated . thereby , three extractions were obtained , joined , and stored under a nitrogen atmosphere until analysis . volumes of 0.75 ml of folin - ciocalteau reagent ( 1 : 10 ) were added to 100 l of the extract of free phenolics , and the mixture was left to stand for 5 min in the dark . afterwards , 0.75 ml of nahco3 60 g / l solution was added to neutralize the reaction . the solution was left to stand for 90 min , and the absorbance was determined at 725 nm . aliquots of 50 l of extract were injected in the hplc equipment ( perkin elmer binary lc pump 250 ; series 200 , uv / vis detector ) using a c18 column ( 25 cm 4.6 mm ; 5 m ) . the mobile phase used was a binary system composed of 0.1% trifluoroacetic acid ( phase a ) and 100% acetonitrile ( phase b ) . starting with 90% phase a and 10% phase b , the mixture was eluted to 55% phase a , 45% phase b in 50 min and to 60% phase a and 40% phase b in the last 10 min . analyses of variance ( anova ) were performed along with student - newman - keuls test for differences between means , using sigmastat 3.5 software . principal component analysis ( pca ) of the ftir extract spectra was performed by using the opus quant ( version 6.5 ) and minitab ( version 14 ) software . the dry extracts were reconstituted in petroleum ether , for carotenoids , and in ethanol , for phenolics , in order to obtain a semisolid consistency of the sample . afterwards , the mixtures were analyzed in a ft ( fourier transform)-infrared spectrometer ( bruker vertex 70 ) , by using the atr ( attenuated total reflectance ) sampling method previously described . briefly , a small sample amount is placed over a znse crystal , where the infrared radiation is propagated and interacts with the sample to obtain the corresponding spectrum , which is averaged from several data acquisitions . the method described by allers and lichten was used for dna extraction , after modifications . all samples of chilli peppers were washed with water , disinfected with sodium hypochlorite at 10% , 30% , and 70% ethanol , exhaustively rinsed with distilled water for 20 min and stored at 70c , under sterile conditions . portions of 100 mg per sample were mixed with 400 l of lysis buffer ( 200 mm tris - hcl ph 6.5 , 70 mm edta ph 8 , 2 mm nacl , 2% pvp 40 , 20 mm sodium metabisulphite , 1% triton x-100 , 24 mm mgcl2/mgso4 , 0.1% spermine , and 0.1% spermidine ) previously heated at 60c . the mixture was agitated and incubated at 60c for 60 min ; then it was transferred to ice , at which time 400 l of 10% pvp was added . the mixture was agitated by inversion of the recipient and left to stand for 60 min at 20c . afterwards , the blend was centrifuged at 4000 rpm for 30 min , and 400 l of each supernatant was transferred into new tubes , in order to add 5 l of 10 mg / ml rnasa for 10 min at room temperature . immediately , the supernatants were precipitated by addition of 200 l of 10 m ammonium acetate and 600 l of cold isopropanol . the mixture was then centrifuged at 4000 rpm for 30 min at 10c , the isopropanol eliminated , and the pellet washed with 1 ml of cold 70% ethanol , followed by centrifugation for 10 min under the same operation conditions . finally , the ethanol was removed , and the pellet was dried for 20 min at room temperature . the pellets were reconstituted into 200 l of 10 mm tris ph 8 , and the dna was precipitated again with 200 l of peg 8000 30%-nacl 1.2 m at 4c for 60 min . the mixture was then centrifuged for 30 min at 4000 rpm and 10c . the supernatants from peg were discarded , and the dna pellet was washed with 1.5 ml of cold 70% ethanol , followed by centrifugation for 15 min at 4000 rpm . the ethanol was eliminated and the dna pellet left to dry , and finally reconstituted into 200 l of 10 mm tris ph 8 . the purity of the extracted dna was determined by absorbance at 260/280 ratio , and values greater than 1.8 were accepted for future procedures . the dna was quantified by its absorbance at 260 nm . for dna amplification , a thermocycler ( bioer xp cycler ) was used under the following operation settings : 1 cycle of 5 min at 94c ; 40 cycles of 1 min at 94c ; 1 min at 36c , 2 min at 72c ; a final extension step of 10 min at 72c . pcr reactions were performed in a volume of 25 l containing 1x pcr buffer , 50 nm of mgcl2 , 200 m dntp , 1.5 units of the enzyme taq dna polymerase ( invitrogen , brazil ) , and 40100 ng/l dna samples . four independent reactions were assayed using 0.4 m oligonucleotide ope18 ( cggcccacgt ) , or 0.2 m oligonucleotide mfg17 ( cgcgttcttg ) , 0.2 m oligonucleotide mfg18 ( cggcccacgt ) , or 0.2 m c51 ( atcaacgtacgt ) and 0.2 m c52 ( gtcgacggacgt ) oligonucleotide mixture ( invitrogen ) [ 4 , 28 ] . the products of dna amplification were analyzed by electrophoresis in 1.5% agarose gel , using tae 1x buffer . the gels were stained with ethidium bromide ( 0.2 l / ml ) and observed with an uv - transilluminator ( stratagene eagle eye ) . in order to increase reproducibility , optimum conditions were established for dna extraction and amplification , without modifying mg2 + concentration , for taq polymerase enzyme and primers as well as for the dna concentration in each reaction . the polymorphic bands and signals of the chromatograms were considered as present or absent in a matrix of similarity . for the chromatogram , only signals above 40 milli - units of absorbance were considered the method of average grouping upgma was used and determined by means of the analysis cluster by using the software paleontological statistics ( past ) 2.10 , taking into account the dice 's similarity coefficient and the euclidean distance . the weight of the different varieties of chilli peppers ranged from 3 to 46 g. in general , samples with the best morphological characteristics ( color , size , texture , and weight ) were those from the northern region , except for the sample jv1 from the south - central area , the latter being the lighter weight . the length was from 4.3 to 9.5 cm , and the width was in the range of 0.1 to 3.34 cm . the three varieties of chilli showed differences in both weight and size , with remarkable greater similarities between the same species ( jalapeo and serrano ) as well as those grown in nearby areas . for instance , the serrano chilli peppers grown in tx , p , and v1 from the central region were quite similar . through statistical analysis , significant differences were found in weight and width between varieties , but not in length between serrano and jalapeo ( i.e. , the same species ) . however , these two peppers showed significant difference with respect to habanero ( i.e. , a different species ) ( see table 2 ) . the content of carotenoids in fresh peppers varied within the interval 0.0830.99 mg / g dry basis ( db ) , while mature peppers were in the range from 0.69 to 13.85 mg / g db . the carotenoid content increased from the fresh to the mature state , as expected accordingly to the natural biosynthesis of pigments as the fruit matures . in accordance with the statistical analysis , of chilli peppers in the fresh stage , there were significant differences between jalapeo and serrano varieties , while analysis of mature peppers , showed no differences between the three cultivars ( see table 3 ) . this is due to the ability of fruits to synthesize carotenoids during the ripening process regardless of the species . regarding the region , fresh jalapeo peppers from tx and p were found to be different than those from v and s , while fresh serrano peppers showed statistical difference in all cases except those from jc and those from si . mature chilli peppers , from si showed differences with respect to those from t , p , v1 and v2 . therefore , no association was found between the concentration of carotenoids and the geographical origin . the results presented above could be affected also by the environmental conditions , such as temperature and relative humidity , as well as the type of soil , which of course are related to the proximity of the different geographical regions . among the identified carotenoids were lutein , -cryptoxanthin , lycopene , and -carotene , with a higher concentration of lutein and lycopene . the latter are part of the main carotenoids present in chilli [ 29 , 31 ] . during the process of maturation , lutein remained constant for jalapeo peppers , whereas it diminished for serrano ones however , it has been reported that lutein disappears in mature peppers by the effect of the synthesis of pigments in chromoplasts . contrarily -cryptoxanthin and the -carotene increase during the ripening process , as reported by marn et al . . according to statistical analysis , no significant differences were found for fresh peppers in concentrations of lutein , lycopene , and -cryptoxanthin between jalapeo and serrano cultivars . nonetheless , there were differences in -carotene content , which is the major compound in several varieties of c. annuum l. . on the other hand , differences were found for mature peppers only between jalapeo and serrano , in this case in lutein and lycopene content ( table 4 ) . the concentration of phenolic compounds in fresh peppers ranged from 30.2 to 128.4 mg / g , whereas in the mature peppers it ranged from 38.6 to 297.8 g/l on dry weight basis . in most of the samples , there was a noteworthy increase in these compounds in mature chilli peppers , in agreement with the results of deepa et al . zhang and hamauzu show that the content of phenolic compounds are significantly higher in the fresh peppers than in the mature ones . given the above , the main factors involved in the variation of phenolic content are the maturation state , as well as the age of the plant , as reported by deepa et al . . when doing a comparison on the total phenolic content within the chilli varieties , significant differences were found between serrano and jalapeo , for both fresh and mature stages , while between jalapeo and serrano as a group no differences were detected with respect to habanero . these results are inconsistent with data reported by oboh et al . , where the phenolics content was significantly higher for the c. annuum l. than for the c. chinense jacq species . concerning the region , differences were found in most of fresh jalapeo peppers samples , except between p and v ( v1 and v2 ) and between si and jco . for the fresh serrano peppers , there were differences between tx and p with respect to jc and v2 . for mature peppers , the region seems to be unimportant ( see table 5 ) . from these results , certain patterns indicate differences between varieties and other between cultivars , in agreement with antonious et al . who reported significant differences between cultivars of c. baccatum l. and c. chinense jacq , and between two genotypes of the latter variety . the phenolic compounds found in chilli peppers were gallic , protocatechuic , ferulic , o - coumaric , p - coumaric , sinapinic , trans - cinnamic and caffeic acids , quercetin , catechin , rutin , and vanillin . many of these compounds belong to the pathway of phenylpropanoids which is characteristic of chilli species . among the varieties of chilli , significant differences were found between serrano and jalapeo in the gallic and p - coumaric acids content , indifferently of maturation stage . regarding protocatechuic and caffeic acids as well as catechin and vanillin , there were differences only for fresh peppers . for the mature stage , the only difference was found in the content of trans - cinnamic acid between the two varieties ( table 6 ) . regarding the place of origin , fresh jalapeo peppers showed differences in the content of sinapic acid between si samples and the rest , in the content of catechin of v samples compared to p and tx samples ( despite the fact that all three belong to the central region ) , and in the content of p - coumaric acid of p compared to v1 , tx , and si . the fresh serrano peppers showed differences in the concentration of gallic acid for samples from si with respect to p and v. in the content of protocatechuic acid for samples from p with respect to the other specimens , and in the content of caffeic acid and rutin for v2 with respect to the other specimens . finally , most regions showed differences in the content of p - coumaric acid , except for v2 compared to p and s , v1 compared to jc and tx , p compared to si , and jc compared to tx , showing no association between geographical areas . among mature jalapeo peppers , t showed differences with respect to si in the content of gallic acid and with respect to v1 in protocatechuic acid . no differences were found between the mature serrano peppers with respect to region . by taking into account the state of maturation , for the rest of the phenolic compounds , there were not many differences as observed by the maturation stage in the content of gallic , protocatechuic , trans - cinnamic and p - coumaric acids ; quercetin , rutin , and vanillin . for the rest of the phenolic compounds , there were no differences that could be explained by the maturation stage . within a jalapeo variety , differences were found between fresh and mature peppers in the content of gallic acid , while no such difference existed in the content of ferulic acid . for mature specimens no differences were found only between the three varieties in the content of protocatechuic acid , caffeic acid , and catechin , while the fresh samples reported differences in the content of p - coumaric acid , catechin , and vanillin . it is clear from the results that variations exist between the three variables and concentrations . some interactions can be explained by the proximity of the geographical areas , as in the case of p , tx , v , and t. other differences may be due to the growing conditions of each geographic region or by the genotype of the chilli , as discussed earlier by silva et al . . these authors studied the content of rutin in a medicinal fruit from brazil and found that the high variability in the concentration and the great genetic variability of the fruit may be associated not only with genetic differences , but also with environmental conditions . the typical ir spectra ( 6002000 cm ) of phenolic extracts from different geographical regions of mexico ( figure 1 ) however , by applying principal component analysis ( pca ) ( figure 2 ) to the second derivative of the spectra , four clusters were found . in the first cluster samples , p appeared with a variation axis which is parallel to the association to a second group of tx and v , that is , with similar chemical characteristics . the third cluster , si and t samples were able to associate and the fourth group to jc samples , but perpendicular to the previous samples , so , the latter three were related to each other , but showed differences with the former . we can say that the associations of geographic location could be due to the environmental conditions , that is the geographical environment determines the concentration of compounds in peppers . by joining these results with the total content of phenolics , and by chromatography , the same association for p , tx , and v in concentration of trans - cinnamic , gallic , p - coumaric , protocatechuic acids , quercetin , and catechin can be found . in the present study , it has been found that ftir spectroscopy combined with the pca method presents great potential for the verification of the geographic origin of phenolic samples . regarding the principal components that were generated , that was too reduced to perform discrimination . such discrimination was realized by using the second derivative of the spectra in the region ( 9001750 cm ) . these results show that this ftir - pca analysis could be used as an alternative , quick , and low - cost method for identification of the geographic origin of mexican chilli peppers ( c. annuum l. ) through their phenolic compounds . for variables regarding type and maturation stage , it was not possible to find differences because the geographical variation , influenced by climatic and growing conditions , was more important than these variables . among the oligonucleotides tested , ope-18 was the most polymorphic rapd oligonucleotide generating a total 10 bands . from the rapd reactions carried out with a mixture of mfg-17 and mfg18 oligonucleotides and c51 to c52 oligonucleotides mixture independently of the oligonucleotide used , serrano and jalapeo chilli peppers exhibit similar rapd profiles , whereas habanero chilli peppers exhibited a particular profile with only 3 or 4 common bands . the presence / absence and similitude matrixes derived from the most informative primer ( ope-18 ) were obtained , and an euclidean distance dendogram was constructed with 10 polymorphic bands ( r = 0.94 ) ( figure 4 ) . c. chinense jacq . ( habanero chilli peppers ) samples were clearly clustered apart from the c. annuum l. ( serrano and jalapeo chilli peppers ) , confirming the clear differences ( coefficient of 2.0 ) observed among rapd profiles . basically , all samples formed three clusters : the first group made by the habanero peppers and the second and third groups do not show a clear separation between cultivars . a previous rapd analysis using 10 germplasm samples of c. annuum l from thailand revealed a higher diversity of rapd profiles those found in mexican samples . moreover , other rapd diversity analysis of c. annuum variety cuneo from northwest italy distinguished five populations . the limited profile diversity and high similitude observed in two mexican varieties studied can be explained by selective pressure derived from culture practices and commercial interests . evidently , rapd confirmed the great morphological diversity observed among c. annuum l varieties around the world , but an extensive study is necessary for molecular intraspecific diversity and population genetics including many c. annuum l varieties collected from different parts of the world . according to the dendrogram coupled to the amplification by oligonucleotide ope-18 and the chromatographic profile of carotenoids in mature peppers ( r = 0.69 ) ( figure 5 ) , the separation between the habanero peppers with respect to jalapeo 's and serrano 's can be observed , although it was not possible to identify between varieties within the same species or to identify an association between regions . . found that the carotenoid content and the use of molecular markers in different genotypes of bananas , provided useful information on kin selection by crossing among different genotypes in order to develop new varieties with functional properties . our results could be influenced only by the rapd because the carotenoid dendrogram did not show differences between species ; however , this separation maintained between the species does not occur in the case of phenols . the present study was able to establish a separation and differentiation between the chilli species c. annuum l. and c. chinense jacq , through the rapd molecular method by using different primers . also , by chromatographic profiles of carotenoids and molecular markers in mature chilli peppers and amplification by oligonucleotide ope 18 , it was possible to distinguish between habanero and serrano / jalapeo varieties , but no differences were found between c. annuum varieties . on the other hand , the pca of phenolic compounds in peppers , showed four groups according to their place of origin . hence this technique can be used to identify chilli varieties from different geographical areas , which might be used for the protection of rights in the variety , diversity and phylogenetic analysis , as well as for the confirmation of hybrids .

the genus capsicum provides antioxidant compounds , such as phenolics and carotenoids , into the diet . in mexico , there is a wide diversity of species and varieties of chilli peppers , a fruit which has local cultural and gastronomic importance . in the present study , the relationship of the carotenoid and phenolic profiles with the rapd fingerprint of three different commercial cultivars of chilli peppers of seven regions of mexico was investigated . through rapd , the species of chilli were differentiated by means of different primers ( ope-18 , mfg-17 , mfg-18 , c51 , and c52 ) . the genetic distance found with ope 18 was in the order of 2.6 . the observed differences were maintained when the chromatographic profile of carotenoids , and the molecular markers were analyzed , which suggest a close relationship between carotenoids and the genetic profile . while the chromatographic profile of phenols and the molecular markers were unable to differentiate between genotypes of chilli peppers . in addition , by using infrared spectroscopy and statistical pca , differences explained by geographic origin were found . thus , this method could be an alternative for identification of chilli species with respect to their geographic origin .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

the greatest genetic variety of c. annuum l. can be found in mexico , where there are chilli peppers of many different shapes , sizes , and colours , and they are among the most frequently consumed products . the genus capsicum is classified into the family of solanaceae , which is constituted by 25 wild and 5 domesticated species ( c. annuum l. , c. frutescens l. , c. chinense jacq , c. baccatum jacq , and c. pubescens l. ) , including more than 200 varieties [ 2 , 3 ] . chilli peppers have been object of study mainly due to containing capsaicin , which produces different pungency levels , as well as carotenoids and phenolic compounds , which are used as natural pigments and antioxidant agents . chilli peppers may have different content and profiles of such compounds , depending on the genotype and variety , maturity of the fruit , and the environmental conditions of the cultures . in order to preserve , manage , and improve the different chilli species , the evaluation of the extent of genetic variation within species , as this has now become a fundamental tool in biology and agriculture . among the main criteria used for that purpose the rapd ( random amplified polymorphic dna ) method is a widely used technique for molecular marking and is based on the amplification of genomic dna fragments by using primers of arbitrary nucleotide sequences ; which in turn detects polymorphisms that can be employed as genetic markers without previous genetic sequences . in addition to being simple , fast , and low - cost , this method does not require radioactive markers and consumes minimum amounts of dna [ 911 ] . however , one of the inconveniences reported about the rapd technique , since it was published for the first time , is the low reproducibility found mainly in low - intensity bands . thereby , the differences in the concentration of dna template between samples are observed as the gain or loss of some bands . correlated the flavonoid content of medicinal plants from brazil with molecular markers obtained by rapd , whereas ercisli et al . previously , the content and composition of fatty acids were tested as taxonomic markers in hippophae rhamnoides l. , showing that these substances , such as linoleic acid , can be used as biochemical markers for this species . another essential technique for determining the biological nature of agriculture products is fourier transform - infrared spectroscopy ( ftir ) , which is a technique that has been widely employed for food characterization because of several important advantages , such as its nondestructive nature and its capacity to yield structural information that constitutes a molecular fingerprint of the sample . a combination of ftir and chemometric techniques like principal component analysis ( pca ) has been employed as a direct and rapid way to discriminate properties in foods , including geographic origin , adulteration , and quality control . the aim of the present study is to contribute to management and improvement of the different chilli species by determining the relationship between molecular and biochemical markers , such as the content of carotenoids and phenolic compounds among different commercial cultivars of chilli peppers from diverse geographic origins . the samples were collected from central and north regions of mexico ( table 1 ) . the chilli peppers were considered fresh when the samples presented bright green colour , and smooth , firm texture . the mature specimens were derived from the green samples , by exposure to sunlight and moisture - free storage in the laboratory for 25 to 35 days . the samples were considered mature when the chilli peppers had a bright red , yellow , or orange colour and less firmness and texture . length , width , and weight of the fruits were determined in accordance to the mexican official standard . briefly , the length was taken from the base to the apex of the fruit without considering the peduncle ; the width was measured at the widest part of the chilli pepper ; for the weight , an analytical balance was used . the extraction of carotenoids from the chilli samples was carried out with 2 g portions of pulp and peel , which were mixed with diatomaceous earth and acetone at 10c . the extracts of carotenoids were reconstituted in 500 l of hplc - grade ethyl acetate and filtered through nylon membranes ( 0.45 m , pore size ) and then introduced into the c18 column ( 25 cm 4.6 mm ; 5 m ) of the hplc equipment ( perkin elmer binary lc pump 250 ; series 200 , uv / vis detector ) . an isocratic elution system was established with acetonitrile - methanol - ethyl acetate ( 73 : 20 : 7 ) working at 0.6 ml / min for 70 min , and the absorbance was measured at 450 nm . a total of 2 g of pulp and peel chilli peppers were mixed with 8 ml of 80% ethanol , and shaken for 10 min at 200 rpm . volumes of 0.75 ml of folin - ciocalteau reagent ( 1 : 10 ) were added to 100 l of the extract of free phenolics , and the mixture was left to stand for 5 min in the dark . starting with 90% phase a and 10% phase b , the mixture was eluted to 55% phase a , 45% phase b in 50 min and to 60% phase a and 40% phase b in the last 10 min . the dry extracts were reconstituted in petroleum ether , for carotenoids , and in ethanol , for phenolics , in order to obtain a semisolid consistency of the sample . afterwards , the mixtures were analyzed in a ft ( fourier transform)-infrared spectrometer ( bruker vertex 70 ) , by using the atr ( attenuated total reflectance ) sampling method previously described . briefly , a small sample amount is placed over a znse crystal , where the infrared radiation is propagated and interacts with the sample to obtain the corresponding spectrum , which is averaged from several data acquisitions . all samples of chilli peppers were washed with water , disinfected with sodium hypochlorite at 10% , 30% , and 70% ethanol , exhaustively rinsed with distilled water for 20 min and stored at 70c , under sterile conditions . afterwards , the blend was centrifuged at 4000 rpm for 30 min , and 400 l of each supernatant was transferred into new tubes , in order to add 5 l of 10 mg / ml rnasa for 10 min at room temperature . the mixture was then centrifuged at 4000 rpm for 30 min at 10c , the isopropanol eliminated , and the pellet washed with 1 ml of cold 70% ethanol , followed by centrifugation for 10 min under the same operation conditions . finally , the ethanol was removed , and the pellet was dried for 20 min at room temperature . the pellets were reconstituted into 200 l of 10 mm tris ph 8 , and the dna was precipitated again with 200 l of peg 8000 30%-nacl 1.2 m at 4c for 60 min . the supernatants from peg were discarded , and the dna pellet was washed with 1.5 ml of cold 70% ethanol , followed by centrifugation for 15 min at 4000 rpm . the ethanol was eliminated and the dna pellet left to dry , and finally reconstituted into 200 l of 10 mm tris ph 8 . the purity of the extracted dna was determined by absorbance at 260/280 ratio , and values greater than 1.8 were accepted for future procedures . pcr reactions were performed in a volume of 25 l containing 1x pcr buffer , 50 nm of mgcl2 , 200 m dntp , 1.5 units of the enzyme taq dna polymerase ( invitrogen , brazil ) , and 40100 ng/l dna samples . for the chromatogram , only signals above 40 milli - units of absorbance were considered the method of average grouping upgma was used and determined by means of the analysis cluster by using the software paleontological statistics ( past ) 2.10 , taking into account the dice 's similarity coefficient and the euclidean distance . the weight of the different varieties of chilli peppers ranged from 3 to 46 g. in general , samples with the best morphological characteristics ( color , size , texture , and weight ) were those from the northern region , except for the sample jv1 from the south - central area , the latter being the lighter weight . the length was from 4.3 to 9.5 cm , and the width was in the range of 0.1 to 3.34 cm . the three varieties of chilli showed differences in both weight and size , with remarkable greater similarities between the same species ( jalapeo and serrano ) as well as those grown in nearby areas . for instance , the serrano chilli peppers grown in tx , p , and v1 from the central region were quite similar . through statistical analysis , significant differences were found in weight and width between varieties , but not in length between serrano and jalapeo ( i.e. however , these two peppers showed significant difference with respect to habanero ( i.e. the content of carotenoids in fresh peppers varied within the interval 0.0830.99 mg / g dry basis ( db ) , while mature peppers were in the range from 0.69 to 13.85 mg / g db . in accordance with the statistical analysis , of chilli peppers in the fresh stage , there were significant differences between jalapeo and serrano varieties , while analysis of mature peppers , showed no differences between the three cultivars ( see table 3 ) . this is due to the ability of fruits to synthesize carotenoids during the ripening process regardless of the species . mature chilli peppers , from si showed differences with respect to those from t , p , v1 and v2 . therefore , no association was found between the concentration of carotenoids and the geographical origin . the results presented above could be affected also by the environmental conditions , such as temperature and relative humidity , as well as the type of soil , which of course are related to the proximity of the different geographical regions . during the process of maturation , lutein remained constant for jalapeo peppers , whereas it diminished for serrano ones however , it has been reported that lutein disappears in mature peppers by the effect of the synthesis of pigments in chromoplasts . according to statistical analysis , no significant differences were found for fresh peppers in concentrations of lutein , lycopene , and -cryptoxanthin between jalapeo and serrano cultivars . nonetheless , there were differences in -carotene content , which is the major compound in several varieties of c. annuum l. . on the other hand , differences were found for mature peppers only between jalapeo and serrano , in this case in lutein and lycopene content ( table 4 ) . in most of the samples , there was a noteworthy increase in these compounds in mature chilli peppers , in agreement with the results of deepa et al . given the above , the main factors involved in the variation of phenolic content are the maturation state , as well as the age of the plant , as reported by deepa et al . when doing a comparison on the total phenolic content within the chilli varieties , significant differences were found between serrano and jalapeo , for both fresh and mature stages , while between jalapeo and serrano as a group no differences were detected with respect to habanero . concerning the region , differences were found in most of fresh jalapeo peppers samples , except between p and v ( v1 and v2 ) and between si and jco . for the fresh serrano peppers , there were differences between tx and p with respect to jc and v2 . for mature peppers , the region seems to be unimportant ( see table 5 ) . who reported significant differences between cultivars of c. baccatum l. and c. chinense jacq , and between two genotypes of the latter variety . the phenolic compounds found in chilli peppers were gallic , protocatechuic , ferulic , o - coumaric , p - coumaric , sinapinic , trans - cinnamic and caffeic acids , quercetin , catechin , rutin , and vanillin . many of these compounds belong to the pathway of phenylpropanoids which is characteristic of chilli species . among the varieties of chilli , significant differences were found between serrano and jalapeo in the gallic and p - coumaric acids content , indifferently of maturation stage . for the mature stage , the only difference was found in the content of trans - cinnamic acid between the two varieties ( table 6 ) . regarding the place of origin , fresh jalapeo peppers showed differences in the content of sinapic acid between si samples and the rest , in the content of catechin of v samples compared to p and tx samples ( despite the fact that all three belong to the central region ) , and in the content of p - coumaric acid of p compared to v1 , tx , and si . the fresh serrano peppers showed differences in the concentration of gallic acid for samples from si with respect to p and v. in the content of protocatechuic acid for samples from p with respect to the other specimens , and in the content of caffeic acid and rutin for v2 with respect to the other specimens . finally , most regions showed differences in the content of p - coumaric acid , except for v2 compared to p and s , v1 compared to jc and tx , p compared to si , and jc compared to tx , showing no association between geographical areas . among mature jalapeo peppers , t showed differences with respect to si in the content of gallic acid and with respect to v1 in protocatechuic acid . no differences were found between the mature serrano peppers with respect to region . by taking into account the state of maturation , for the rest of the phenolic compounds , there were not many differences as observed by the maturation stage in the content of gallic , protocatechuic , trans - cinnamic and p - coumaric acids ; quercetin , rutin , and vanillin . for the rest of the phenolic compounds , there were no differences that could be explained by the maturation stage . within a jalapeo variety , differences were found between fresh and mature peppers in the content of gallic acid , while no such difference existed in the content of ferulic acid . for mature specimens no differences were found only between the three varieties in the content of protocatechuic acid , caffeic acid , and catechin , while the fresh samples reported differences in the content of p - coumaric acid , catechin , and vanillin . some interactions can be explained by the proximity of the geographical areas , as in the case of p , tx , v , and t. other differences may be due to the growing conditions of each geographic region or by the genotype of the chilli , as discussed earlier by silva et al . these authors studied the content of rutin in a medicinal fruit from brazil and found that the high variability in the concentration and the great genetic variability of the fruit may be associated not only with genetic differences , but also with environmental conditions . the typical ir spectra ( 6002000 cm ) of phenolic extracts from different geographical regions of mexico ( figure 1 ) however , by applying principal component analysis ( pca ) ( figure 2 ) to the second derivative of the spectra , four clusters were found . the third cluster , si and t samples were able to associate and the fourth group to jc samples , but perpendicular to the previous samples , so , the latter three were related to each other , but showed differences with the former . by joining these results with the total content of phenolics , and by chromatography , the same association for p , tx , and v in concentration of trans - cinnamic , gallic , p - coumaric , protocatechuic acids , quercetin , and catechin can be found . in the present study , it has been found that ftir spectroscopy combined with the pca method presents great potential for the verification of the geographic origin of phenolic samples . such discrimination was realized by using the second derivative of the spectra in the region ( 9001750 cm ) . these results show that this ftir - pca analysis could be used as an alternative , quick , and low - cost method for identification of the geographic origin of mexican chilli peppers ( c. annuum l. ) through their phenolic compounds . from the rapd reactions carried out with a mixture of mfg-17 and mfg18 oligonucleotides and c51 to c52 oligonucleotides mixture independently of the oligonucleotide used , serrano and jalapeo chilli peppers exhibit similar rapd profiles , whereas habanero chilli peppers exhibited a particular profile with only 3 or 4 common bands . the presence / absence and similitude matrixes derived from the most informative primer ( ope-18 ) were obtained , and an euclidean distance dendogram was constructed with 10 polymorphic bands ( r = 0.94 ) ( figure 4 ) . ( habanero chilli peppers ) samples were clearly clustered apart from the c. annuum l. ( serrano and jalapeo chilli peppers ) , confirming the clear differences ( coefficient of 2.0 ) observed among rapd profiles . according to the dendrogram coupled to the amplification by oligonucleotide ope-18 and the chromatographic profile of carotenoids in mature peppers ( r = 0.69 ) ( figure 5 ) , the separation between the habanero peppers with respect to jalapeo 's and serrano 's can be observed , although it was not possible to identify between varieties within the same species or to identify an association between regions . found that the carotenoid content and the use of molecular markers in different genotypes of bananas , provided useful information on kin selection by crossing among different genotypes in order to develop new varieties with functional properties . our results could be influenced only by the rapd because the carotenoid dendrogram did not show differences between species ; however , this separation maintained between the species does not occur in the case of phenols . the present study was able to establish a separation and differentiation between the chilli species c. annuum l. and c. chinense jacq , through the rapd molecular method by using different primers . also , by chromatographic profiles of carotenoids and molecular markers in mature chilli peppers and amplification by oligonucleotide ope 18 , it was possible to distinguish between habanero and serrano / jalapeo varieties , but no differences were found between c. annuum varieties . on the other hand , the pca of phenolic compounds in peppers , showed four groups according to their place of origin . hence this technique can be used to identify chilli varieties from different geographical areas , which might be used for the protection of rights in the variety , diversity and phylogenetic analysis , as well as for the confirmation of hybrids .

skeletal muscles are composed of a variety of fibers which are traditionally classified as fast and slow depending on their contractile parameters , such as time to peak in the isometric twitch or maximum shortening velocity , or as glycolytic and oxidative depending on their metabolic properties . the current nomenclature , based on myosin heavy chain ( mhc ) isoform composition , includes 4 major fiber types , called type 1 or slow and type 2a , 2x , and 2b or fast , respectively , each of them with specific contractile properties . the differences between fast and slow fibers , however , are not restricted only to myofibrillar proteins ( myosin isoforms ) and to metabolic enzymes ( predominance of glycolitic versus oxidative activities ) , but also involves other subcellular systems . importantly the kinetics of ca mobilization are profoundly different in slow versus fast twitch fibers [ 47 ] . intracellular ca concentrations ( [ ca]i ) and ca release / reuptake from intracellular stores ( i.e. , the sarcoplasmic reticulum , sr ) are controlled by the sarcotubular system , a highly organized system of membranes formed by the association of invaginations of the sarcolemma , i.e. , the transverse ( t)-tubules , with the terminal cisternae of the sr [ 8 , 9 ] . t - tubules contain voltage - gated l - type ca channel ( or dihydropyridine receptors , dhprs ) which are mechanically coupled to ca release channels of the sr , the ryanodine receptors type-1 interaction between dhpr and ryr1 occurs at intracellular junctions called ca release units ( crus ) or triads , which mediate excitation - contraction ( ec ) coupling [ 12 , 13 ] . crus contain several other proteins beside dhprs and ryr1s : among them calsequestrin ( casq ) , the main intraluminal ca binding protein of the sr [ 14 , 15 ] , which is located in terminal cisternae of the junctional sr in close proximity to ryrs [ 16 , 17 ] . in skeletal muscles , casq exists in two isoforms known as casq1 ( or skeletal ) and casq2 ( or cardiac ) . both isoforms can be found in slow fibers , whereas only casq1 is expressed in fast fibers [ 18 , 19 ] . it has been reported that the total casq content is greater in fast than in slow fibers . a recent quantitative analysis on single fibers from rat points to a concentration of 36 m in fast fibers ( only casq1 ) versus 10 m in slow fibers ( casq1 and casq2 ) . owing to its properties ( medium - low affinity , but high capacity ) , casq provides a large sr pool of releasable ca , while maintaining sr intraluminal concentrations of free ca low enough to facilitate the work of sarcoendoplasmic reticulum ca atp - ase ( serca ) pumps . casq in skeletal muscle fibers plays an important dual role : ( i ) to buffer ca inside the sr thanks to the large number of acidic residues which allows each casq molecule to bind up to 6080 ca ions ; ( ii ) to modulate ca release from the sr via a tradin / junctin - mediated interaction with ryr1 [ 2325 ] . there is evidence that casq1 has different polymerization rate and ca buffering properties than casq2 , suggesting the possibility that ryr1 is modulated differently by casq1 and 2 in fast- and slow - twitch fibers . murphy and colleagues recently suggested that casq2 is more efficient than casq1 in reducing sr ca leak , a property also shown in cardiac muscle . mice lacking casq1 were viable and fertile . nevertheless , lack of casq1 induced significant functional and structural modifications in skeletal fibers : casq1 ablation reduced dramatically the total sr ca content ( of about 70% ) in fast twitch from flexor digitorum brevis ( fdb ) muscles . however , ca transients evoked by a single stimulus was surprisingly not dramatically reduced so that twitch peak force was preserved . this apparent minor functional impairment can be in part explained by morphological adaptations taking place in fast fibers , that is , profound remodelling of crus which forms multiple layers of junctional sr and t - tubules bearing an approximately doubled number of ryrs . both functional and structural changes were more evident in extensor digitorum longus ( edl ) and in fdb , containing predominantly fast twitch fibers , than in soleus muscle , a predominantly slow twitch muscle . there are reasons to believe that the impact of casq1 ablation is more evident in edl and in fdb than in soleus , because casq2 is expressed in casq1-null mice and quite abundant in slow twitch fibers . to explain the differential impact of casq1 ablation in fast versus slow fibers , we may also have to consider important functional differences : in fast fibers greater amounts of ca are released after each action potential , sr volume is greater , and sr is filled only to 35% of its maximal capacity . in contrast sr of slow fibers is filled to its maximal capacity . in view of the possible diversity in ca handling between slow and fast muscles and to investigate how complete ablation of casq will affect the different muscle types we studied edl and soleus muscle in mice lacking both casq isoforms [ 29 , 30 ] , generated by cross - breeding preexisting casq1-null and casq2-null mice [ 28 , 31 ] . interestingly , our results show that edl and soleus are differently affected by complete ablation of casq , as edl , but not soleus , becomes unable to maintain tension during prolonged tetanic contractions , while soleus , but not edl , displays the early onset of a myopathic phenotype . casq1-null and casq2-null mice were generated as previously described [ 28 , 31 ] . double ( d)casq - null mice lacking both casq isoforms were generated by cross - breeding the preexisting casq1-null and casq2-null mice . c57bl/6j mice were used as wild - type ( wt ) controls and obtained from charles river italia . mice were housed in microisolator cages , temperature 22c , 12 hr light / dark cycle , with free access to water and food . mice were killed by an overdose of the anaesthetic ethylic ether , and their muscles were rapidly dissected . all experiments were conducted according to the national institutes of health guide for the care and use of laboratory animals and were approved by the ethical committee of the university of chieti and of the department of anatomy and physiology , university of padova . preparation of total homogenates from wt , casq1-null and dcasq - null muscles ( hind limb , edl , and soleus ) , and western blot analysis were performed as previously described in 46 -month- old mice . the antibody used was a rabbit polyclonal antibody reactive with both isoforms of casq ( affinity bioreagents , usa ) . the rate of spontaneous mortality under standard housing conditions was assessed during the entire life span using the kaplan - meier method in a subpopulation of mice which were not utilized for other experiments . strength developed by wt , casq1-null , and dcasq - null male mice of 6 months of age was measured during instinctive grasp with a grip - strength - test protocol . the mouse was held by the tail in proximity to a trapeze bar connected with the shaft of a shimpo fgv 0.5x force transducer ( metrotec group , san sebastin spain ) . once the mouse had firmly grabbed the trapeze , a gentle pull was exerted on the tail . the measurement of the peak force generated by each mouse with fore and hind limbs was repeated three times with appropriate intervals to avoid fatigue , and the average of the highest peak force values was normalized to the body mass . edl and soleus muscles were carefully dissected from wt , casq1-null , and dcasq - null at 46 months of age . muscles were fixed at rt in 3.5% glutaraldehyde in 0.1 m sodium cacodylate buffer , ph 7.2 for 2 h and kept in fixative before further use . small bundles of fixed fibers were postfixed in 2% oso4 in 0.1 m sodium cacodylate buffer for 2 h and block stained in aqueous saturated uranyl acetate . after dehydration , specimens were embedded in an epoxy resin ( epon 812 ) . for histological analysis , longitudinal and cross - oriented semithin sections ( 300 nm ) were cut with a leica ultracut r microtome ( leica microsystem , vienna , austria ) using a diatome diamond knife ( diatomeltd . , the sections were viewed on a leica dmlb fluorescence microscope ( leica microsystem , vienna , austria ) . quantitative analysis of damaged fibers ( data in table 1 ) was performed on histology images . for em , ultrathin sections ( 35 nm ) were cut and , after staining in 4% uranyl acetate and lead citrate , examined with a morgagni series 268d electron microscope ( fei company , brno , czech republic ) , equipped with megaview iii digital camera . edl and soleus muscles were dissected from the hind limbs of wt and knock - out male mice ( 4 months old ) in warm oxygenated krebs solution and mounted between a force transducer ( si - h force transducer world precision instruments , inc . , sarasota , fl , usa ) and a micromanipulator - controlled shaft in a small chamber where oxygenated krebs solution was continuously circulated . the stimulation conditions were optimized , and muscle length was increased until force development during tetanus was maximal . the responses to a single stimulus ( twitch ) or to a series of stimuli at various rates producing unfused or fused tetani were recorded . time - to - peak tension , time - to - half relaxation , time - to - base tension , and peak - tension were measured in single twitches . tension was measured in completely fused maximal tetani of different duration ( 0.52 s ) at the peak and just after the last stimulus . for analysis of force and contraction kinetics comparison between the three groups ( wt , casq1-null , and dcasq - null mice ) was carried out using anova followed by a post hoc test ( newman - keuls test ) , while for weight and grip test the statistical significance was assessed using the unpaired student 's t - test . the expression of the two casq isoforms in wt , casq1-null , and dcasq - null mice was assessed by western blots of homogenates prepared from either all hind limb muscles ( figure 1(a ) ) or separately from edl and soleus ( figures 1(b ) and 1(c ) , resp . ) . analysis showed that : ( a ) in casq1-null muscles casq1 is missing , whereas casq2 is still present , more in soleus ( slow twitch ) and less in edl ( fast twitch ) ; ( b ) in dcasq - null muscles both isoforms are absent . dcasq - null mice were viable and fertile , appeared to develop and breed normally , and did not present a clear overt phenotype . however , male mice carrying the double - null mutation displayed a greatly increased mortality rate compared to wt animals , even more pronounced than that of casq1-null male mice ( figure 2 ; see also [ 32 , 34 ] for more detail ) : more than 50% of male mice died before reaching the age of 6 months . rate of spontaneous mortality of casq1-null and dcasq - null females was not significantly different from that of male and female wt ( figure 2 ) . we measured the average body weight of wt , casq1-null , and dcasq - null adult male mice ( 46 months of age ) : dcasq - null mice were on the average significantly smaller than wt and slightly smaller than casq1-null mice ( figure 3(a ) ) . to assess basic neuromuscular function of living mice , we used the grip strength test , which provides a simple way to test the global muscle performance during maximal isometric contraction of short duration . figure 3(b ) shows that mice lacking either casq1 or both casq isoforms developed a significantly lower force output than wt . no significant difference was detectable between mice lacking only casq1 and both casq isoforms using this method . we performed structural analysis of edl and soleus muscles from adult male ( 46 months of age ) wt , casq1-null , and dcasq - null mice using a combination of sectioning for histology and em ( figure 4 ) . this examination revealed that a significant percentage of fibers in dcasq - null soleus exhibit severe morphological alterations ( table 1 ) . this structural damage , which was not found in edl at this age ( 46 months ) , disrupts the regular cross - striation of skeletal fibers and affects large portions of the fiber interior ( figure 4(e ) , asterisk ) . whereas structural alterations are quite variable in appearance , we classified abnormal fibers in two main classes ( table 1 ) : ( a ) those presenting large areas loosing cross - striation , or unstructured cores , but no contractures ( figure 4(f ) ) ; ( b ) those containing also areas of contracture ( figure 4(e ) , asterisk ) . fibers containing unstructured cores were frequent in soleus from dcasq - null mice , but rare in casq1-null soleus and totally absent in wt ( table 1 , column ( a ) ) . fiber presenting contracture cores , on the other hand , were never found in soleus muscles from wt and casq1-null mice , whereas again they were quite frequent in soleus fibers from dcasq - null ( table 1 , column ( b ) ) . overall , ~35% of soleus fibers from dcasq - null mice showed structural alterations ( table 1 , column ( c ) ) . in order to assess the effects of the complete removal of casq on the contractile performance of fast- and slow - twitch muscles , we dissected edl and soleus muscles from wt , casq1-null , and dcasq - null mice and studied their function ex vivo . the altered kinetics profile of the contractile cycle previously described in casq1-null muscle was also evident in dcasq - null muscles . the changes included a significant prolongation of time - to - peak tension ( i.e. from the stimulus to the tension peak ) in edl compared to wt , but also to casq1-null ( figure 5(a ) ) . no prolongation of time - to - peak tension was seen in soleus ( figure 5(b ) ) . in addition , time to base ( i.e. , time elapsing from stimulus to the return to base line at the end of relaxation ) was significantly prolonged in both edl and soleus muscles of knock - out mice compared to wt . the latter effect was more pronounced in dcasq - null than in casq1-null mice ( figures 5(c ) and 5(d ) ) . isometric tension was determined in short fused isometric tetani with a duration just sufficient to reach peak tension ( 500 ms in edl and 1 s in soleus ) and found significantly reduced in edl , but not in soleus muscles of casq1-null and dcasq - null mice ( not shown ) . however , the most interesting result was obtained when the duration of the tetanus was prolonged up to 2 seconds ( figure 6 ) : in both casq1- and dcasq - null edl the residual tension declined dramatically ( by about 80% ) ( figures 6(a ) and 6(b ) ) , while in soleus only a minor decline of developed tension occurred in the absence of casq ( figures 6(c ) and 6(d ) ) . specifically , the tension decline of soleus during a 2 s tetanus was approximately 15% in casq1-null and 25% in dcasq - null . a careful inspection of the traces showed in figures 6(a ) and 6(c ) revealed that the kinetics of the rising phase of the isometric tetani were faster when casq is missing both in edl and in soleus . an increased early phase of ca release has been previously reported in fibers lacking casq isolated from dcasq - null mice . this effect can be related to the alterations of the twitch response . in agreement with the above observations , the amplitude of the twitch ( measured as peak tension , not shown ) was preserved , and the duration of the twitch ( measured as time - to - peak and time - to - base line , figure 5 ) was prolonged , thus allowing a faster tension development during repeated high frequency stimulation . two isoforms , casq1 and casq2 , are expressed in skeletal muscle fibers [ 18 , 19 ] , with casq1 and casq2 being more abundant in fast- and slow - twitch fibers , respectively . the impact of their removal in different fiber types has not been investigated yet . in view of the possible diversity in ca handling between slow and fast muscles and to investigate how complete ablation of casq will affect the two different muscle types , we studied edl and soleus muscle in mice lacking both casq isoforms . the comparison of the structural and functional effect of the complete ablation of casq revealed two main distinctive features : ( 1 ) soleus , but not edl , shows a number of fibers with signs of structural degeneration ( figure 4 ) ; ( 2 ) edl is unable to maintain active tension during a prolonged tetanus , while soleus is only marginally affected ( figure 6 ) . previous studies have shown that casq1-null mice are susceptible to spontaneous mortality and trigger mh - like episodes when exposed to either heat or anesthesia [ 32 , 34 ] . casq2-null mice display frequent episodes of catecholaminergic polymorphic ventricular tachycardia ( cpvt ) , which can be triggered by a catecholamine challenge with the -adrenergic agonist isoproterenol . dcasq - null mice are viable , but present high frequency of spontaneous death in male mice , even higher than that previously registered in casq1-null animals ( figure 2 ) . the specific reason for the increased spontaneous mortality rate of dcasq - null mice is still under investigation , since cpvt in mice lacking casq2 is not lethal . grip strength test confirmed a significant impairment of the overall neuromuscular function ( figure 3(b ) ) , similar to that of casq1-null mice . the lack of a significant difference in grip strength between casq1- and dcasq - null mice probably reflects the fact that murine muscles show a great predominance of fast fibers expressing exclusively casq1 . finally , dcasq - null mice show a reduced body weight which can be likely ascribed to a decrease in skeletal muscle mass due to a myopathic phenotype . we have recently reported that isolated muscles ( edl ) , muscle fibers ( from fdbs ) , and myotubes lacking casq1 present elevated basal cytosolic ca at body temperature [ 30 , 32 ] . this abnormally high resting cytosolic ca causes abnormal development of muscle tension ( contracture ) when body temperature is raised above physiological values ( 3941c ) . this reaction to heat explains why casq1-null mice are susceptible to trigger lethal malignant hyperthermia ( mh ) like episodes when exposed to either high environmental temperatures or halogenated anesthetics . in the present study we show that ~30% of soleus fibers from dcasq - null mice present clear evidence of the early onset of a myopathic phenotype , which is not as evident in edl fibers at the same age ( 46 months , figure 4 ) . these alterations , which resemble those described in other murine models of mh and central core disease ( ccd ) [ 36 , 37 ] , were not seen in soleus fibers of casq1-null mice , suggesting that the abundant expression of casq2 in slow fibers ( figure 1 ) prevents the onset of the pathology . preliminary data from our laboratory shows that also edl of casq1-null mice ( where a minor amount of casq2 is expressed ) will eventually develop a similar myopathy with increasing age . this considered , the difference between the two muscles is that soleus develops a myopathy at an earlier stage . the reason can be tentatively found in the different capabilities of edl and soleus to deal with removal of ca from cytosol and with sr ca leak . in support to this view , it has been recently suggested that casq2 in slow muscle fibers may be important to prevent ca leakage . the sr of slow fibers is saturated with ca at resting myoplasmic ca concentration , while the sr of fast fibers is only about one - third saturated with ca under equivalent conditions [ 21 , 39 ] . such difference implies that the rate of sr ca uptake in fast fibers is predominantly controlled by myoplasmic ca , while in slow fibers is more likely limited by the ca concentration within the sr lumen . the intraluminal ca concentration is likely increased when the buffering action of casq is missing in the soleus of dcasq - null mice , causing serious challenge to reuptake . this would in turn result in excessive sr ca leak and [ ca]i , which will ultimately lead to the structural decay . the dramatic prolongation of the time to base ( i.e. , relaxation duration ) of soleus lacking both casq isoforms is in agreement with the impaired ca reuptake . the complete removal of casq in double - null mice decreases the ability of the sr to store ca both in fast and in slow muscle fibers . slow soleus fibers , but not fast edl fibers , are able to sustain tension during a prolonged contractions ( figure 6 ) , due to specific feature of the ca kinetics as discussed here below . the ryr - mediated ca release from the sr is approximately two times greater in fast compared to slow fibers . the larger size of crus and the higher density of ryr1 and dhpr in fast fibers is instrumental to this [ 40 , 41 ] . a greater ca release is needed in edl fast fibers compared to soleus slow fibers as the number of cytosolic ca binding sites is greater . in the first place , the troponin - c ( tnc ) isoform expressed in fast - twitch fibers presents two low affinity ca binding sites , whereas there is only one in the slow - twitch tnc isoform and , in addition , other cytosolic ca binding proteins must be saturated during the contractile cycle . among them , there are parvalbumin , present at a concentration of 400500 mol / liter in fast fibers but not in slow fibers and containing two ca binding sites , and serca1 , with a concentration of 120 mol / liter and two ca binding sites . during a twitch ( or the initial phase of a tetanus ) the ca released can efficiently saturate tnc and serca and other minor ca buffers like calmodulin , and the amount of 300400 moles is likely to be sufficient , in agreement with published evidence [ 40 , 43 ] . during a tetanic train of stimuli , more ca enters the cytosol through repeated releases , although the amplitude of the subsequent releases progressively decreases with the fifth release being only 10% of the first release [ 40 , 43 ] . in order to sustain tension for more than one second , it is necessary to saturate also parvalbumin , a process occurring with a slower kinetics related to calcium replacement for magnesium : fast fibers deprived of casq fails to maintain a prolonged tetanic tension , probably because the ca released is not sufficient to saturate all cytosolic calcium - binding proteins , parvalbumin among them , with the result of reducing the ca available for tnc , and therefore for tension generation . conversely , a quite different scenario takes place in a typical slow fibers of soleus where the lower serca density , the single low binding site of slow tnc , and , above all , the absence of parvalbumin would prevent the tension decline observed in edl . in this contest , very likely the minor reduction ( ~25% ) in tetanic tension recorded in dcasq - null soleus ( figures 6(c ) and 6(d ) ) could be almost completely ascribed to the presence of damaged fibers , which likely have compromised contractile function , and not to an insufficient amount of ca to activate contraction . whereas in edl casq is necessary to provide the large amount of ca required for a maximal sustained contraction , slow - twitch fibers are only moderately affected by the absence of casq during prolonged tetani . however , casq presence in soleus seems necessary to help ca reuptake , reduce ca leakage , and control myoplasmic ca , which otherwise will eventually lead to the early onset of a myopathy . in this aspect , slow fibers are reminiscent of cardiac myocytes where casq role is essential to control diastolic ca leakage . these findings add novel information , which may help to better understand the differences in ca handling of fast and slow fibers and also offer new insights to unlock mechanisms leading to myopathies such as mh and ccd .

we compared structure and function of edl and soleus muscles in adult ( 46 m ) mice lacking both calsequestrin ( casq ) isoforms , the main sr ca2 + -binding proteins . lack of casq induced ultrastructural alterations in ~30% of soleus fibers , but not in edl . twitch time parameters were prolonged in both muscles , although tension was not reduced . however , when stimulated for 2 sec at 100 hz , soleus was able to sustain contraction , while in edl active tension declined by 7080% . the results presented in this paper unmask a differential effect of casq1&2 ablation in fast versus slow fibers . casq is essential in edl to provide large amount of ca2 + released from the sr during tetanic stimulation . in contrast , soleus deals much better with lack of casq because slow fibers require lower ca2 + amounts and slower cycling to function properly . nevertheless , soleus suffers more severe structural damage , possibly because sr ca2 + leak is more pronounced .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

the differences between fast and slow fibers , however , are not restricted only to myofibrillar proteins ( myosin isoforms ) and to metabolic enzymes ( predominance of glycolitic versus oxidative activities ) , but also involves other subcellular systems . , the sarcoplasmic reticulum , sr ) are controlled by the sarcotubular system , a highly organized system of membranes formed by the association of invaginations of the sarcolemma , i.e. , the transverse ( t)-tubules , with the terminal cisternae of the sr [ 8 , 9 ] . t - tubules contain voltage - gated l - type ca channel ( or dihydropyridine receptors , dhprs ) which are mechanically coupled to ca release channels of the sr , the ryanodine receptors type-1 interaction between dhpr and ryr1 occurs at intracellular junctions called ca release units ( crus ) or triads , which mediate excitation - contraction ( ec ) coupling [ 12 , 13 ] . crus contain several other proteins beside dhprs and ryr1s : among them calsequestrin ( casq ) , the main intraluminal ca binding protein of the sr [ 14 , 15 ] , which is located in terminal cisternae of the junctional sr in close proximity to ryrs [ 16 , 17 ] . in skeletal muscles , casq exists in two isoforms known as casq1 ( or skeletal ) and casq2 ( or cardiac ) . both isoforms can be found in slow fibers , whereas only casq1 is expressed in fast fibers [ 18 , 19 ] . it has been reported that the total casq content is greater in fast than in slow fibers . a recent quantitative analysis on single fibers from rat points to a concentration of 36 m in fast fibers ( only casq1 ) versus 10 m in slow fibers ( casq1 and casq2 ) . owing to its properties ( medium - low affinity , but high capacity ) , casq provides a large sr pool of releasable ca , while maintaining sr intraluminal concentrations of free ca low enough to facilitate the work of sarcoendoplasmic reticulum ca atp - ase ( serca ) pumps . casq in skeletal muscle fibers plays an important dual role : ( i ) to buffer ca inside the sr thanks to the large number of acidic residues which allows each casq molecule to bind up to 6080 ca ions ; ( ii ) to modulate ca release from the sr via a tradin / junctin - mediated interaction with ryr1 [ 2325 ] . murphy and colleagues recently suggested that casq2 is more efficient than casq1 in reducing sr ca leak , a property also shown in cardiac muscle . mice lacking casq1 were viable and fertile . nevertheless , lack of casq1 induced significant functional and structural modifications in skeletal fibers : casq1 ablation reduced dramatically the total sr ca content ( of about 70% ) in fast twitch from flexor digitorum brevis ( fdb ) muscles . however , ca transients evoked by a single stimulus was surprisingly not dramatically reduced so that twitch peak force was preserved . this apparent minor functional impairment can be in part explained by morphological adaptations taking place in fast fibers , that is , profound remodelling of crus which forms multiple layers of junctional sr and t - tubules bearing an approximately doubled number of ryrs . both functional and structural changes were more evident in extensor digitorum longus ( edl ) and in fdb , containing predominantly fast twitch fibers , than in soleus muscle , a predominantly slow twitch muscle . there are reasons to believe that the impact of casq1 ablation is more evident in edl and in fdb than in soleus , because casq2 is expressed in casq1-null mice and quite abundant in slow twitch fibers . to explain the differential impact of casq1 ablation in fast versus slow fibers , we may also have to consider important functional differences : in fast fibers greater amounts of ca are released after each action potential , sr volume is greater , and sr is filled only to 35% of its maximal capacity . in contrast sr of slow fibers is filled to its maximal capacity . in view of the possible diversity in ca handling between slow and fast muscles and to investigate how complete ablation of casq will affect the different muscle types we studied edl and soleus muscle in mice lacking both casq isoforms [ 29 , 30 ] , generated by cross - breeding preexisting casq1-null and casq2-null mice [ 28 , 31 ] . interestingly , our results show that edl and soleus are differently affected by complete ablation of casq , as edl , but not soleus , becomes unable to maintain tension during prolonged tetanic contractions , while soleus , but not edl , displays the early onset of a myopathic phenotype . double ( d)casq - null mice lacking both casq isoforms were generated by cross - breeding the preexisting casq1-null and casq2-null mice . preparation of total homogenates from wt , casq1-null and dcasq - null muscles ( hind limb , edl , and soleus ) , and western blot analysis were performed as previously described in 46 -month- old mice . the antibody used was a rabbit polyclonal antibody reactive with both isoforms of casq ( affinity bioreagents , usa ) . edl and soleus muscles were carefully dissected from wt , casq1-null , and dcasq - null at 46 months of age . muscles were fixed at rt in 3.5% glutaraldehyde in 0.1 m sodium cacodylate buffer , ph 7.2 for 2 h and kept in fixative before further use . small bundles of fixed fibers were postfixed in 2% oso4 in 0.1 m sodium cacodylate buffer for 2 h and block stained in aqueous saturated uranyl acetate . , the sections were viewed on a leica dmlb fluorescence microscope ( leica microsystem , vienna , austria ) . edl and soleus muscles were dissected from the hind limbs of wt and knock - out male mice ( 4 months old ) in warm oxygenated krebs solution and mounted between a force transducer ( si - h force transducer world precision instruments , inc . tension was measured in completely fused maximal tetani of different duration ( 0.52 s ) at the peak and just after the last stimulus . for analysis of force and contraction kinetics comparison between the three groups ( wt , casq1-null , and dcasq - null mice ) was carried out using anova followed by a post hoc test ( newman - keuls test ) , while for weight and grip test the statistical significance was assessed using the unpaired student 's t - test . the expression of the two casq isoforms in wt , casq1-null , and dcasq - null mice was assessed by western blots of homogenates prepared from either all hind limb muscles ( figure 1(a ) ) or separately from edl and soleus ( figures 1(b ) and 1(c ) , resp . ) analysis showed that : ( a ) in casq1-null muscles casq1 is missing , whereas casq2 is still present , more in soleus ( slow twitch ) and less in edl ( fast twitch ) ; ( b ) in dcasq - null muscles both isoforms are absent . however , male mice carrying the double - null mutation displayed a greatly increased mortality rate compared to wt animals , even more pronounced than that of casq1-null male mice ( figure 2 ; see also [ 32 , 34 ] for more detail ) : more than 50% of male mice died before reaching the age of 6 months . rate of spontaneous mortality of casq1-null and dcasq - null females was not significantly different from that of male and female wt ( figure 2 ) . we measured the average body weight of wt , casq1-null , and dcasq - null adult male mice ( 46 months of age ) : dcasq - null mice were on the average significantly smaller than wt and slightly smaller than casq1-null mice ( figure 3(a ) ) . to assess basic neuromuscular function of living mice , we used the grip strength test , which provides a simple way to test the global muscle performance during maximal isometric contraction of short duration . figure 3(b ) shows that mice lacking either casq1 or both casq isoforms developed a significantly lower force output than wt . no significant difference was detectable between mice lacking only casq1 and both casq isoforms using this method . we performed structural analysis of edl and soleus muscles from adult male ( 46 months of age ) wt , casq1-null , and dcasq - null mice using a combination of sectioning for histology and em ( figure 4 ) . this structural damage , which was not found in edl at this age ( 46 months ) , disrupts the regular cross - striation of skeletal fibers and affects large portions of the fiber interior ( figure 4(e ) , asterisk ) . whereas structural alterations are quite variable in appearance , we classified abnormal fibers in two main classes ( table 1 ) : ( a ) those presenting large areas loosing cross - striation , or unstructured cores , but no contractures ( figure 4(f ) ) ; ( b ) those containing also areas of contracture ( figure 4(e ) , asterisk ) . fibers containing unstructured cores were frequent in soleus from dcasq - null mice , but rare in casq1-null soleus and totally absent in wt ( table 1 , column ( a ) ) . fiber presenting contracture cores , on the other hand , were never found in soleus muscles from wt and casq1-null mice , whereas again they were quite frequent in soleus fibers from dcasq - null ( table 1 , column ( b ) ) . overall , ~35% of soleus fibers from dcasq - null mice showed structural alterations ( table 1 , column ( c ) ) . in order to assess the effects of the complete removal of casq on the contractile performance of fast- and slow - twitch muscles , we dissected edl and soleus muscles from wt , casq1-null , and dcasq - null mice and studied their function ex vivo . from the stimulus to the tension peak ) in edl compared to wt , but also to casq1-null ( figure 5(a ) ) . no prolongation of time - to - peak tension was seen in soleus ( figure 5(b ) ) . , time elapsing from stimulus to the return to base line at the end of relaxation ) was significantly prolonged in both edl and soleus muscles of knock - out mice compared to wt . the latter effect was more pronounced in dcasq - null than in casq1-null mice ( figures 5(c ) and 5(d ) ) . isometric tension was determined in short fused isometric tetani with a duration just sufficient to reach peak tension ( 500 ms in edl and 1 s in soleus ) and found significantly reduced in edl , but not in soleus muscles of casq1-null and dcasq - null mice ( not shown ) . however , the most interesting result was obtained when the duration of the tetanus was prolonged up to 2 seconds ( figure 6 ) : in both casq1- and dcasq - null edl the residual tension declined dramatically ( by about 80% ) ( figures 6(a ) and 6(b ) ) , while in soleus only a minor decline of developed tension occurred in the absence of casq ( figures 6(c ) and 6(d ) ) . specifically , the tension decline of soleus during a 2 s tetanus was approximately 15% in casq1-null and 25% in dcasq - null . a careful inspection of the traces showed in figures 6(a ) and 6(c ) revealed that the kinetics of the rising phase of the isometric tetani were faster when casq is missing both in edl and in soleus . in agreement with the above observations , the amplitude of the twitch ( measured as peak tension , not shown ) was preserved , and the duration of the twitch ( measured as time - to - peak and time - to - base line , figure 5 ) was prolonged , thus allowing a faster tension development during repeated high frequency stimulation . two isoforms , casq1 and casq2 , are expressed in skeletal muscle fibers [ 18 , 19 ] , with casq1 and casq2 being more abundant in fast- and slow - twitch fibers , respectively . in view of the possible diversity in ca handling between slow and fast muscles and to investigate how complete ablation of casq will affect the two different muscle types , we studied edl and soleus muscle in mice lacking both casq isoforms . the comparison of the structural and functional effect of the complete ablation of casq revealed two main distinctive features : ( 1 ) soleus , but not edl , shows a number of fibers with signs of structural degeneration ( figure 4 ) ; ( 2 ) edl is unable to maintain active tension during a prolonged tetanus , while soleus is only marginally affected ( figure 6 ) . dcasq - null mice are viable , but present high frequency of spontaneous death in male mice , even higher than that previously registered in casq1-null animals ( figure 2 ) . the specific reason for the increased spontaneous mortality rate of dcasq - null mice is still under investigation , since cpvt in mice lacking casq2 is not lethal . the lack of a significant difference in grip strength between casq1- and dcasq - null mice probably reflects the fact that murine muscles show a great predominance of fast fibers expressing exclusively casq1 . in the present study we show that ~30% of soleus fibers from dcasq - null mice present clear evidence of the early onset of a myopathic phenotype , which is not as evident in edl fibers at the same age ( 46 months , figure 4 ) . these alterations , which resemble those described in other murine models of mh and central core disease ( ccd ) [ 36 , 37 ] , were not seen in soleus fibers of casq1-null mice , suggesting that the abundant expression of casq2 in slow fibers ( figure 1 ) prevents the onset of the pathology . this considered , the difference between the two muscles is that soleus develops a myopathy at an earlier stage . the reason can be tentatively found in the different capabilities of edl and soleus to deal with removal of ca from cytosol and with sr ca leak . the sr of slow fibers is saturated with ca at resting myoplasmic ca concentration , while the sr of fast fibers is only about one - third saturated with ca under equivalent conditions [ 21 , 39 ] . such difference implies that the rate of sr ca uptake in fast fibers is predominantly controlled by myoplasmic ca , while in slow fibers is more likely limited by the ca concentration within the sr lumen . the intraluminal ca concentration is likely increased when the buffering action of casq is missing in the soleus of dcasq - null mice , causing serious challenge to reuptake . , relaxation duration ) of soleus lacking both casq isoforms is in agreement with the impaired ca reuptake . the complete removal of casq in double - null mice decreases the ability of the sr to store ca both in fast and in slow muscle fibers . slow soleus fibers , but not fast edl fibers , are able to sustain tension during a prolonged contractions ( figure 6 ) , due to specific feature of the ca kinetics as discussed here below . the ryr - mediated ca release from the sr is approximately two times greater in fast compared to slow fibers . the larger size of crus and the higher density of ryr1 and dhpr in fast fibers is instrumental to this [ 40 , 41 ] . a greater ca release is needed in edl fast fibers compared to soleus slow fibers as the number of cytosolic ca binding sites is greater . in the first place , the troponin - c ( tnc ) isoform expressed in fast - twitch fibers presents two low affinity ca binding sites , whereas there is only one in the slow - twitch tnc isoform and , in addition , other cytosolic ca binding proteins must be saturated during the contractile cycle . among them , there are parvalbumin , present at a concentration of 400500 mol / liter in fast fibers but not in slow fibers and containing two ca binding sites , and serca1 , with a concentration of 120 mol / liter and two ca binding sites . during a tetanic train of stimuli , more ca enters the cytosol through repeated releases , although the amplitude of the subsequent releases progressively decreases with the fifth release being only 10% of the first release [ 40 , 43 ] . in order to sustain tension for more than one second , it is necessary to saturate also parvalbumin , a process occurring with a slower kinetics related to calcium replacement for magnesium : fast fibers deprived of casq fails to maintain a prolonged tetanic tension , probably because the ca released is not sufficient to saturate all cytosolic calcium - binding proteins , parvalbumin among them , with the result of reducing the ca available for tnc , and therefore for tension generation . conversely , a quite different scenario takes place in a typical slow fibers of soleus where the lower serca density , the single low binding site of slow tnc , and , above all , the absence of parvalbumin would prevent the tension decline observed in edl . in this contest , very likely the minor reduction ( ~25% ) in tetanic tension recorded in dcasq - null soleus ( figures 6(c ) and 6(d ) ) could be almost completely ascribed to the presence of damaged fibers , which likely have compromised contractile function , and not to an insufficient amount of ca to activate contraction . whereas in edl casq is necessary to provide the large amount of ca required for a maximal sustained contraction , slow - twitch fibers are only moderately affected by the absence of casq during prolonged tetani . however , casq presence in soleus seems necessary to help ca reuptake , reduce ca leakage , and control myoplasmic ca , which otherwise will eventually lead to the early onset of a myopathy . in this aspect , slow fibers are reminiscent of cardiac myocytes where casq role is essential to control diastolic ca leakage . these findings add novel information , which may help to better understand the differences in ca handling of fast and slow fibers and also offer new insights to unlock mechanisms leading to myopathies such as mh and ccd .

the first indication that sulfur amino acid metabolism is linked to atherosclerosis came from observations in 1953 demonstrating that pathogenic cholesterol concentrations and experimental atherogenesis in monkeys can be inhibited by dietary methionine . since the early 60s elevated hcy levels in blood ( hyperhomocysteinemia ) caused by different deficiencies of sulfur amino acid metabolism were reported to be associated with vascular disease and , in particular , with atherosclerotic plaque formation [ 2 , 3 ] . today , hcy is recognized by many studies as a strong , independent and causal risk factor for atherosclerosis [ 48 ] , although there is still controversy on the underlying metabolic connections . in addition to its association with vascular diseases , hcy is also linked to neurological disorders , aging , and all - cause mortality . understanding the pathological mechanisms triggered by hcy is , therefore , essential for understanding its role in several disease states . numerous mechanisms have been proposed that explain pathological changes associated with elevated hcy levels ( reviewed in ) . several of them , for example , protein homocysteinylation and oxidative stress , are directly triggered by hcy . however , not hcy , but rather adohcy , an immediate precursor of hcy ( figure 1 ) , emerged as a more sensitive indicator of cardiovascular disease during the last decade [ 13 , 14 ] . supporting the potentially pathogenic role of adohcy , studies in yeast showed that indeed adohcy is more toxic than hcy to cells that are deficient in hcy catabolism . adohcy is synthesized as a universal byproduct of adomet - dependent methyltransferase reactions ( figure 1 ) . it is a strong competitive inhibitor of many adomet - dependent methyltransferases and , therefore , has to be removed to sustain these reactions . the only eukaryotic enzyme capable of adohcy catabolism , s - adenosyl - l - homocysteine hydrolase ( sah1 in yeast , ahcy in mammals ) , catalyzes the reversible hydrolysis of adohcy to hcy and adenosine . the equilibrium of s - adenosyl - l - homocysteine hydrolase - catalyzed reaction lies far in the direction of synthesis , and both hcy and adenosine have to be quickly metabolized in order to drive the net hydrolysis of adohcy . therefore , accumulation of hydrolytic products of the s - adenosyl - l - homocysteine hydrolase - catalyzed reaction , in particular hcy , results in adohcy synthesis and accumulation showing that adohcy is not only the precursor , but also the product of hcy metabolism in vivo [ 1820 ] . changes at the epigenetic level are the most extensively studied consequences of methylation deficiency [ 2124 ] . however , phospholipid methylation that requires three sequential adomet - dependent methylation steps to synthesize one molecule of phosphatidylcholine ( pc ) from phosphatidylethanolamine ( pe ) , the predominant way for pc synthesis in yeast , in particular , in the absence of choline and ethanolamine in the culture medium , is the major consumer of adomet . phospholipid methylation is also the major consumer of adomet in mice , since the loss of phosphatidylethanolamine n - methyltransferase ( pemt ) in pemt knockout mice leads to a 50% decrease in plasma hcy levels . reexamination of methylation metabolism in humans also revealed that phospholipid methylation , but not creatine synthesis , as was assumed previously , accounts for the major part of adomet being utilized in the human body . while pe methylation is the predominant way to synthesize phospholipids in yeast , phospholipid synthesis by the de novo methylation pathway is primarily present in the liver in mammals , where it constitutes 30% of pc production and account for estimated 10 mol and 1,65 mmol pemt - derived pc secreted into bile per day in mice and humans , respectively [ 26 , 27 ] . however , other mammalian tissues and cells are also capable of phospholipid methylation including brain , skeletal muscle , adipose tissues , fibroblasts , arterial smooth muscle cells , endothelial cells , macrophages , and erythrocytes [ 2837 ] . the evolutionary conservation of phospholipid methylation suggests its essential role in some specific functions in different cell types . for instance , phospholipid methylation is enhanced in hypertrophied myocardium , correlates with the level of -adrenergic receptors [ 38 , 39 ] and is stimulated by isoproterenol , a potent cardiac stimulant . in contrast , phospholipid methylation is inhibited by quinidine , an antiarrhythmic drug that causes repression of myocardial contractility . phospholipid methylation was also observed in microsome preparations from aorta [ 42 , 43 ] and was suggested to affect membrane fluidity and function of membrane calcium channels in aorta [ 42 , 43 ] as well as in heart . moreover , phospholipid methylation appears to be coupled to ca influx and von willebrand factor release in endothelial cells . in accordance , it was shown that increased methylation of phospholipids is required for an influx of ca and subsequent release of histamine in mast cells . furthermore , ca influx was correlated with the release of arachidonic acid in rabbit neutrophils and human fibroblasts , which also appears to require phospholipid methylation [ 32 , 45 ] . requirement of phospholipid methylation for polyunsaturated fatty acid metabolism was also observed in the brain . it was reported that developing , remyelinating , and diabetic brain exhibits increased synthesis of pc by the de novo methylation pathway in comparison with normal adult brain [ 47 , 48 ] . phospholipid methylation was shown to be linked to diabetes [ 4951 ] and neurological disorders [ 52 , 53 ] also in other studies . it was shown very recently that phospholipid methylation is required for lipid droplet formation and stability in 3t3-l1 adipocytes , and high - fat challenge induces pemt expression in adipose tissue . moreover , pemt and the cdp - choline pathway for pc synthesis are both required for the secretion of very - low - density lipoproteins [ 5557 ] . while cells lacking the rate - limiting enzyme of the cdp - choline pathway , ctp : phosphocholine cytidylyltransferase , do not survive , deficiency of phospholipid methylation in pemt mice under choline deprivation results in development of hepatic steatosis followed by steatohepatitis and hyperacute liver failure and is lethal within 5 days . moreover , deficiency in phospholipid methylation , but not in the synthesis of pc by the cdp - choline pathway , protects from diet - induced obesity in mice due to increased energy utilization suggesting that pemt plays a role in whole energy metabolism and is linked to insulin signaling . finally , an isoform of phosphatidylethanolamine n - methyltransferase , pemt2 , appears to be involved in the control of hepatocyte cell division , since its inactivation is associated with several types of liver cell proliferation including tumorigenesis . sensitivity of phospholipid methylation to adohcy accumulation [ 16 , 18 , 63 ] as well as numerous correlations reported for phospholipid methylation pathway suggests that interference with this reaction in hcy - associated pathology may lead to widespread defects , what indeed seems to be the case . in particular , elevated hcy levels were found to trigger deregulation of lipid metabolism in yeast and mammalian cells [ 18 , 64 ] . a mechanism of deregulation of lipid metabolism and lipid - associated cellular functions in hyperhomocysteinemia mediated by adohcy accumulation and subsequent inhibition of phospholipid methylation homocysteine is a sulfur - containing amino acid , which does not occur in proteins , but is found at the intersection of methylation and transsulfuration metabolism ( figure 1 , reviewed in ) . hcy is formed during methionine metabolism by s - adenosyl - l - homocysteine hydrolase that catalyzes the reversible hydrolysis of adohcy to hcy and adenosine . to be kept in the methylation cycle , hcy has to be remethylated to methionine , which can be further activated to adomet and used by over 50 adomet - dependent methyltransferases that release adohcy as a by - product after the methyl transfer reaction . the ratio of adomet to adohcy , that is , the ratio of the substrate versus the specific inhibitor of adomet - dependent methyltransferases , is indicative of the cellular methylation potential . in addition to its remethylation to methionine , hcy can be subjected to transsulfuration leading to the synthesis of cysteine , which is also a precursor of glutathione , an essential cellular defense molecule in oxidative stress response . an alternative way for hcy metabolism is the reversal of the reaction catalyzed by s - adenosyl - l - homocysteine hydrolase . this occurs upon accumulation of the hydrolytic products of the reaction , in particular hcy , and leads to adohcy synthesis and accumulation [ 19 , 6769 ] . thus , elevated hcy levels via accumulation of adohcy lead to the disruption of the methylation cycle and , potentially , to methylation deficiency . deficiency in cystathionine -synthase ( cbs ) , the first and rate - limiting enzyme of the transsulfuration pathway ( figure 1 ) , is the major cause of severe hyperhomocysteinemia followed by genetic defects of folate and cobalamin metabolism that is involved in hcy remethylation . these pathological conditions lead to the plasma hcy levels of more than 100 mol / l and are rare in comparison with mild hyperhomocysteinemia that is caused by dietary deficiencies of the vitamin cofactors required for hcy catabolism - folic acid , vitamins b6 and b12 , and characterized by the plasma hcy levels of 1525 folic acid and vitamin b6 are required for the activity of methionine synthase catalyzing 5-methyltetrahydrofolate - dependent remethylation of hcy to methionine ( figure 1 ) . while vitamin supplementation appeared to be a straightforward strategy to reduce however , it was observed that vitamins , while capable of lowering elevated plasma hcy levels , do not reduce the rates of vascular events . several potential mechanisms that might explain this result by offsetting the positive effect of hcy - lowering therapy were subsequently proposed . these include promotion of cell proliferation by folic acid through its role in the synthesis of thymidine , increase of the methylation potential leading to changes in gene expression , and increase in the levels of asymmetric dimethylarginine that inhibit the activity of nitric oxide synthase . an additional possibility is that , not hcy , but rather a related metabolite could be a trigger of some pathological changes associated with elevated hcy levels . possibly explaining the failure of hcy - lowering vitamins to reduce vascular events , it was recently reported that supplementation with b - vitamins including folate does not efficiently lower plasma adohcy levels , presumably due to elevation of adomet - dependent methylation . in yeast , the synthesis of pc from pe by the de novo phospholipid methylation pathway is particularly sensitive to adohcy accumulation [ 18 , 63 ] . both inhibition of s - adenosyl - l - homocysteine hydrolase and hcy supplementation results in adohcy accumulation and inhibition of phospholipid methylation in yeast . however , not only phospholipid methylation , but also a methylation - independent branch of lipid metabolism , namely , tag synthesis , is affected by adohcy accumulation in yeast : yeast cells deficient in adohcy catabolism or supplemented with hcy massively accumulate tag . supporting the causal role of impaired phospholipid methylation in the deregulation of tag metabolism in response to adohcy accumulation , it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of pe to pc , cho2 and opi3 , also accumulate tag . therefore , tag accumulation under these conditions suggests accumulation of fatty acids and their redirection from phospholipid to tag synthesis in methylation deficiency in yeast . in addition to tag metabolism , transcriptional regulation of phospholipid biosynthesis is also affected in yeast mutants deficient in adohcy catabolism . impaired phospholipid methylation in sah1-depleted cells unable to hydrolyze adohcy or in cho2 and opi3 mutants leads to upregulation of genes , which have an inositol - sensitive upstream regulatory sequence ( uasino ) in their promoter regions , indicating accumulation of the phospholipid precursor , phosphatidic acid , in the er . acc1 encoding acetyl - coa carboxylase , the first and rate - limiting enzyme of fatty acid biosynthesis , is also a subject to uasino - mediated regulation , suggesting upregulation of the de novo fatty acid biosynthesis in response to adohcy accumulation . moreover , sah1 depletion also affects sterol synthesis in yeast , leading to 4-fold elevated squalene levels and suggesting accumulation of early precursors of ergosterol biosynthesis under these conditions ( tehlivets , kohlwein , unpublished ) . taken together , inhibition of phospholipid methylation induced by adohcy accumulation appears to lead to upregulation of fatty acid , tag , and sterol biosynthetic pathways in yeast . adohcy inhibits phosphatidylethanolamine n - methyltransferase in vitro and in vivo also in mammals [ 28 , 37 , 74 ] . similarly as in yeast , deficiency of phospholipid methylation in pemt knockout mice leads to a rapid decrease of the hepatic pc / pe ratio and accumulation of tag in the liver , in the absence of choline supplementation . however , tag accumulation in the livers of these animals appears to be at least in part due to decreased tag secretion from hepatocytes . elevated levels of hcy are as well linked to deregulation of lipid metabolism in mammals . cbs knockout mice exhibit severe hyperhomocysteinemia and accumulate adohcy in all tissues tested [ 68 , 69 ] . these mutant animals show elevated tag and nonesterified fatty acid levels in the liver and serum and develop hepatic steatosis [ 76 , 77 ] . another genetic disorder that results in moderately elevated hcy levels , methylenetetrahydrofolate reductase ( mthfr ) deficiency , leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [ 78 , 79 ] . dietary - induced hyperhomocysteinemia in mice also causes fatty liver , further supporting the role of hcy in deregulation of lipid metabolism in mammals . in these mice as well as in the cbs knockout mice lipid accumulates in liver rather than in serum [ 64 , 76 ] . preferable accumulation of lipids in the liver and , possibly , other tissues in hyperhomocysteinemia suggests that other mechanisms than those associated with elevation of circulating lipids are responsible for the development of cardiovascular disease under these conditions . indeed , conventional risk factors including hypercholesterolemia accounts only for approximately 50% of all cases of cardiovascular disease , while 40% of patiens diagnosed with premature coronary artery disease , peripheral vascular disease or venous thrombosis exhibit hyperhomocysteinemia . in accordance , unlike typical lipid - rich atherosclerotic plagues , vascular lesions associated with hyperhomocysteinemia are lipid - poor , fibrous plaques [ 81 , 82 ] , greatly outnumbering fatty atherosclerotic lesions . in animal models of hyperhomocysteinemia atherosclerotic lesions are rare . they are found only in the mthfr knockout mice that exhibit aortic lipid accumulation reminiscent of early atherosclerotic lesions [ 2 , 78 , 83 ] , however , not in , for example , cbs knockout mice . this discrepancy might be due to disruption of two different hcy utilizing pathways in these animals . while 5-methyltetrahydrofolate - dependent hcy remethylation occurs in all mammalian cells , transsulfuration of hcy occurs primarily in the liver and kidney . thus , impairment of 5-methyltetrahydrofolate - dependent hcy remethylation in mthfr knockout mice may differently affect hcy metabolism in comparison to the deficiency in the first step of hcy transsulfuration in cbs knockout mice . the observation that dietary ( methionine or hcy supplementation ) or genetically ( cbs gene deletion ) induced hyperhomocysteinemia in apoe - deficient ( apoe ) mice leads to development of larger and more advanced atherosclerotic lesions clearly demonstrates a causal relationship between elevated hcy levels and atherosclerosis . in contrast , lack of pemt was shown to reduce significantly plasma vldl and to attenuate atherosclerosis in both pemt / ldlr mice deficient in pemt and ldl receptors as well as in pemt / apoe mice [ 84 , 85 ] . elevated hcy levels induce endoplasmic reticulum ( er ) stress and activate the unfolded protein response ( upr ) in a variety of mammalian cells . these include cultured human hepatocytes , vascular endothelial and aortic smooth muscle cells as well as liver cells of the cbs knockout mice [ 64 , 8688 ] . furthermore , elevated hcy levels lead to activation of the sterol regulatory element - binding proteins ( srebps ) , which function to activate genes encoding enzymes in cholesterol , fatty acid , and tag metabolism and uptake , both in cultured mammalian cell lines as well as in the livers of the cbs knockout mice [ 64 , 86 ] . er stress appears to play a direct role in the activation of tag and cholesterol biosynthesis , since overexpression of the er chaperone grp78/bip was reported to inhibit hcy - induced srebp-1 gene expression in mammalian cell cultures as well as in mice and lead to reduction of the hepatic steatosis in leptin - deficient ( ob / ob ) mice . srebp-1 overcomes translation inhibition induced by upr through an internal ribosome entry site ( irs ) , similarly to grp78 . confirming the causal role of hcy in upr induction and deregulation of lipid metabolism , a decrease of elevated plasma hcy levels is accompanied by a decrease in hepatic lipids and er stress response . a strong correlation between lipid metabolism , er stress response and elevated hcy levels is also evident form a literature mining approach . further demonstration of close relationship between er stress and hcy metabolism came from the observation that mthfr involved in hcy remethylation is induced in response to er stress . evolutionary conservation of the relationship between hcy and upr is shown by the induction of er stress and activation of upr in response to hcy supplementation in yeast . finally , demonstrating its pathophysiological role , er stress was shown to be strongly associated with accelerated atherosclerosis in hyperhomocysteinemic apoe - deficient mice , liver diseases as well as hyperglycemia - induced atherosclerosis . the upr , as a conserved cellular stress response pathway , is aimed at restoring normal er and secretory function as well as membrane trafficking upon impaired protein folding in the er . the de novo methylation and the cdp - choline phospholipid biosynthetic pathways produce phospholipid species with distinct fatty acyl chain composition in yeast : the de novo phospholipid methylation pathway produces more unsaturated phospholipids [ 98 , 99 ] . similarly , the pemt - generated pc pool in mammals is also enriched in unsaturated fatty acids [ 100 , 101 ] . supporting the role of pemt in metabolism of unsaturated fatty acids pem knockout mice were reported to accumulate more saturated pc molecular species in the liver compared with the control littermates and to exhibit dramatically reduced concentrations of polyunsaturated fatty acids in the plasma and in hepatic pc , independently of choline status . thus , beyond its role as a compensatory pathway for pc biosynthesis under conditions of choline deprivation , phospholipid methylation plays a crucial role in unsaturated fatty acid metabolism both in yeast and in mammals . the observation that deficiency of phospholipid methylation in cho2 and opi3 yeast mutants is synthetically lethal in the absence of a functional upr suggests an essential requirement of upr in response to impaired phospholipid methylation . thus , hcy accumulation , which was shown to lead to adohcy - mediated inhibition of phospholipid methylation in yeast , may lead to accumulation of saturated fatty acids in membrane phospholipids a potential pathological mechanism that might be shared by both yeast and mammals . indeed , accumulation of saturated fatty acids in membrane phospholipids interferes with er structure and integrity , induces er stress and leads to cell death in mammalian cell cultures [ 104 , 105 ] . accordingly , decreased membrane phospholipid desaturation due to stearoyl - coa desaturase 1 knockdown induces upr in hela cells . vice versa , overexpression of stearoyl - coa desaturase attenuates palmitate - induced er stress and protects from lipoapoptosis [ 107109 ] . treatment with the molecular chaperone 4-phenyl butyrate , which is capable of stabilization of protein conformation , improvement of er folding capacity and facilitation of protein trafficking , leads to abolishment of upr induction in yeast subjected to lipid - induced er stress . this finding suggests that accumulation of saturated fatty acids in membrane phospholipids first leads to changes in the membrane environment followed by induction of er stress and accumulation of misfolded protein(s ) that , in turn , activate upr . potential mechanisms involved in saturated fatty acid - induced upr include depletion of er ca stores leading to decreased er chaperone activity and protein misfolding , as well as interference with er - to - golgi trafficking . recently , proteomic studies showed that carboxypeptidase e , a key enzyme involved in processing and sorting of insulin , is involved in induction of er stress in -cells in response to palmitate treatment . degradation of carboxypeptidase e in palmitate - induced er stress is mediated by palmitate metabolism and ca flux . , atf6 , and perk or modulate the binding of the er sensors to the er chaperone grp78 causing its dissociation and activation of upr pathways . supporting the hypothesis that hcy interferes with phospholipid acyl chain composition it was observed in humans that elevated plasma adohcy levels are negatively correlated with both pc content and the level of polyunsaturated fatty acids in pc , but not in pe , in red blood cells in alzheimer 's patients . elevated plasma hcy levels were also shown to be associated with a decrease in polyunsaturated ( docosahexaenoic ) fatty acids in the plasma of healthy humans and in the plasma and erythrocytes of cystic fibrosis patients ; these individuals exhibit increased hcy and adohcy levels as well as altered pe and pc metabolism . taken together , deficiency of phospholipid methylation caused by adohcy accumulation in hcy - associated pathology appears to lead to an increase in saturated pc molecular species in er membranes followed by er stress , protein misfolding , induction of upr , and activation of lipid metabolism ( figure 2 ) . upregulation of lipid biosynthesis , which apparently should serve to compensate for suboptimal composition of membrane lipids , leads , however , to accumulation of fatty acids , tag and sterols in the absence of functional phospholipid methylation . in addition to a role in formation of a specific pool of pc molecular species , phospholipid methylation is crucial for maintenance of a distinct pc / pe ratio important for cell integrity when dietary choline - supply is blunted . decrease of the pc / pe ratio was reported to result in increased cell permeability of hepatocytes from pemt mice fed choline deficient diet leading to liver damage . similarly , decrease of pc or increase in pe was shown to lead to cell damage and/or death in several other mammalian cell types [ 117119 ] . finally , observation of different outcomes in hyperhomocysteinemic apoe mice and both in pemt / ldlr and pemt / apoe mice suggests mechanisms besides inhibition of phospholipid methylation , for example , adohcy - dependent modulation of gene expression that may also contribute to the development of hcy - dependent atherosclerosis . the metabolism of homocysteine , consequences of its accumulation as well as associated adohcy - triggered inhibition of adomet - dependent methylation are complex . in this paper a novel mechanism of hcy - triggered deregulation of lipid metabolism and upr induction that is mediated through an adohcy - dependent inhibition of phospholipid methylation and based on experimental evidence derived from both yeast and mammalian systems is proposed . s - adenosyl - l - homocysteine hydrolase is recognized since many years as a target for antiviral drug design . inhibitors that block adohcy hydrolysis are efficient against many types of viruses including ebola and show other effects of pharmacological importance [ 120122 ] . however , they are associated with high cytotoxicity due to interference with central metabolic pathways [ 121 , 122 ] . while using these inhibitors to study the effects of adohcy accumulation appears to be straightforward , ability of some of nucleoside inhibitors of s - adenosyl - l - homocysteine hydrolase to undergo metabolic phosphorylation to nucleotides may account for a part of their biological activities [ 121 , 122 ] . inhibitors that would be able to block selectively homocysteine and adenosine conversion to adohcy are not available . however , based on current understanding of regulation of homocysteine and methionine metabolism ( i ) selective blockage of adohcy synthesis from homocysteine and adenosine that will relieve not only inhibition of phospholipid methylation but also many other adomet - dependent methyltransferase reactions , combined with ( ii ) vitamin b6 supplementation in order to accelerate homocystein catabolism by transsulfuration pathway , may serve as a way to reduce adohcy in hyperhomocysteinemia without elevation of adomet . is adohcy - mediated accumulation of saturated fatty acids in membrane lipids indeed the way by which elevated hcy levels induce er stress ? what role do specific lipid precursors have in regulation of lipid metabolism in upr ? what is the impact of adohcy accumulation on other methylation reactions unrelated to lipid metabolism ? what is the role of deficient phospholipid methylation in homocysteine - associated pathology beyond deregulation of fatty acid , tag , sterol metabolism , and upr induction ? elucidation of the molecular mechanisms triggered by elevated hcy levels will undoubtfully improve our understanding of its pathological role in numerous diseases .

homocysteine ( hcy ) has been recognized for the past five decades as a risk factor for atherosclerosis . however , the role of hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by hcy accumulation is poorly understood . due to the reversal of the physiological direction of the reaction catalyzed by s - adenosyl - l - homocysteine hydrolase hcy accumulation leads to the synthesis of s - adenosyl - l - homocysteine ( adohcy ) . adohcy is a strong product inhibitor of s - adenosyl - l - methionine ( adomet)-dependent methyltransferases , and to date more than 50 adomet - dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids , proteins and lipids have been identified . phospholipid methylation is the major consumer of adomet , both in mammals and in yeast . adohcy accumulation induced either by hcy supplementation or due to s - adenosyl - l - homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast . moreover , yeast cells accumulating adohcy also massively accumulate triacylglycerols ( tag ) . similarly , hcy supplementation was shown to lead to increased tag and sterol synthesis as well as to the induction of the unfolded protein response ( upr ) in mammalian cells . in this review a model of deregulation of lipid metabolism in response to accumulation of adohcy in hcy - associated pathology is proposed .

1. Introduction 2. Role of Homocysteine in the Methylation Cycle 3. AdoHcy-Triggered Deregulation of Lipid Metabolism in Yeast 4. Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals 5. Role of the Unfolded Protein Response in Hyperhomocysteinemia and Atherosclerosis 6. Deregulation of Fatty Acid Metabolism in Response to AdoHcy Accumulation 7. Concluding Remarks

today , hcy is recognized by many studies as a strong , independent and causal risk factor for atherosclerosis [ 48 ] , although there is still controversy on the underlying metabolic connections . understanding the pathological mechanisms triggered by hcy is , therefore , essential for understanding its role in several disease states . numerous mechanisms have been proposed that explain pathological changes associated with elevated hcy levels ( reviewed in ) . supporting the potentially pathogenic role of adohcy , studies in yeast showed that indeed adohcy is more toxic than hcy to cells that are deficient in hcy catabolism . adohcy is synthesized as a universal byproduct of adomet - dependent methyltransferase reactions ( figure 1 ) . it is a strong competitive inhibitor of many adomet - dependent methyltransferases and , therefore , has to be removed to sustain these reactions . the only eukaryotic enzyme capable of adohcy catabolism , s - adenosyl - l - homocysteine hydrolase ( sah1 in yeast , ahcy in mammals ) , catalyzes the reversible hydrolysis of adohcy to hcy and adenosine . the equilibrium of s - adenosyl - l - homocysteine hydrolase - catalyzed reaction lies far in the direction of synthesis , and both hcy and adenosine have to be quickly metabolized in order to drive the net hydrolysis of adohcy . therefore , accumulation of hydrolytic products of the s - adenosyl - l - homocysteine hydrolase - catalyzed reaction , in particular hcy , results in adohcy synthesis and accumulation showing that adohcy is not only the precursor , but also the product of hcy metabolism in vivo [ 1820 ] . however , phospholipid methylation that requires three sequential adomet - dependent methylation steps to synthesize one molecule of phosphatidylcholine ( pc ) from phosphatidylethanolamine ( pe ) , the predominant way for pc synthesis in yeast , in particular , in the absence of choline and ethanolamine in the culture medium , is the major consumer of adomet . phospholipid methylation is also the major consumer of adomet in mice , since the loss of phosphatidylethanolamine n - methyltransferase ( pemt ) in pemt knockout mice leads to a 50% decrease in plasma hcy levels . reexamination of methylation metabolism in humans also revealed that phospholipid methylation , but not creatine synthesis , as was assumed previously , accounts for the major part of adomet being utilized in the human body . while pe methylation is the predominant way to synthesize phospholipids in yeast , phospholipid synthesis by the de novo methylation pathway is primarily present in the liver in mammals , where it constitutes 30% of pc production and account for estimated 10 mol and 1,65 mmol pemt - derived pc secreted into bile per day in mice and humans , respectively [ 26 , 27 ] . however , other mammalian tissues and cells are also capable of phospholipid methylation including brain , skeletal muscle , adipose tissues , fibroblasts , arterial smooth muscle cells , endothelial cells , macrophages , and erythrocytes [ 2837 ] . phospholipid methylation was also observed in microsome preparations from aorta [ 42 , 43 ] and was suggested to affect membrane fluidity and function of membrane calcium channels in aorta [ 42 , 43 ] as well as in heart . requirement of phospholipid methylation for polyunsaturated fatty acid metabolism was also observed in the brain . it was shown very recently that phospholipid methylation is required for lipid droplet formation and stability in 3t3-l1 adipocytes , and high - fat challenge induces pemt expression in adipose tissue . while cells lacking the rate - limiting enzyme of the cdp - choline pathway , ctp : phosphocholine cytidylyltransferase , do not survive , deficiency of phospholipid methylation in pemt mice under choline deprivation results in development of hepatic steatosis followed by steatohepatitis and hyperacute liver failure and is lethal within 5 days . moreover , deficiency in phospholipid methylation , but not in the synthesis of pc by the cdp - choline pathway , protects from diet - induced obesity in mice due to increased energy utilization suggesting that pemt plays a role in whole energy metabolism and is linked to insulin signaling . sensitivity of phospholipid methylation to adohcy accumulation [ 16 , 18 , 63 ] as well as numerous correlations reported for phospholipid methylation pathway suggests that interference with this reaction in hcy - associated pathology may lead to widespread defects , what indeed seems to be the case . in particular , elevated hcy levels were found to trigger deregulation of lipid metabolism in yeast and mammalian cells [ 18 , 64 ] . a mechanism of deregulation of lipid metabolism and lipid - associated cellular functions in hyperhomocysteinemia mediated by adohcy accumulation and subsequent inhibition of phospholipid methylation homocysteine is a sulfur - containing amino acid , which does not occur in proteins , but is found at the intersection of methylation and transsulfuration metabolism ( figure 1 , reviewed in ) . hcy is formed during methionine metabolism by s - adenosyl - l - homocysteine hydrolase that catalyzes the reversible hydrolysis of adohcy to hcy and adenosine . to be kept in the methylation cycle , hcy has to be remethylated to methionine , which can be further activated to adomet and used by over 50 adomet - dependent methyltransferases that release adohcy as a by - product after the methyl transfer reaction . the ratio of adomet to adohcy , that is , the ratio of the substrate versus the specific inhibitor of adomet - dependent methyltransferases , is indicative of the cellular methylation potential . in addition to its remethylation to methionine , hcy can be subjected to transsulfuration leading to the synthesis of cysteine , which is also a precursor of glutathione , an essential cellular defense molecule in oxidative stress response . an alternative way for hcy metabolism is the reversal of the reaction catalyzed by s - adenosyl - l - homocysteine hydrolase . this occurs upon accumulation of the hydrolytic products of the reaction , in particular hcy , and leads to adohcy synthesis and accumulation [ 19 , 6769 ] . thus , elevated hcy levels via accumulation of adohcy lead to the disruption of the methylation cycle and , potentially , to methylation deficiency . deficiency in cystathionine -synthase ( cbs ) , the first and rate - limiting enzyme of the transsulfuration pathway ( figure 1 ) , is the major cause of severe hyperhomocysteinemia followed by genetic defects of folate and cobalamin metabolism that is involved in hcy remethylation . these pathological conditions lead to the plasma hcy levels of more than 100 mol / l and are rare in comparison with mild hyperhomocysteinemia that is caused by dietary deficiencies of the vitamin cofactors required for hcy catabolism - folic acid , vitamins b6 and b12 , and characterized by the plasma hcy levels of 1525 folic acid and vitamin b6 are required for the activity of methionine synthase catalyzing 5-methyltetrahydrofolate - dependent remethylation of hcy to methionine ( figure 1 ) . these include promotion of cell proliferation by folic acid through its role in the synthesis of thymidine , increase of the methylation potential leading to changes in gene expression , and increase in the levels of asymmetric dimethylarginine that inhibit the activity of nitric oxide synthase . an additional possibility is that , not hcy , but rather a related metabolite could be a trigger of some pathological changes associated with elevated hcy levels . possibly explaining the failure of hcy - lowering vitamins to reduce vascular events , it was recently reported that supplementation with b - vitamins including folate does not efficiently lower plasma adohcy levels , presumably due to elevation of adomet - dependent methylation . in yeast , the synthesis of pc from pe by the de novo phospholipid methylation pathway is particularly sensitive to adohcy accumulation [ 18 , 63 ] . both inhibition of s - adenosyl - l - homocysteine hydrolase and hcy supplementation results in adohcy accumulation and inhibition of phospholipid methylation in yeast . however , not only phospholipid methylation , but also a methylation - independent branch of lipid metabolism , namely , tag synthesis , is affected by adohcy accumulation in yeast : yeast cells deficient in adohcy catabolism or supplemented with hcy massively accumulate tag . supporting the causal role of impaired phospholipid methylation in the deregulation of tag metabolism in response to adohcy accumulation , it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of pe to pc , cho2 and opi3 , also accumulate tag . impaired phospholipid methylation in sah1-depleted cells unable to hydrolyze adohcy or in cho2 and opi3 mutants leads to upregulation of genes , which have an inositol - sensitive upstream regulatory sequence ( uasino ) in their promoter regions , indicating accumulation of the phospholipid precursor , phosphatidic acid , in the er . acc1 encoding acetyl - coa carboxylase , the first and rate - limiting enzyme of fatty acid biosynthesis , is also a subject to uasino - mediated regulation , suggesting upregulation of the de novo fatty acid biosynthesis in response to adohcy accumulation . moreover , sah1 depletion also affects sterol synthesis in yeast , leading to 4-fold elevated squalene levels and suggesting accumulation of early precursors of ergosterol biosynthesis under these conditions ( tehlivets , kohlwein , unpublished ) . taken together , inhibition of phospholipid methylation induced by adohcy accumulation appears to lead to upregulation of fatty acid , tag , and sterol biosynthetic pathways in yeast . similarly as in yeast , deficiency of phospholipid methylation in pemt knockout mice leads to a rapid decrease of the hepatic pc / pe ratio and accumulation of tag in the liver , in the absence of choline supplementation . however , tag accumulation in the livers of these animals appears to be at least in part due to decreased tag secretion from hepatocytes . elevated levels of hcy are as well linked to deregulation of lipid metabolism in mammals . another genetic disorder that results in moderately elevated hcy levels , methylenetetrahydrofolate reductase ( mthfr ) deficiency , leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [ 78 , 79 ] . dietary - induced hyperhomocysteinemia in mice also causes fatty liver , further supporting the role of hcy in deregulation of lipid metabolism in mammals . in these mice as well as in the cbs knockout mice lipid accumulates in liver rather than in serum [ 64 , 76 ] . preferable accumulation of lipids in the liver and , possibly , other tissues in hyperhomocysteinemia suggests that other mechanisms than those associated with elevation of circulating lipids are responsible for the development of cardiovascular disease under these conditions . while 5-methyltetrahydrofolate - dependent hcy remethylation occurs in all mammalian cells , transsulfuration of hcy occurs primarily in the liver and kidney . thus , impairment of 5-methyltetrahydrofolate - dependent hcy remethylation in mthfr knockout mice may differently affect hcy metabolism in comparison to the deficiency in the first step of hcy transsulfuration in cbs knockout mice . in contrast , lack of pemt was shown to reduce significantly plasma vldl and to attenuate atherosclerosis in both pemt / ldlr mice deficient in pemt and ldl receptors as well as in pemt / apoe mice [ 84 , 85 ] . elevated hcy levels induce endoplasmic reticulum ( er ) stress and activate the unfolded protein response ( upr ) in a variety of mammalian cells . these include cultured human hepatocytes , vascular endothelial and aortic smooth muscle cells as well as liver cells of the cbs knockout mice [ 64 , 8688 ] . furthermore , elevated hcy levels lead to activation of the sterol regulatory element - binding proteins ( srebps ) , which function to activate genes encoding enzymes in cholesterol , fatty acid , and tag metabolism and uptake , both in cultured mammalian cell lines as well as in the livers of the cbs knockout mice [ 64 , 86 ] . er stress appears to play a direct role in the activation of tag and cholesterol biosynthesis , since overexpression of the er chaperone grp78/bip was reported to inhibit hcy - induced srebp-1 gene expression in mammalian cell cultures as well as in mice and lead to reduction of the hepatic steatosis in leptin - deficient ( ob / ob ) mice . confirming the causal role of hcy in upr induction and deregulation of lipid metabolism , a decrease of elevated plasma hcy levels is accompanied by a decrease in hepatic lipids and er stress response . further demonstration of close relationship between er stress and hcy metabolism came from the observation that mthfr involved in hcy remethylation is induced in response to er stress . evolutionary conservation of the relationship between hcy and upr is shown by the induction of er stress and activation of upr in response to hcy supplementation in yeast . finally , demonstrating its pathophysiological role , er stress was shown to be strongly associated with accelerated atherosclerosis in hyperhomocysteinemic apoe - deficient mice , liver diseases as well as hyperglycemia - induced atherosclerosis . the upr , as a conserved cellular stress response pathway , is aimed at restoring normal er and secretory function as well as membrane trafficking upon impaired protein folding in the er . similarly , the pemt - generated pc pool in mammals is also enriched in unsaturated fatty acids [ 100 , 101 ] . supporting the role of pemt in metabolism of unsaturated fatty acids pem knockout mice were reported to accumulate more saturated pc molecular species in the liver compared with the control littermates and to exhibit dramatically reduced concentrations of polyunsaturated fatty acids in the plasma and in hepatic pc , independently of choline status . thus , beyond its role as a compensatory pathway for pc biosynthesis under conditions of choline deprivation , phospholipid methylation plays a crucial role in unsaturated fatty acid metabolism both in yeast and in mammals . the observation that deficiency of phospholipid methylation in cho2 and opi3 yeast mutants is synthetically lethal in the absence of a functional upr suggests an essential requirement of upr in response to impaired phospholipid methylation . thus , hcy accumulation , which was shown to lead to adohcy - mediated inhibition of phospholipid methylation in yeast , may lead to accumulation of saturated fatty acids in membrane phospholipids a potential pathological mechanism that might be shared by both yeast and mammals . indeed , accumulation of saturated fatty acids in membrane phospholipids interferes with er structure and integrity , induces er stress and leads to cell death in mammalian cell cultures [ 104 , 105 ] . this finding suggests that accumulation of saturated fatty acids in membrane phospholipids first leads to changes in the membrane environment followed by induction of er stress and accumulation of misfolded protein(s ) that , in turn , activate upr . , atf6 , and perk or modulate the binding of the er sensors to the er chaperone grp78 causing its dissociation and activation of upr pathways . elevated plasma hcy levels were also shown to be associated with a decrease in polyunsaturated ( docosahexaenoic ) fatty acids in the plasma of healthy humans and in the plasma and erythrocytes of cystic fibrosis patients ; these individuals exhibit increased hcy and adohcy levels as well as altered pe and pc metabolism . taken together , deficiency of phospholipid methylation caused by adohcy accumulation in hcy - associated pathology appears to lead to an increase in saturated pc molecular species in er membranes followed by er stress , protein misfolding , induction of upr , and activation of lipid metabolism ( figure 2 ) . upregulation of lipid biosynthesis , which apparently should serve to compensate for suboptimal composition of membrane lipids , leads , however , to accumulation of fatty acids , tag and sterols in the absence of functional phospholipid methylation . similarly , decrease of pc or increase in pe was shown to lead to cell damage and/or death in several other mammalian cell types [ 117119 ] . finally , observation of different outcomes in hyperhomocysteinemic apoe mice and both in pemt / ldlr and pemt / apoe mice suggests mechanisms besides inhibition of phospholipid methylation , for example , adohcy - dependent modulation of gene expression that may also contribute to the development of hcy - dependent atherosclerosis . the metabolism of homocysteine , consequences of its accumulation as well as associated adohcy - triggered inhibition of adomet - dependent methylation are complex . in this paper a novel mechanism of hcy - triggered deregulation of lipid metabolism and upr induction that is mediated through an adohcy - dependent inhibition of phospholipid methylation and based on experimental evidence derived from both yeast and mammalian systems is proposed . s - adenosyl - l - homocysteine hydrolase is recognized since many years as a target for antiviral drug design . however , they are associated with high cytotoxicity due to interference with central metabolic pathways [ 121 , 122 ] . while using these inhibitors to study the effects of adohcy accumulation appears to be straightforward , ability of some of nucleoside inhibitors of s - adenosyl - l - homocysteine hydrolase to undergo metabolic phosphorylation to nucleotides may account for a part of their biological activities [ 121 , 122 ] . however , based on current understanding of regulation of homocysteine and methionine metabolism ( i ) selective blockage of adohcy synthesis from homocysteine and adenosine that will relieve not only inhibition of phospholipid methylation but also many other adomet - dependent methyltransferase reactions , combined with ( ii ) vitamin b6 supplementation in order to accelerate homocystein catabolism by transsulfuration pathway , may serve as a way to reduce adohcy in hyperhomocysteinemia without elevation of adomet . what role do specific lipid precursors have in regulation of lipid metabolism in upr ? what is the impact of adohcy accumulation on other methylation reactions unrelated to lipid metabolism ? what is the role of deficient phospholipid methylation in homocysteine - associated pathology beyond deregulation of fatty acid , tag , sterol metabolism , and upr induction ? elucidation of the molecular mechanisms triggered by elevated hcy levels will undoubtfully improve our understanding of its pathological role in numerous diseases .

glaucoma is the second most frequent cause of permanent blindness in industrial developed countries . it is caused by an irreversible damage of the optical nerve connected with degeneration of retinal ganglia cells , axons ( neural fibres ) , and gliocells ( providing nutrition for the axons ) . if not diagnosed in early stage , the damage of the optical nerve becomes permanent , which in the final stage may lead to blindness . the number of people with open - angle and angle - closure glaucoma in 2020 is estimated to 79.6 million . development of new diagnostic approaches that would enable early detection of glaucoma development is therefore very desirable . one important view is that these new methods are to be applicable in routine diagnostic process , they should be robust and fast , particularly for screening programs . during the last years , there is a clear tendency in medicine to combine ( fuse ) data from different diagnostic sources ( e.g. , x - ray , ct , pet ) . similar situation is also in the area of ophthalmologic diagnostic methods . besides registration and fusion of standard photos of the eye fundus and data from confocal ophtalmoscope ( e.g. , ) , the images obtained under various imaging conditions are investigated , as different modalities . one of the promising approaches that could support early diagnosis of the glaucoma is the use of combined retinal autofluorescence ( af ) images and infrared ( ir ) images . this method utilizes information about hyperfluorescent zones in af image ( which is a symptom of glaucoma in its early stage ) and information about position of the optical nerve head from ir images . obviously , multimodal registration is needed and one has to find a specific registration framework , including also the proper image preprocessing . such a framework is a trade - off between registration accuracy and computational demands as shown in previous papers [ 68 ] . in this article , we propose a registration method ( section 3 ) for the autofluorescent and infrared retinal images ( section 2 ) ; its evaluation on an available image set is also presented ( section 4 ) . the direct application of the registered images is presented by means of pixel level image fusion ( section 5 ) . the images , for which the algorithms have been designed , were acquired at the department of ophthalmology , friedrich - alexander university of erlangen - nurnberg , germany . the laser scanning ophthalmoscope used for the acquisition was the heidelberg retina angiograph2 ( hra2 ) , which serves for angiographic examination of human retina . several modes of the examination are available , for example , fluorescein angiography , indocyanine green angiography , infrared imaging . all images , provided by either of the modalities , may be denoted as scanning laser ophthalmoscopic ( slo ) images . in af mode , the retina is illuminated by a narrow blue light laser beam ( = 488 nm ) in a raster manner . this beam excites the lipofuscin that consequently emits light with a longer wavelength ( around 500 nm ) . the emission intensity depends on the amount of lipofuscin accumulation in retinal pigment epithelium ( rpe ) . there are some studies showing the correlation between the lipofuscin accumulations around the optic disc ( od ) and the stage of the optic disc atrophy . . the higher level of autofluorescence is predominantly observed at the border of the atrophic zone alpha , according to the classification used by ophthalmologists . in ir mode , hra2 device uses infrared laser light with wavelength = 820 nm to illuminate the tissue in raster manner . during scanning an example of an ir image is shown on figure 1(b ) . because the af and ir images are recorded in time sequence , the patient 's eye can move , which causes a spatial shift between the images or even inside a gradually scanned image . to be able to fuse the information from both images , a multimodal registration ( e.g. , [ 12 , 13 ] ) this is partly simplified by identical image resolution in both modalities ( 10 m / pixel and size 512 512 pixels for low resolution mode and 5 m / pixel , size 1024 1024 pixels for high resolution mode ) . general view of registration methods applied to various types of image data ( ct , pet , mri , etc . ) can be found in several review papers [ 1417 ] or books [ 18 , 19 ] . specific methods for the retinal image registration are also described in several application papers [ 12 , 13 , 2025 ] . zana and klein describe multimodal method based on segmentation of vessel tree in fluorescent retinal angiography combined with fundus photograph ( fp ) . the bifurcation points of the vessel tree are detected and used as features for the registration process . in , can describes an algorithm for constructing a mosaic consisting of several curved fundus photographs . similar method is mentioned in stewart et al . where authors designed feature correspondence registration for retina mosaicing . presented a new method for fusion of fp and fluorescein angiography images after registration also based on bifurcation points . chanwimaluang et al . used the vascular tree extraction in monomodal registration to create a mosaic image . extracted bifurcation points only in the reference image and used self - organizing maps for minimizing the difference between gradient pixel intensities to find the parameters of the affine transforms . there are several problems of this registration approach : determination of the landmarks in case of hyperfluorescence , haemorrhages , or drusens . other problem is connected with landmark correspondence accented in a case of multimodality images , absence of landmarks for small blood vessels at the image periphery , and in a case of low - quality images ( blurred or noisy ) . in wachowiak robustness of a particle swarm optimization in comparison with evolutionary search is discussed . in rosin et al . , the method concerning multimodal registration of confocal slo and color fp images is presented . it uses mutual information ( mi ) metric and pyramidal approach in combination with simulated annealing and gradient descent optimizers . quality of registration was judged subjectively on the scale : 0-poor , a similar approach has been used in the present work as well as in the previous paper . image registration can be treated as the optimization process of finding the parameter vector 0 of the spatial transformation t aligning the content of the images : ( 1)o = arg{max c(f , t(g ) ) } , where f denotes the reference ( fixed ) image , g is the transformed ( moving ) image , and c is an optimization criterion which evaluates the registration quality . based on the prior knowledge of image properties , all parts of the registration process should be chosen carefully to ensure registration robustness . the components of the registration framework include the following.preprocessing for example , noise suppression and edge enhancement.criterion metrics : selection among mean squared differences , normalized cross - correlation , gradient difference , and mutual information.spatial transformation : rigid or flexible transformation.optimization strategy : for example , gradient - based or population - based methods.interpolation : needed to match the discretization grids of both images : for example , nearest neighbor , bilinear , radial basis functions , and so forth . in the following , we will briefly comment on each of them with respect to the concrete problem to be solved . criterion metrics : selection among mean squared differences , normalized cross - correlation , gradient difference , and mutual information . . interpolation : needed to match the discretization grids of both images : for example , nearest neighbor , bilinear , radial basis functions , and so forth . the first step is the image preprocessing . because of the low signal - to - noise ratio ( snr ) , noise suppression method should be performed , but possibly without blurring the edges which carry information needed for registration . for that reason , we used the anisotropic diffusion introduced by perona and malik . the approach of this method is that a gaussian smoothed image is considered as a single time slice of the solution to the heat equation : ( 2)g(x , y , t)t = c(|g|)g(x , y , t ) , where g(x , y , 0 ) = f(x , y ) is the input image . the function c(|g| ) is a function that reduces the conductance at the areas of large gradient |g| . we used the following form : ( 3)c(|g| ) = e|g|2/22 , which introduces a new conductance parameter , controlling sensitivity of the smoothing process . the behavior of this function for tested values of is depicted on figure 2 . the second parameter of anisotropic diffusion is time t and the last parameter is the number of iterations . for the ir and af images , we found as suitable values for = 1.5 , t = 0.125 , and iteration = 10 . for higher values of , the smoothing effect was too high and the resulting images were not suitable for consequential gradient computation ( see section 3.2 ) . the effect of this filter is shown on figure 3 . the choice of the similarity metric plays a crucial role in the registration process . because the shift in x , y axes will be the parameters with the main influence to the value of metrics , we investigated the behavior of different kind of metrics as a function of x , y shift : the normalized cross - correlation ( ncc ) , the mean of squared differences ( msd ) , and the mutual information ( mi ) as defined in . as expected , the absolute gradient of the filtered image turned out more convenient for registration , using any of the criteria , because it causes more distinct peak of the maximum ( see figure 4 for comparison ) and this peak is global in contrast to local maximum on the metric surface obtained for nongradient images it is a numerical approximation of the gradient absolute value : ( 4)|f| = ( fxi^)2 + ( fyj^)2 . as can be seen from figure 4 there is clear visibility of the positive influence to ncc metric in case that the gradient images are used . the above - mentioned metrics were evaluated for the defined shifts pixel by pixel , using several pairs of af and ir images . one can see that the ncc and msd similarity metrics have quite similar surfaces . more detailed examination showed that the ncc metric plane has sharper global maximum for majority of the tested images . it also has less local extremes , which is welcome in optimization . comparing ncc and mi metric planes , the mi metric shows also good properties for optimization . but , considering the computational complexity , we decided to use the ncc metric , according to the definition ( 5)ncc(f , g ) = i=1nfigii=1nfi2i=1ngi2 , where fi and gi represent the pixel values of gradient images and n is number of pixels in the overlapping region . the spatial transform t represents the spatial mapping of pixel values from the moving image space to points in the fixed image space . the type of the transform has to be chosen suitably in order to compensate the distortion between images . considering the acquisition process one is due to the patient movement between the af and ir image acquisition , which can be shift and small rotation . the scaling parameters were also included because of the optic of the eye ; the slightly different behavior in reflection and refraction for light with different wavelengths . therefore , we considered only translations in both axes ( tx , ty ) , rotation ( ) , and scaling ( sx , sy ) as the transform parameters ( see ( 6 ) ) . obviously , there can be more complicated discrepancies between images , caused by movement of the eye during the scanning process . these kinds of distortion would need flexible transformations . to make our method practicable ( i.e. , to speed up the computations ) , we restrict it to the class of image pairs that are not corrupted during the single - image scanning and thus need affine transform with 5 parameters . the chosen transform can be represented by the transformation matrix for homogeneous coordinates : ( 6)t = ( sxcos()sysin()txsxsin()sycos()ty001 ) . the registered image data of the moving image are computed for the coordinates in the target pixels of the fixed image . therefore , interpolation takes place after each new ( possibly optimal ) spatial transform is computed . the nearest neighbor , bilinear , and the choice of the interpolation technique is more critical in cases where high enlargement of the moving image is employed , which is not our case . we observed that the choice of any of the three interpolation techniques had no visible influence on the registration results . to achieve high computational speed , the nearest neighbor technique the registration consists of searching the optimal transform parameters in multidimensional space ; in our case , 5-parametric space . in spite of image preprocessing one class of the methods that can cope with this problem is based on the population evolution , where more solutions are considered at one iteration . each solution has its own similarity metric value . during optimization process ( evolution ) the samples with a high metric value are replaced with new solutions according to a defined ( heuristic ) rule . this rule , called alternation heuristic , can also change during this process . one of the successful population evolution methods in retina image registration is the controlled random search ( crs ) algorithm . the basic version of crs is shown in algorithm 1 . it uses a defined heuristic rule h(p ) to generate a new sample in population . if this new point in the search space has a better similarity metric value , it replaces the worst sample in population . the heuristic rule h(p ) can be based on evolutional strategy , simplex reflection , or differential evolution and can also alternate between them during the registration process [ 28 , 29 ] . the above - mentioned heuristic rules were used also in our case , with switching between them randomly with equal probabilities . several parameters for the crs method has to be set before registration and had to be determined experimentally . maximum number of iterations if convergence is not reached : was set to 2000.function convergence tolerance : defined as ( 7)|vivn/2| < , where vi and vn/2 are the metric values for the first and middle population members of sorted - out population . n is the population size.population size : number of members ( or active solutions ) ; it was set to 100 at the coarse level and 400 at the fine level of registration.population to stop : the number of solutions in the population that satisfy the condition . this parameter was set to n/2 : half of the population size n. the used registration approach utilizes a multiscale approach , widely used to improve speed , accuracy , and robustness . the basic idea is that registration is first performed at a coarse scale ( low - spatial resolution ) . the spatial mapping determined at the coarse level is then used to initialize registration at the next finer scale . this process is repeated recursively at finer levels until it reaches the finest possible scale . the advantage is that the registration with respect to large - scale features is achieved first and then small corrections are made for finer details . on the other hand , this multiscale strategy fails if a false match is identified on a coarser level . maximum number of iterations if convergence is not reached : was set to 2000 . function convergence tolerance : defined as ( 7)|vivn/2| < , where vi and vn/2 are the metric values for the first and middle population members of sorted - out population . population size : number of members ( or active solutions ) ; it was set to 100 at the coarse level and 400 at the fine level of registration . population to stop : the number of solutions in the population that satisfy the condition . this parameter was set to n/2 : half of the population size n. for af and ir images , we set only one coarse level with decimal subsampling , where only translations tx and ty are considered as the optimization parameters . at the next finest scale , the proposed approach with the above parameters was tested on our database of 131 ophthalmologic image pairs ( af and ir images ) . these images were obtained in low ( 512 512 pixels ) or high ( 1024 1024 pixels ) resolution mode . high - resolution images were decimated to low resolution , taking every second pixel , to speed up the computation . the crs optimization was run 5 times in each case of 30 image pairs ( randomly selected from 131 image pairs ) to test the sensitivity to random initialization . unfortunately , verification of registration algorithms accuracy is not trivial , because the correct geometrical alignment is not known . our verification was based on the visual inspection of the edge images and the mosaic images . examples of registration results are shown in figures 6 and 7 together with the edge and mosaic images , used for visual evaluation . each registered pair was classified according to as poor , moderate , good , and excellent , or , more precisely , as follows.excellent : the best quality with no visible discrepancy between both images.good : small misalignment between the images in the range of 1 to 5 pixels.moderate : higher misalignment between the images in the range of 6 to 15 pixels.poor : registration with significant misalignment . good : small misalignment between the images in the range of 1 to 5 pixels . moderate : higher misalignment between the images in the range of 6 to 15 pixels . the possible pixel misalignments were examined primarily in the area around the optical disc because this area is most important in glaucoma diagnosis . two experts ( a , b ) evaluated independently the registration results using the edge and mosaic images simultaneously . pixel level image fusion means merging the information from several images into a single image . there are many different areas of image fusion , namely , thermal and visual image fusion ; remote sensing [ 31 , 32 ] ; medical imaging , particularly mri t1-weighted , t2-weighted , and proton density images , spect / pet - ct images ; and also multimodal retinal images [ 3 , 20 ] . the aim of the af - ir image fusion process is to provide a single image with extended information content for the glaucoma diagnosis process . generally , image fusion is particularly useful for reducing the workload of human operators because it offers different information content in one image . the greatest benefit of the image fusion is achieved if the component images contain complementary information , which applies to af - ir image pairs . the af image contains information about the zones with high autofluorescent activity and more visible periphery blood vessels while the ir image carries the information about the optical disc border , its structure , and blood vessels inside od . the important point is the knowledge of the mutual positions of the autofluorescence zones with respect to optic disc border . without fusion , the physician must move his eye between images and it may be difficult to recognize the relationship among patterns and objects , particularly , od border and af zones . to prevent this , we tested two pixel - wise fusion processes that are very fast , using artificial color mapping applied on the two mentioned gray - scale component images . this approach is similar to the method used in , where the fusion was used for color fundus and angiography images . this scheme arises from the biologically motivated pixel - wise fusion methods ( hvs stands for the human visual system ) . for our af - ir fusion application , consecutively , the red channel is computed as a difference between ir and af images , which enhances the information present in ir image . the green channel is the af image itself , because the zones with higher autofluorescency play an important role in early diagnosis of glaucoma and the fusion is thus performed with visually emphasized af components . this scheme is based on a method used for gray - scale video ( tv ) and infrared ( ir ) images as in . the common component of the images is computed as the morphological intersection : ( 8)fg = min{f(i , j ) , g(i , j)}. the characteristic components f * or g * of each image remain after subtraction of the common component : ( 9)f * = ffg , g * = gfg . the characteristic component can be emphasized in the fused image h by subtracting each of them from the other image , so that the rgb components are then defined as in : ( 10)h = ( rgb)=(fg*gf*0 ) . we modified this definition in the similar way as in the hvs method to enhance the af zones in the fused image : ( 11)h = ( rgb)=(fg*fgf * ) , where f represents the af image , g represents the ir channel . for all channels , the normalization to 256 levels the example results of the image fusion are shown on figures 9 and 10 , where the original ( registered ) af and ir images are shown together with the corresponding fusion results . to discuss the results of the registration phase , let us concentrate to table 1 . one can see that even considering only the class 1 , the percentage of good registration is satisfactory . classes 2 and 3 include images where the misalignment errors were visible at the periphery of the images and were in the range of several pixels . the application described in previous works [ 5 , 36 ] uses only the area around the optical disc for analysis of the autofluorescent zones . therefore , it is important to register precisely the central part ( od with its surroundings ) . in that sense , class 2 can also be considered well registered and the percentage of successfully registered images is thus 93.1% and 90.8% , respectively ( including classes 1 and 2 ) . by detailed analysis of images in class 3 , we can conclude that the rare wrong registration results arise from blurred ir images , images with small overlap and for images with small but complex distortions , where more generic flexible registration is needed . investigating the images with high registration errors in class 4 , we can conclude that significant misalignment is found in pairs of images where the ir image is blurred and has a low contrast and the af image is also blurred or dark . also , in some cases , a visible strong flexible distortion is present . as for the preliminary evaluation of the fusion , one can see that the results of the fusion process are very similar for hvs and tv - ir fusions methods . considering the aim of the fusing ( i.e. , human analysis of the af zones with respect to od border ) , the tv - ir scheme seems to be more convenient because of higher contrast of af zones ( see figure 9 ) . a robust framework for registration of af and ir images was proposed and experimentally verified , including the image preprocessing . the framework is the part of custom - made software , which is currently used at the department of ophthalmology , friedrich - alexander university of erlangen - nurnberg , germany . a reasonable trade - off between the speed of computation and registration accuracy was achieved . the computation time for a single registration is about 1 minute at intel , pentium m , 1.7 mhz . it has been found in clinical trial that the tv - ir - fused images can serve well as a support for the diagnosis and for physician during segmentation process .

this article deals with registration and fusion of multimodal opththalmologic images obtained by means of a laser scanning device ( heidelberg retina angiograph ) . the registration framework has been designed and tested for combination of autofluorescent and infrared images . this process is a necessary step for consecutive pixel level fusion and analysis utilizing information from both modalities . two fusion methods are presented and compared .

1. INTRODUCTION 2. IMAGE DATA 3. IMAGE REGISTRATION 4. REGISTRATION RESULTS 5. PIXEL LEVEL IMAGE FUSION 6. DISCUSSION 7. CONCLUSION

glaucoma is the second most frequent cause of permanent blindness in industrial developed countries . if not diagnosed in early stage , the damage of the optical nerve becomes permanent , which in the final stage may lead to blindness . the number of people with open - angle and angle - closure glaucoma in 2020 is estimated to 79.6 million . development of new diagnostic approaches that would enable early detection of glaucoma development is therefore very desirable . one important view is that these new methods are to be applicable in routine diagnostic process , they should be robust and fast , particularly for screening programs . during the last years , there is a clear tendency in medicine to combine ( fuse ) data from different diagnostic sources ( e.g. , x - ray , ct , pet ) . similar situation is also in the area of ophthalmologic diagnostic methods . besides registration and fusion of standard photos of the eye fundus and data from confocal ophtalmoscope ( e.g. , ) , the images obtained under various imaging conditions are investigated , as different modalities . one of the promising approaches that could support early diagnosis of the glaucoma is the use of combined retinal autofluorescence ( af ) images and infrared ( ir ) images . this method utilizes information about hyperfluorescent zones in af image ( which is a symptom of glaucoma in its early stage ) and information about position of the optical nerve head from ir images . obviously , multimodal registration is needed and one has to find a specific registration framework , including also the proper image preprocessing . such a framework is a trade - off between registration accuracy and computational demands as shown in previous papers [ 68 ] . in this article , we propose a registration method ( section 3 ) for the autofluorescent and infrared retinal images ( section 2 ) ; its evaluation on an available image set is also presented ( section 4 ) . the direct application of the registered images is presented by means of pixel level image fusion ( section 5 ) . the images , for which the algorithms have been designed , were acquired at the department of ophthalmology , friedrich - alexander university of erlangen - nurnberg , germany . the laser scanning ophthalmoscope used for the acquisition was the heidelberg retina angiograph2 ( hra2 ) , which serves for angiographic examination of human retina . several modes of the examination are available , for example , fluorescein angiography , indocyanine green angiography , infrared imaging . all images , provided by either of the modalities , may be denoted as scanning laser ophthalmoscopic ( slo ) images . in af mode , the retina is illuminated by a narrow blue light laser beam ( = 488 nm ) in a raster manner . this beam excites the lipofuscin that consequently emits light with a longer wavelength ( around 500 nm ) . the emission intensity depends on the amount of lipofuscin accumulation in retinal pigment epithelium ( rpe ) . there are some studies showing the correlation between the lipofuscin accumulations around the optic disc ( od ) and the stage of the optic disc atrophy . . in ir mode , hra2 device uses infrared laser light with wavelength = 820 nm to illuminate the tissue in raster manner . because the af and ir images are recorded in time sequence , the patient 's eye can move , which causes a spatial shift between the images or even inside a gradually scanned image . to be able to fuse the information from both images , a multimodal registration ( e.g. , [ 12 , 13 ] ) this is partly simplified by identical image resolution in both modalities ( 10 m / pixel and size 512 512 pixels for low resolution mode and 5 m / pixel , size 1024 1024 pixels for high resolution mode ) . can be found in several review papers [ 1417 ] or books [ 18 , 19 ] . specific methods for the retinal image registration are also described in several application papers [ 12 , 13 , 2025 ] . zana and klein describe multimodal method based on segmentation of vessel tree in fluorescent retinal angiography combined with fundus photograph ( fp ) . the bifurcation points of the vessel tree are detected and used as features for the registration process . in , can describes an algorithm for constructing a mosaic consisting of several curved fundus photographs . similar method is mentioned in stewart et al . where authors designed feature correspondence registration for retina mosaicing . presented a new method for fusion of fp and fluorescein angiography images after registration also based on bifurcation points . chanwimaluang et al . extracted bifurcation points only in the reference image and used self - organizing maps for minimizing the difference between gradient pixel intensities to find the parameters of the affine transforms . there are several problems of this registration approach : determination of the landmarks in case of hyperfluorescence , haemorrhages , or drusens . in wachowiak robustness of a particle swarm optimization in comparison with evolutionary search is discussed . in rosin et al . , the method concerning multimodal registration of confocal slo and color fp images is presented . quality of registration was judged subjectively on the scale : 0-poor , a similar approach has been used in the present work as well as in the previous paper . image registration can be treated as the optimization process of finding the parameter vector 0 of the spatial transformation t aligning the content of the images : ( 1)o = arg{max c(f , t(g ) ) } , where f denotes the reference ( fixed ) image , g is the transformed ( moving ) image , and c is an optimization criterion which evaluates the registration quality . based on the prior knowledge of image properties , all parts of the registration process should be chosen carefully to ensure registration robustness . the components of the registration framework include the following.preprocessing for example , noise suppression and edge enhancement.criterion metrics : selection among mean squared differences , normalized cross - correlation , gradient difference , and mutual information.spatial transformation : rigid or flexible transformation.optimization strategy : for example , gradient - based or population - based methods.interpolation : needed to match the discretization grids of both images : for example , nearest neighbor , bilinear , radial basis functions , and so forth . . the first step is the image preprocessing . for that reason , we used the anisotropic diffusion introduced by perona and malik . the approach of this method is that a gaussian smoothed image is considered as a single time slice of the solution to the heat equation : ( 2)g(x , y , t)t = c(|g|)g(x , y , t ) , where g(x , y , 0 ) = f(x , y ) is the input image . the function c(|g| ) is a function that reduces the conductance at the areas of large gradient |g| . we used the following form : ( 3)c(|g| ) = e|g|2/22 , which introduces a new conductance parameter , controlling sensitivity of the smoothing process . the second parameter of anisotropic diffusion is time t and the last parameter is the number of iterations . for higher values of , the smoothing effect was too high and the resulting images were not suitable for consequential gradient computation ( see section 3.2 ) . the choice of the similarity metric plays a crucial role in the registration process . because the shift in x , y axes will be the parameters with the main influence to the value of metrics , we investigated the behavior of different kind of metrics as a function of x , y shift : the normalized cross - correlation ( ncc ) , the mean of squared differences ( msd ) , and the mutual information ( mi ) as defined in . as expected , the absolute gradient of the filtered image turned out more convenient for registration , using any of the criteria , because it causes more distinct peak of the maximum ( see figure 4 for comparison ) and this peak is global in contrast to local maximum on the metric surface obtained for nongradient images it is a numerical approximation of the gradient absolute value : ( 4)|f| = ( fxi^)2 + ( fyj^)2 . as can be seen from figure 4 there is clear visibility of the positive influence to ncc metric in case that the gradient images are used . the above - mentioned metrics were evaluated for the defined shifts pixel by pixel , using several pairs of af and ir images . one can see that the ncc and msd similarity metrics have quite similar surfaces . more detailed examination showed that the ncc metric plane has sharper global maximum for majority of the tested images . it also has less local extremes , which is welcome in optimization . comparing ncc and mi metric planes , the mi metric shows also good properties for optimization . the spatial transform t represents the spatial mapping of pixel values from the moving image space to points in the fixed image space . considering the acquisition process one is due to the patient movement between the af and ir image acquisition , which can be shift and small rotation . the scaling parameters were also included because of the optic of the eye ; the slightly different behavior in reflection and refraction for light with different wavelengths . therefore , we considered only translations in both axes ( tx , ty ) , rotation ( ) , and scaling ( sx , sy ) as the transform parameters ( see ( 6 ) ) . obviously , there can be more complicated discrepancies between images , caused by movement of the eye during the scanning process . these kinds of distortion would need flexible transformations . the chosen transform can be represented by the transformation matrix for homogeneous coordinates : ( 6)t = ( sxcos()sysin()txsxsin()sycos()ty001 ) . the registered image data of the moving image are computed for the coordinates in the target pixels of the fixed image . therefore , interpolation takes place after each new ( possibly optimal ) spatial transform is computed . we observed that the choice of any of the three interpolation techniques had no visible influence on the registration results . to achieve high computational speed , the nearest neighbor technique the registration consists of searching the optimal transform parameters in multidimensional space ; in our case , 5-parametric space . in spite of image preprocessing one class of the methods that can cope with this problem is based on the population evolution , where more solutions are considered at one iteration . during optimization process ( evolution ) the samples with a high metric value are replaced with new solutions according to a defined ( heuristic ) rule . this rule , called alternation heuristic , can also change during this process . if this new point in the search space has a better similarity metric value , it replaces the worst sample in population . the heuristic rule h(p ) can be based on evolutional strategy , simplex reflection , or differential evolution and can also alternate between them during the registration process [ 28 , 29 ] . several parameters for the crs method has to be set before registration and had to be determined experimentally . maximum number of iterations if convergence is not reached : was set to 2000.function convergence tolerance : defined as ( 7)|vivn/2| < , where vi and vn/2 are the metric values for the first and middle population members of sorted - out population . the basic idea is that registration is first performed at a coarse scale ( low - spatial resolution ) . this process is repeated recursively at finer levels until it reaches the finest possible scale . the advantage is that the registration with respect to large - scale features is achieved first and then small corrections are made for finer details . on the other hand , this multiscale strategy fails if a false match is identified on a coarser level . maximum number of iterations if convergence is not reached : was set to 2000 . function convergence tolerance : defined as ( 7)|vivn/2| < , where vi and vn/2 are the metric values for the first and middle population members of sorted - out population . population size : number of members ( or active solutions ) ; it was set to 100 at the coarse level and 400 at the fine level of registration . population to stop : the number of solutions in the population that satisfy the condition . at the next finest scale , the proposed approach with the above parameters was tested on our database of 131 ophthalmologic image pairs ( af and ir images ) . these images were obtained in low ( 512 512 pixels ) or high ( 1024 1024 pixels ) resolution mode . high - resolution images were decimated to low resolution , taking every second pixel , to speed up the computation . unfortunately , verification of registration algorithms accuracy is not trivial , because the correct geometrical alignment is not known . our verification was based on the visual inspection of the edge images and the mosaic images . examples of registration results are shown in figures 6 and 7 together with the edge and mosaic images , used for visual evaluation . each registered pair was classified according to as poor , moderate , good , and excellent , or , more precisely , as follows.excellent : the best quality with no visible discrepancy between both images.good : small misalignment between the images in the range of 1 to 5 pixels.moderate : higher misalignment between the images in the range of 6 to 15 pixels.poor : registration with significant misalignment . good : small misalignment between the images in the range of 1 to 5 pixels . moderate : higher misalignment between the images in the range of 6 to 15 pixels . the possible pixel misalignments were examined primarily in the area around the optical disc because this area is most important in glaucoma diagnosis . two experts ( a , b ) evaluated independently the registration results using the edge and mosaic images simultaneously . pixel level image fusion means merging the information from several images into a single image . the aim of the af - ir image fusion process is to provide a single image with extended information content for the glaucoma diagnosis process . generally , image fusion is particularly useful for reducing the workload of human operators because it offers different information content in one image . the af image contains information about the zones with high autofluorescent activity and more visible periphery blood vessels while the ir image carries the information about the optical disc border , its structure , and blood vessels inside od . the important point is the knowledge of the mutual positions of the autofluorescence zones with respect to optic disc border . without fusion , the physician must move his eye between images and it may be difficult to recognize the relationship among patterns and objects , particularly , od border and af zones . this approach is similar to the method used in , where the fusion was used for color fundus and angiography images . this scheme arises from the biologically motivated pixel - wise fusion methods ( hvs stands for the human visual system ) . the green channel is the af image itself , because the zones with higher autofluorescency play an important role in early diagnosis of glaucoma and the fusion is thus performed with visually emphasized af components . this scheme is based on a method used for gray - scale video ( tv ) and infrared ( ir ) images as in . the common component of the images is computed as the morphological intersection : ( 8)fg = min{f(i , j ) , g(i , j)}. the characteristic component can be emphasized in the fused image h by subtracting each of them from the other image , so that the rgb components are then defined as in : ( 10)h = ( rgb)=(fg*gf*0 ) . we modified this definition in the similar way as in the hvs method to enhance the af zones in the fused image : ( 11)h = ( rgb)=(fg*fgf * ) , where f represents the af image , g represents the ir channel . to discuss the results of the registration phase , let us concentrate to table 1 . one can see that even considering only the class 1 , the percentage of good registration is satisfactory . the application described in previous works [ 5 , 36 ] uses only the area around the optical disc for analysis of the autofluorescent zones . in that sense , class 2 can also be considered well registered and the percentage of successfully registered images is thus 93.1% and 90.8% , respectively ( including classes 1 and 2 ) . by detailed analysis of images in class 3 , we can conclude that the rare wrong registration results arise from blurred ir images , images with small overlap and for images with small but complex distortions , where more generic flexible registration is needed . investigating the images with high registration errors in class 4 , we can conclude that significant misalignment is found in pairs of images where the ir image is blurred and has a low contrast and the af image is also blurred or dark . , human analysis of the af zones with respect to od border ) , the tv - ir scheme seems to be more convenient because of higher contrast of af zones ( see figure 9 ) . a robust framework for registration of af and ir images was proposed and experimentally verified , including the image preprocessing . the framework is the part of custom - made software , which is currently used at the department of ophthalmology , friedrich - alexander university of erlangen - nurnberg , germany . the computation time for a single registration is about 1 minute at intel , pentium m , 1.7 mhz . it has been found in clinical trial that the tv - ir - fused images can serve well as a support for the diagnosis and for physician during segmentation process .

hepatocellular carcinoma ( hcc ) is a primary liver cancer derived from hepatocytes . in more than 80% cases the early detection of hcc has been attempted using fluorodeoxyglucose positron emission tomography ( fdg pet ) . the accumulation of fluorin - fdg ( f - fdg ) in hcc is variable due to the varying degrees of activity of the enzyme well - differentiated hcc , and low grades tumor have more or less similar degree of activity as compared to the normal liver cells , and thus results in small difference in f - fdg accumulation , and therefore , f - fdg pet picks up only 5070% of the lesion in these patients . it is possible to increase the contrast between the hcc cells , and hepatocytes on f - fdg pet using digital contrast enhancement techniques . almost all contrast enhancement technique increases the contrast of the targeted lesion at the cost of decreasing contrast in other areas of the image . it is a challenging task to find the optimum balance between the acceptable enhancements of the targeted lesion while maintaining the existing contrast in other areas of the image . very few digital contrast enhancement techniques have been tried to improve the low contrast in hcc . we conducted a prototype study to select one technique from a set of contrast enhancement techniques available in the literature . during the prototype study , we realized that many techniques can serve our purpose . however , there is need to standardize the input parameters of any selected technique for the best result . in this study , we selected a contrast enhancement technique that increases the contrast in both the darker , and brighter region of the image simultaneously , and optimized the value of its input parameters for the best possible contrast for f - fdg pet images in hcc . no ethical approval was required for the study involved in the digital processing of pet images acquired for a clinical indication . patients fasted for at least 4 h before undergoing f - fdg pet - computed tomography ( pet - ct ) . mmol / l prior to the intravenous injection of 370 mbq of f - fdg . the data acquisition were performed at 4560 min later with an integrated pet - ct system ( biograph mct , siemens medical solutions , erlangen , germany ) . ct was acquired with covering the patient from skull base to mid - thigh which was matched to pet bed size . immediately after ct scanning pet acquisition was done for 2 min / bed , and on a matrix size of 128 128 . images were reconstructed using the ordered subset expectation maximization algorithm with 2 iterations , and 8 subsets , and full width half maximum of 5 mm . finally , the attenuation corrected pet , ct and fused pet - ct images were available for review . an experienced nuclear medicine physician adjusted the contrast of a transverse slice of f - fdg pet image containing hcc tumor with the help of a most widely used brightness - contrast tool . once the best possible contrast in the image in his opinion was achieved , then the slice was saved in the jpeg 2000 format . a total of nine f fdg pet studies referred the case of suspected hcc were selected , and hence nine jpeg images were saved [ figure 1 ] , and their 2 order entropy values are given in table 1 . the nine original images entropy of all nine original images digital contrast enhancement of these images was carried by standardizing the value of the parameter ( m and e ) of the contrast enhancement function t ( r ) represented by equation ( 1 ) . in the equation ( 1 ) , r is the numerical value stored at a pixel location in the image , e determines the slope of the linear portion of the curve represented by the equation ( 1 ) . the transform function t ( r ) compresses the input levels below , and above m into a narrow range of darker and brighter levels , respectively , and thereby improves contrast . jpeg image in rgb format were converted into the gray level , and then the image data were converted into double data type before applying equation ( 1 ) . we have used im2double , an inbuilt function available in the matlab , to convert the image to double data type . m from 60 to 180 in the steps of 1 and for each value of m , the value of e was varied from 1 to 7 again in the steps of 1 . the same process was repeated for all the nine images and in this way 9 847 = 7623 images were visually , and quantitatively analyzed for the global contrast enhancement with respect to the original input image . the global contrast enhancement of these images was assessed by the following contrast evaluation metrics : we calculated 2 order entropy based on co - occurrence matrix using the distance of one pixel in the horizontal direction . 2 order entropy gives the information about the image richness in detail taking the spatial relation of the pixel value into account . we calculated the edge at each pixel position using the standard matlab edge function , and sobel operator , and edge content ( ec ) by dividing the sum of the value of edge by the matrix size of the image . the performance of canny operator have been reported as the best , however , computationally most expensive among other such edge detection operators such as sobel , laplacian , and laplacian of gaussian . otherwise , in case of smooth images , sobel gives much faster results similar to canny operator . the input image provided by the clinical team was the best images from their end ; it was the smooth image as can be seen from the figure 1 . we calculated the value of absolute mean brightness error ( ambe ) , which is the deviation of the mean intensity of the enhanced image from the mean intensity of the original image by finding the absolute difference between the mean intensity of the output , and the input image . the mean intensity was calculated as the sum of all pixel values divided by a number of pixels in the image . saturation evaluation metric ( sem ) measures the saturation by computing the number of saturated pixels ( black or white pixels which were not saturated before ) after applying contrast enhancement , we calculated the value of saturation metric by dividing the number of saturated pixels ( black or white pixels which were not saturated before ) by the size of the matrix that is number of pixels in the image . the calculated values of 2 order entropy , abme , ec , and sem for each of the 7623 images were stored . we defined criteria for the selection of the image having best possible contrast among the 847 images . we defined the best possible contrast image as the image whose 2 order entropy , and ec , have high values , while ambe and sem , have low values , respectively . the input parameter ( m and e ) that resulted in best possible contrast image was considered as standardized input parameters of the contrast enhancement function . the plot of 2 order entropy , ec , ambe , and sem versus output image parameters ( m and e ) was carefully analyzed to locate the approximate position on the plot for best possible contrast image . in next step , we drew a separate plot by taking 10 data points around this point and determined the standardized parameter . patients fasted for at least 4 h before undergoing f - fdg pet - computed tomography ( pet - ct ) . mmol / l prior to the intravenous injection of 370 mbq of f - fdg . the data acquisition were performed at 4560 min later with an integrated pet - ct system ( biograph mct , siemens medical solutions , erlangen , germany ) . ct was acquired with covering the patient from skull base to mid - thigh which was matched to pet bed size . immediately after ct scanning pet acquisition was done for 2 min / bed , and on a matrix size of 128 128 . images were reconstructed using the ordered subset expectation maximization algorithm with 2 iterations , and 8 subsets , and full width half maximum of 5 mm . finally , the attenuation corrected pet , ct and fused pet - ct images were available for review . an experienced nuclear medicine physician adjusted the contrast of a transverse slice of f - fdg pet image containing hcc tumor with the help of a most widely used brightness - contrast tool . once the best possible contrast in the image in his opinion was achieved , then the slice was saved in the jpeg 2000 format . a total of nine f fdg pet studies referred the case of suspected hcc were selected , and hence nine jpeg images were saved [ figure 1 ] , and their 2 order entropy values are given in table 1 . the nine original images entropy of all nine original images digital contrast enhancement of these images was carried by standardizing the value of the parameter ( m and e ) of the contrast enhancement function t ( r ) represented by equation ( 1 ) . in the equation ( 1 ) , r is the numerical value stored at a pixel location in the image , e determines the slope of the linear portion of the curve represented by the equation ( 1 ) . m into a narrow range of darker and brighter levels , respectively , and thereby improves contrast . jpeg image in rgb format were converted into the gray level , and then the image data were converted into double data type before applying equation ( 1 ) . we have used im2double , an inbuilt function available in the matlab , to convert the image to double data type . m from 60 to 180 in the steps of 1 and for each value of m , the value of e was varied from 1 to 7 again in the steps of 1 . the same process was repeated for all the nine images and in this way 9 847 = 7623 images were visually , and quantitatively analyzed for the global contrast enhancement with respect to the original input image . the global contrast enhancement of these images was assessed by the following contrast evaluation metrics : we calculated 2 order entropy based on co - occurrence matrix using the distance of one pixel in the horizontal direction . 2 order entropy gives the information about the image richness in detail taking the spatial relation of the pixel value into account . we calculated the edge at each pixel position using the standard matlab edge function , and sobel operator , and edge content ( ec ) by dividing the sum of the value of edge by the matrix size of the image . the performance of canny operator have been reported as the best , however , computationally most expensive among other such edge detection operators such as sobel , laplacian , and laplacian of gaussian . otherwise , in case of smooth images , sobel gives much faster results similar to canny operator . the input image provided by the clinical team was the best images from their end ; it was the smooth image as can be seen from the figure 1 . we calculated the value of absolute mean brightness error ( ambe ) , which is the deviation of the mean intensity of the enhanced image from the mean intensity of the original image by finding the absolute difference between the mean intensity of the output , and the input image . the mean intensity was calculated as the sum of all pixel values divided by a number of pixels in the image . saturation evaluation metric ( sem ) measures the saturation by computing the number of saturated pixels ( black or white pixels which were not saturated before ) after applying contrast enhancement , we calculated the value of saturation metric by dividing the number of saturated pixels ( black or white pixels which were not saturated before ) by the size of the matrix that is number of pixels in the image . the calculated values of 2 order entropy , abme , ec , and sem for each of the 7623 images were stored . we defined criteria for the selection of the image having best possible contrast among the 847 images . we defined the best possible contrast image as the image whose 2 order entropy , and ec , have high values , while ambe and sem , have low values , respectively . the input parameter ( m and e ) that resulted in best possible contrast image was considered as standardized input parameters of the contrast enhancement function . the plot of 2 order entropy , ec , ambe , and sem versus output image parameters ( m and e ) was carefully analyzed to locate the approximate position on the plot for best possible contrast image . in next step , we drew a separate plot by taking 10 data points around this point and determined the standardized parameter . we calculated 2 order entropy based on co - occurrence matrix using the distance of one pixel in the horizontal direction . 2 order entropy gives the information about the image richness in detail taking the spatial relation of the pixel value into account . we calculated the edge at each pixel position using the standard matlab edge function , and sobel operator , and edge content ( ec ) by dividing the sum of the value of edge by the matrix size of the image . the performance of canny operator have been reported as the best , however , computationally most expensive among other such edge detection operators such as sobel , laplacian , and laplacian of gaussian . otherwise , in case of smooth images , sobel gives much faster results similar to canny operator . the input image provided by the clinical team was the best images from their end ; it was the smooth image as can be seen from the figure 1 . we calculated the value of absolute mean brightness error ( ambe ) , which is the deviation of the mean intensity of the enhanced image from the mean intensity of the original image by finding the absolute difference between the mean intensity of the output , and the input image . the mean intensity was calculated as the sum of all pixel values divided by a number of pixels in the image . saturation evaluation metric ( sem ) measures the saturation by computing the number of saturated pixels ( black or white pixels which were not saturated before ) after applying contrast enhancement , we calculated the value of saturation metric by dividing the number of saturated pixels ( black or white pixels which were not saturated before ) by the size of the matrix that is number of pixels in the image . the calculated values of 2 order entropy , abme , ec , and sem for each of the 7623 images were stored . we defined criteria for the selection of the image having best possible contrast among the 847 images . we defined the best possible contrast image as the image whose 2 order entropy , and ec , have high values , while ambe and sem , have low values , respectively . the input parameter ( m and e ) that resulted in best possible contrast image was considered as standardized input parameters of the contrast enhancement function . the plot of 2 order entropy , ec , ambe , and sem versus output image parameters ( m and e ) was carefully analyzed to locate the approximate position on the plot for best possible contrast image . in next step , we drew a separate plot by taking 10 data points around this point and determined the standardized parameter . for one image , we had 847 data point to be plotted , and there were nine images . for each data points , there were four values namely : 2 order entropy , ec , ambe , and sem . the plotted data of a representative image appears as an envelope depicting the trend , and the range of variations as shown in figure 2 . the plotted data of the individual nine images were zoomed , and the following conclusions were drawn after review : with the increase in the value of m the upper , and lower limit of 2 order entropy and ec also increased , attained a maximum value at some point , after that it becomes constant . the upper and lower limit of the ambe decreased with increase in the value of m , and after some point it also attained a maximum value , and remained constant thereafter . the value of sem initially increased very slowly with an increase in the value of m , and after a certain value of m , it increased steeply . m , the trendline of contrast enhancement metrics are not the same ; in fact in some cases , they are opposite . the result of one input image showed a different trend in terms of ambe , and sem . the data plot of a representative image . ( b ) absolute mean brightness error on the y - axis , ( c ) edge content on the y - axis , and ( d ) saturation evaluation metric on the y - axis . the x - axis has threshold value ( m ) from 60 to 180 and at each threshold there were seven data points ( e ) making total 847 data points we wrote attained a maximum value at some point in the previous paragraph because for all nine images these points ( maximum value for 2 order entropy , ec , ambe , and sem ) were different , and moreover these values were varying at each threshold that is shown in the zoomed view of figure 2 , by taking only first 49 data point [ figure 3 ] . data plot of a representative image considering only 49 data values from the beginning of the data plot shown in figure 1 . here the x - axis has threshold value ( m ) from 60 to 66 and at each threshold there were seven data points ( e ) making total 49 data points table 2 shows the 2 order entropy , ec , ambe , and sem values , of images having the best possible contrast . 2 ) , that is , its mean intensity was greater than the input image by 16.49 this indicates that very few pixels in the image either became white or black , as compared to the unprocessed image . the value of ambe , and sem together indicates that the overall or global brightness of the images have increased , and also very few pixels became white or black as compared to unprocessed image , and hence the contrast in the image improved . table 2 also demonstrates that a particular combination of m and e value might not be suitable for all f - fdg pet images in hcc because for each image we found different values of m and e. the best possible contrast in seven images was obtained at a threshold value of m between 150 , and 160 , and in another two images at 138 and 175 . the input parameters ( m and e ) that resulted in best image and their corresponding values of 2 order entropy , ec , abme , and sem the difference in entropy , and ec of the output images from their corresponding input images is shown in table 3 . the difference in ec is almost zero for all the nine images when the values are rounded to two decimal points , but there was the difference in the values , when the data were considered to six decimal points . this difference might not be appreciable visually , but this also indicates that the contrast enhancement produced by this technique has not deteriorated the edges . the difference in entropy was also very minimal ( rounded data up to two decimal points ) , indicating that after processing , the information content of the images has reduced , however , it is very difficult to appreciate this reduction in information content visually . the image produced as a result of the application of the optimized value of input parameter m and e has been shown along with the original input image in figures 4 and 5 . from these figures , it is clear that the contrast in the output image is more than that of the input image , and simultaneously , it has not distorted the information in the other areas . deviation of entropy and ec of the output images from that of the input images 18fluorin - fluorodeoxyglucose positron emission tomography images of a patient with hepatocellular carcinoma . ( a ) original image , ( b ) image having highest value of entropy ( c ) image having highest value of edge content ( slightly more than the original image ) , ( d ) image having lowest value of absolute mean brightness error , ( e ) lowest value of saturation evaluation metric , and ( f ) image with best contrast based on high value of entropy and edge content and low value of absolute mean brightness error and saturation evaluation metric . visually it is clear that the selected transformation function in this study has produced better contrast between the tumor and normal liver cell than the original image original image along with processed image with optimum selected value of m and e for four different patients ( a - d ) . the images clearly show that the contrast of the image has improved significantly and has not distorted any information available in original image in this study , we attempted to evaluate the optimum parameters for digital contrast enhancement of f - fdg pet images in hcc . we found the different trend lines for 2 order entropy , ec , ambe , and sem at m = 60 , the darker pixels ( pixel value < 60 ) will be compressed into narrow range of darker level . and pixels having values above 60 ( set of few black , gray , and white pixels ) will be compressed into a narrow range of brighter pixels . thus , the edge of the tumor that lies in the darker region will improve ( because at threshold value 60 , the difference between the black and white pixel intensities will increase , thus the contrast will improve ) , and entropy , and ec will be more at m = 60 , and e = 1 . as the slope increases , there will be the more smooth transition from black to white , compressed , narrow range of black , and white pixel will relax in both the direction , and therefore , the entropy and ec will decrease . at m = 152 , pixel values below 152 ( the set of black , gray , and white pixel will be compressed into the black regions ) and above 152 ( the set of white pixels will be compressed into narrow range of whiter level ) . since our tumor is in black region , compression of pixel below 152 into narrow range of black will decrease the sharp transition as in the case of m = 60 , and therefore entropy , and ec will be less in comparison to that of m = 60 . however , as the slope will increase entropy and ec will improve , because of the relaxation in the compressed , narrow range of black , and white pixels . the best possible contrast in seven out of nine images was obtained at threshold value m between 150 and 160 , including both 150 , and 160 . in another two images , best possible contrast was found at 138 , and 175 . this means , that there is a minimal shift in brightness level after processing the image , and the output images are a little bit brighter than their corresponding input images . this indicates that , after the process very few pixels have either become white , or black as compared to unprocessed images , that is . this study also demonstrates that a particular combination of m and e , might not result in an image having the best possible contrast for all pet images ; because for each image we have found the different value of m and e. this is because each image is unique , and requires the unique value of m and e to produce best possible contrast . our result suggests that a default parameter may not be the best for all kind of images , a finding that agrees with other authors . we found that the histograms of all the nine input images were concentrated in the brighter side , that is . therefore , we can say that the selection of threshold from the region of concentrated histogram might provide a best possible contrast in the image . however , to be more precise , we need to increase the number of input images . our criteria for selection of parameters m and e that produce best possible contrast in an image was high value of entropy , ec , and low value of ambe , sem , as previously suggested by saleem et al . however , we did not find a single processed image ( from a set of 847 images ) that had a highest value of entropy , highest value of ec , lowest value of ambe , and lowest value of sem . this may be because with the increase in ambe , the value of sem also increases , the entropy increases with more variability in the data in spatial domain , and with the increase in entropy , it is not necessary that ec will also increase . therefore , we made a compromise in the selection of the standardized parameter . we restricted the value of m in the range of 60180 , because in a prototype study we found that the images processed at m > 180 had maximum number of saturated pixels producing an image having no details . to the best of our knowledge , till date the reported sensitivity , and specificity of pet based detection of hcc is based on the most widely used contrast enhancement tool , and very few attempts have been made to improve the contrast between the hcc tumor , and hepatocytes . one study was recently attempted to improve the contrast by using the principle of stochastic resonance but it can not be compared with the present study because the method of contrast enhancement is completely different . at this point , our study is different from others that the contrast enhancement technique with standardized input parameters has improved the contrast of those images which were adjusted for best possible contrast by reporting physician with existing routinely used contrast enhancement tool . the nine images included in this study were having histogram concentrated on the brighter side . there may be images whose histogram might be concentrated on the darker side or in the middle part of the histogram . this study lacks the inclusion of input image having the various types of histogram and images having a different spatial distribution of pixel values . although the sample size was small , the strength of this study is that with one original image , 847 output images were generated using various combinations of m and e ( any one of them can result in possible contrast ) the best ways to standardize the input parameters , leaving least possibility of missing best possible contrast achievable in the output image . therefore , the clinical endorsement for the improvement in the contrast of these images is required . our future plan is to clinically evaluate this digital contrast enhancement technique by selecting more than fifty images of having different spatial patterns of pixels values . the use of above mentioned digital contrast enhancement technique increases the overall contrast of the f - fdg pet images in hcc and also the contrast between the hcc tumor and hepatocytes . further clinical validation of this encouraging technique is required .

purpose : the role of 18fluorodeoxyglucose positron emission tomography ( pet ) is limited for detection of primary hepatocellular carcinoma ( hcc ) due to low contrast to the tumor , and normal hepatocytes ( background ) . the aim of the present study was to improve the contrast between the tumor and background by standardizing the input parameters of a digital contrast enhancement technique.materials and methods : a transverse slice of pet image was adjusted for the best possible contrast , and saved in jpeg 2000 format . we processed this image with a contrast enhancement technique using 847 possible combinations of input parameters ( threshold m and slope e ) . the input parameters which resulted in an image having a high value of 2nd order entropy , and edge content , and low value of absolute mean brightness error , and saturation evaluation metrics , were considered as standardized input parameters . the same process was repeated for total nine pet - computed tomography studies , thus analyzing 7623 images.results:the selected digital contrast enhancement technique increased the contrast between the hcc tumor and background . in seven out of nine images , the standardized input parameters m had values between 150 and 160 , and for other two images values were 138 and 175 , respectively . the value of slope e was 4 in 4 images , 3 in 3 images and 1 in 2 images . it was found that it is important to optimize the input parameters for the best possible contrast for each image ; a particular value was not sufficient for all the hcc images.conclusion:the use of above digital contrast enhancement technique improves the tumor to background ratio in pet images of hcc and appears to be useful . further clinical validation of this finding is warranted .

INTRODUCTION MATERIALS AND METHODS Positron emission tomography-computed tomography acquisition protocol Digital image enhancement 2 Edge content based contrast metrics Absolute mean brightness error Saturation evaluation metric RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

hepatocellular carcinoma ( hcc ) is a primary liver cancer derived from hepatocytes . in more than 80% cases the early detection of hcc has been attempted using fluorodeoxyglucose positron emission tomography ( fdg pet ) . the accumulation of fluorin - fdg ( f - fdg ) in hcc is variable due to the varying degrees of activity of the enzyme well - differentiated hcc , and low grades tumor have more or less similar degree of activity as compared to the normal liver cells , and thus results in small difference in f - fdg accumulation , and therefore , f - fdg pet picks up only 5070% of the lesion in these patients . it is possible to increase the contrast between the hcc cells , and hepatocytes on f - fdg pet using digital contrast enhancement techniques . almost all contrast enhancement technique increases the contrast of the targeted lesion at the cost of decreasing contrast in other areas of the image . it is a challenging task to find the optimum balance between the acceptable enhancements of the targeted lesion while maintaining the existing contrast in other areas of the image . very few digital contrast enhancement techniques have been tried to improve the low contrast in hcc . however , there is need to standardize the input parameters of any selected technique for the best result . in this study , we selected a contrast enhancement technique that increases the contrast in both the darker , and brighter region of the image simultaneously , and optimized the value of its input parameters for the best possible contrast for f - fdg pet images in hcc . no ethical approval was required for the study involved in the digital processing of pet images acquired for a clinical indication . patients fasted for at least 4 h before undergoing f - fdg pet - computed tomography ( pet - ct ) . an experienced nuclear medicine physician adjusted the contrast of a transverse slice of f - fdg pet image containing hcc tumor with the help of a most widely used brightness - contrast tool . once the best possible contrast in the image in his opinion was achieved , then the slice was saved in the jpeg 2000 format . a total of nine f fdg pet studies referred the case of suspected hcc were selected , and hence nine jpeg images were saved [ figure 1 ] , and their 2 order entropy values are given in table 1 . the nine original images entropy of all nine original images digital contrast enhancement of these images was carried by standardizing the value of the parameter ( m and e ) of the contrast enhancement function t ( r ) represented by equation ( 1 ) . the transform function t ( r ) compresses the input levels below , and above m into a narrow range of darker and brighter levels , respectively , and thereby improves contrast . m from 60 to 180 in the steps of 1 and for each value of m , the value of e was varied from 1 to 7 again in the steps of 1 . the same process was repeated for all the nine images and in this way 9 847 = 7623 images were visually , and quantitatively analyzed for the global contrast enhancement with respect to the original input image . the global contrast enhancement of these images was assessed by the following contrast evaluation metrics : we calculated 2 order entropy based on co - occurrence matrix using the distance of one pixel in the horizontal direction . we calculated the edge at each pixel position using the standard matlab edge function , and sobel operator , and edge content ( ec ) by dividing the sum of the value of edge by the matrix size of the image . the input image provided by the clinical team was the best images from their end ; it was the smooth image as can be seen from the figure 1 . we calculated the value of absolute mean brightness error ( ambe ) , which is the deviation of the mean intensity of the enhanced image from the mean intensity of the original image by finding the absolute difference between the mean intensity of the output , and the input image . saturation evaluation metric ( sem ) measures the saturation by computing the number of saturated pixels ( black or white pixels which were not saturated before ) after applying contrast enhancement , we calculated the value of saturation metric by dividing the number of saturated pixels ( black or white pixels which were not saturated before ) by the size of the matrix that is number of pixels in the image . the calculated values of 2 order entropy , abme , ec , and sem for each of the 7623 images were stored . we defined criteria for the selection of the image having best possible contrast among the 847 images . we defined the best possible contrast image as the image whose 2 order entropy , and ec , have high values , while ambe and sem , have low values , respectively . the input parameter ( m and e ) that resulted in best possible contrast image was considered as standardized input parameters of the contrast enhancement function . the plot of 2 order entropy , ec , ambe , and sem versus output image parameters ( m and e ) was carefully analyzed to locate the approximate position on the plot for best possible contrast image . patients fasted for at least 4 h before undergoing f - fdg pet - computed tomography ( pet - ct ) . finally , the attenuation corrected pet , ct and fused pet - ct images were available for review . an experienced nuclear medicine physician adjusted the contrast of a transverse slice of f - fdg pet image containing hcc tumor with the help of a most widely used brightness - contrast tool . once the best possible contrast in the image in his opinion was achieved , then the slice was saved in the jpeg 2000 format . a total of nine f fdg pet studies referred the case of suspected hcc were selected , and hence nine jpeg images were saved [ figure 1 ] , and their 2 order entropy values are given in table 1 . the nine original images entropy of all nine original images digital contrast enhancement of these images was carried by standardizing the value of the parameter ( m and e ) of the contrast enhancement function t ( r ) represented by equation ( 1 ) . m into a narrow range of darker and brighter levels , respectively , and thereby improves contrast . m from 60 to 180 in the steps of 1 and for each value of m , the value of e was varied from 1 to 7 again in the steps of 1 . the same process was repeated for all the nine images and in this way 9 847 = 7623 images were visually , and quantitatively analyzed for the global contrast enhancement with respect to the original input image . the global contrast enhancement of these images was assessed by the following contrast evaluation metrics : we calculated 2 order entropy based on co - occurrence matrix using the distance of one pixel in the horizontal direction . 2 order entropy gives the information about the image richness in detail taking the spatial relation of the pixel value into account . we calculated the edge at each pixel position using the standard matlab edge function , and sobel operator , and edge content ( ec ) by dividing the sum of the value of edge by the matrix size of the image . the performance of canny operator have been reported as the best , however , computationally most expensive among other such edge detection operators such as sobel , laplacian , and laplacian of gaussian . the input image provided by the clinical team was the best images from their end ; it was the smooth image as can be seen from the figure 1 . we calculated the value of absolute mean brightness error ( ambe ) , which is the deviation of the mean intensity of the enhanced image from the mean intensity of the original image by finding the absolute difference between the mean intensity of the output , and the input image . saturation evaluation metric ( sem ) measures the saturation by computing the number of saturated pixels ( black or white pixels which were not saturated before ) after applying contrast enhancement , we calculated the value of saturation metric by dividing the number of saturated pixels ( black or white pixels which were not saturated before ) by the size of the matrix that is number of pixels in the image . the calculated values of 2 order entropy , abme , ec , and sem for each of the 7623 images were stored . we defined criteria for the selection of the image having best possible contrast among the 847 images . we defined the best possible contrast image as the image whose 2 order entropy , and ec , have high values , while ambe and sem , have low values , respectively . the input parameter ( m and e ) that resulted in best possible contrast image was considered as standardized input parameters of the contrast enhancement function . the plot of 2 order entropy , ec , ambe , and sem versus output image parameters ( m and e ) was carefully analyzed to locate the approximate position on the plot for best possible contrast image . we calculated the edge at each pixel position using the standard matlab edge function , and sobel operator , and edge content ( ec ) by dividing the sum of the value of edge by the matrix size of the image . the input image provided by the clinical team was the best images from their end ; it was the smooth image as can be seen from the figure 1 . we calculated the value of absolute mean brightness error ( ambe ) , which is the deviation of the mean intensity of the enhanced image from the mean intensity of the original image by finding the absolute difference between the mean intensity of the output , and the input image . saturation evaluation metric ( sem ) measures the saturation by computing the number of saturated pixels ( black or white pixels which were not saturated before ) after applying contrast enhancement , we calculated the value of saturation metric by dividing the number of saturated pixels ( black or white pixels which were not saturated before ) by the size of the matrix that is number of pixels in the image . the calculated values of 2 order entropy , abme , ec , and sem for each of the 7623 images were stored . we defined criteria for the selection of the image having best possible contrast among the 847 images . we defined the best possible contrast image as the image whose 2 order entropy , and ec , have high values , while ambe and sem , have low values , respectively . the input parameter ( m and e ) that resulted in best possible contrast image was considered as standardized input parameters of the contrast enhancement function . the plot of 2 order entropy , ec , ambe , and sem versus output image parameters ( m and e ) was carefully analyzed to locate the approximate position on the plot for best possible contrast image . for one image , we had 847 data point to be plotted , and there were nine images . for each data points , there were four values namely : 2 order entropy , ec , ambe , and sem . the plotted data of the individual nine images were zoomed , and the following conclusions were drawn after review : with the increase in the value of m the upper , and lower limit of 2 order entropy and ec also increased , attained a maximum value at some point , after that it becomes constant . the upper and lower limit of the ambe decreased with increase in the value of m , and after some point it also attained a maximum value , and remained constant thereafter . the value of sem initially increased very slowly with an increase in the value of m , and after a certain value of m , it increased steeply . m , the trendline of contrast enhancement metrics are not the same ; in fact in some cases , they are opposite . ( b ) absolute mean brightness error on the y - axis , ( c ) edge content on the y - axis , and ( d ) saturation evaluation metric on the y - axis . the x - axis has threshold value ( m ) from 60 to 180 and at each threshold there were seven data points ( e ) making total 847 data points we wrote attained a maximum value at some point in the previous paragraph because for all nine images these points ( maximum value for 2 order entropy , ec , ambe , and sem ) were different , and moreover these values were varying at each threshold that is shown in the zoomed view of figure 2 , by taking only first 49 data point [ figure 3 ] . here the x - axis has threshold value ( m ) from 60 to 66 and at each threshold there were seven data points ( e ) making total 49 data points table 2 shows the 2 order entropy , ec , ambe , and sem values , of images having the best possible contrast . 2 ) , that is , its mean intensity was greater than the input image by 16.49 this indicates that very few pixels in the image either became white or black , as compared to the unprocessed image . the value of ambe , and sem together indicates that the overall or global brightness of the images have increased , and also very few pixels became white or black as compared to unprocessed image , and hence the contrast in the image improved . table 2 also demonstrates that a particular combination of m and e value might not be suitable for all f - fdg pet images in hcc because for each image we found different values of m and e. the best possible contrast in seven images was obtained at a threshold value of m between 150 , and 160 , and in another two images at 138 and 175 . the input parameters ( m and e ) that resulted in best image and their corresponding values of 2 order entropy , ec , abme , and sem the difference in entropy , and ec of the output images from their corresponding input images is shown in table 3 . the difference in ec is almost zero for all the nine images when the values are rounded to two decimal points , but there was the difference in the values , when the data were considered to six decimal points . the difference in entropy was also very minimal ( rounded data up to two decimal points ) , indicating that after processing , the information content of the images has reduced , however , it is very difficult to appreciate this reduction in information content visually . the image produced as a result of the application of the optimized value of input parameter m and e has been shown along with the original input image in figures 4 and 5 . from these figures , it is clear that the contrast in the output image is more than that of the input image , and simultaneously , it has not distorted the information in the other areas . deviation of entropy and ec of the output images from that of the input images 18fluorin - fluorodeoxyglucose positron emission tomography images of a patient with hepatocellular carcinoma . ( a ) original image , ( b ) image having highest value of entropy ( c ) image having highest value of edge content ( slightly more than the original image ) , ( d ) image having lowest value of absolute mean brightness error , ( e ) lowest value of saturation evaluation metric , and ( f ) image with best contrast based on high value of entropy and edge content and low value of absolute mean brightness error and saturation evaluation metric . visually it is clear that the selected transformation function in this study has produced better contrast between the tumor and normal liver cell than the original image original image along with processed image with optimum selected value of m and e for four different patients ( a - d ) . the images clearly show that the contrast of the image has improved significantly and has not distorted any information available in original image in this study , we attempted to evaluate the optimum parameters for digital contrast enhancement of f - fdg pet images in hcc . we found the different trend lines for 2 order entropy , ec , ambe , and sem at m = 60 , the darker pixels ( pixel value < 60 ) will be compressed into narrow range of darker level . thus , the edge of the tumor that lies in the darker region will improve ( because at threshold value 60 , the difference between the black and white pixel intensities will increase , thus the contrast will improve ) , and entropy , and ec will be more at m = 60 , and e = 1 . as the slope increases , there will be the more smooth transition from black to white , compressed , narrow range of black , and white pixel will relax in both the direction , and therefore , the entropy and ec will decrease . since our tumor is in black region , compression of pixel below 152 into narrow range of black will decrease the sharp transition as in the case of m = 60 , and therefore entropy , and ec will be less in comparison to that of m = 60 . the best possible contrast in seven out of nine images was obtained at threshold value m between 150 and 160 , including both 150 , and 160 . in another two images , best possible contrast was found at 138 , and 175 . this study also demonstrates that a particular combination of m and e , might not result in an image having the best possible contrast for all pet images ; because for each image we have found the different value of m and e. this is because each image is unique , and requires the unique value of m and e to produce best possible contrast . our result suggests that a default parameter may not be the best for all kind of images , a finding that agrees with other authors . therefore , we can say that the selection of threshold from the region of concentrated histogram might provide a best possible contrast in the image . our criteria for selection of parameters m and e that produce best possible contrast in an image was high value of entropy , ec , and low value of ambe , sem , as previously suggested by saleem et al . however , we did not find a single processed image ( from a set of 847 images ) that had a highest value of entropy , highest value of ec , lowest value of ambe , and lowest value of sem . this may be because with the increase in ambe , the value of sem also increases , the entropy increases with more variability in the data in spatial domain , and with the increase in entropy , it is not necessary that ec will also increase . we restricted the value of m in the range of 60180 , because in a prototype study we found that the images processed at m > 180 had maximum number of saturated pixels producing an image having no details . to the best of our knowledge , till date the reported sensitivity , and specificity of pet based detection of hcc is based on the most widely used contrast enhancement tool , and very few attempts have been made to improve the contrast between the hcc tumor , and hepatocytes . one study was recently attempted to improve the contrast by using the principle of stochastic resonance but it can not be compared with the present study because the method of contrast enhancement is completely different . at this point , our study is different from others that the contrast enhancement technique with standardized input parameters has improved the contrast of those images which were adjusted for best possible contrast by reporting physician with existing routinely used contrast enhancement tool . this study lacks the inclusion of input image having the various types of histogram and images having a different spatial distribution of pixel values . although the sample size was small , the strength of this study is that with one original image , 847 output images were generated using various combinations of m and e ( any one of them can result in possible contrast ) the best ways to standardize the input parameters , leaving least possibility of missing best possible contrast achievable in the output image . therefore , the clinical endorsement for the improvement in the contrast of these images is required . our future plan is to clinically evaluate this digital contrast enhancement technique by selecting more than fifty images of having different spatial patterns of pixels values . the use of above mentioned digital contrast enhancement technique increases the overall contrast of the f - fdg pet images in hcc and also the contrast between the hcc tumor and hepatocytes . further clinical validation of this encouraging technique is required .

dysfunctional alternative complement pathway activation reduces host cell protection and results in inappropriate complement activation of platelets and endothelial cells.1,2 atypical hemolytic uremic syndrome can cause multiorgan failure and severe neurologic changes . we present a case of severe atypical hemolytic uremic syndrome with significant neurologic damage amidst multiorgan failure , including severe renal failure . terminal complement inhibition with eculizumab safely reversed the neurologic changes and eliminated the need for dialysis . while eculizumab is not yet approved by the us food and drug administration for atypical hemolytic uremic syndrome , this case report is one of several illustrating its safety and efficacy . a fifty - year - old female with a history of rheumatoid arthritis was transferred to our intensive care unit with sepsis , pancolitis , acute renal failure , and thrombocytopenia . three days prior to transfer , she had presented with malaise , bloody diarrhea , and abdominal pain . her home medications included an estrogen patch , lansoprazole , and twice - weekly etanercept . stool studies , including tests for escherichia coli 0157:h7 and clostridium difficile , were all negative . prior to transfer , she was on ceftriaxone , ciprofloxacin , and dexamethasone for colitis . at our facility , she remained on broad - spectrum antimicrobials , including fluconazole , ciprofloxacin , metronidazole , and pipercillin - tazobactum . her initial laboratory investigations revealed a count of 12.7 gm / dl hemoglobin , white blood cells of 31.4 10e9/l , creatinine of 3.8 mg / dl , and platelets of 28 10e9/l . were later noted : c3 was 35 ( 83184 ) mg / dl and c4 was 6 ( 1759 ) mg / dl . adamts13 activity was adequate at 63% ( drawn prior to treatment ) . by hospital day 5 , there were occasional schistocytes on the peripheral smear . on the day of transfer , she underwent total abdominal colectomy with end ileostomy . during surgery she received 2 units ( u ) of packed red blood cells , 2 u of fresh frozen plasma , and 2 u of pooled platelets . after surgery , her platelets rose , and continued to rise during the first five days of the admission , without additional transfusions . the rising platelets after colectomy suggested previous platelet consumption by a thrombotic microangiopathic process in the colon . this was later confirmed by the pathology report , which described diffuse ischemic colitis with scattered fibrin microthrombi along the mesentery , consistent with microangiopathy . with mental and respiratory decline , she remained intubated . by hospital day 3 , she did not respond to commands and was generally unresponsive to voice and painful stimuli for the next 11 days . by hospital day 4 , there were reports of seizure activity . a noncontrast computed tomography scan of the brain was unremarkable ; she was too unstable to leave the intensive care unit for a magnetic resonance imaging scan of the brain . electroencephalographic findings revealed no definite epileptiform activity , but were consistent with diffuse cerebral dysfunction . her clinical picture , with severe renal failure , profound neurologic changes , severe hemolysis , thrombotic microangiopathy , and low complement levels , were all suggestive of atypical hemolytic uremic syndrome . because atypical hemolytic uremic syndrome is characterized by chronic uncontrolled systemic complement activation,2 the anti - c5 monoclonal antibody , eculizumab , was administered intravenously at a dose of 900 mg on hospital day 6 . therapeutic plasma exchange was also administered for a few hours before her first dose of eculizumab . because treatment with eculizumab increases the risk of infection with neisseria meningitidis , the meningococcal vaccine was given prior to the first dose , and 14 days of ciprofloxacin was ordered for meningococcal prophylaxis.3 four days after receiving her first dose of eculizumab , the patient s lactate dehydrogenase dropped to 837 u / l and her complement levels rose : c3 rose to 81 ( 83184 ) mg / dl and c4 rose to 16 ( 1759 ) mg / dl , as shown in figures 1 and 2 . seven days into treatment , she was opening her eyes , tracking , and localizing to painful stimuli . the day after her second dose , her lactate dehydrogenase decreased to 681 u / l . two days after the second dose she was nodding appropriately , following commands , and interacting with her family . three days after the second dose , her lactate dehydrogenase decreased to 498 u / l and her dialysis frequency was reduced to three times per week . four days after her second dose of eculizumab , she was ready to be weaned from the ventilator ; within several days , she no longer required oxygen . a week after her third dose of eculizumab , her lactate dehydrogenase was down to 349 u / l ( figure 1 ) . three days after her third dose , she was urinating up to 875 ml , and her cognition and memory had recovered to baseline . after the fourth dose of eculizumab , her urine output reached 1451 ml , and dialysis was discontinued . three days after her sixth dose , her lactate dehydrogenase , haptoglobin , c3 , and c4 were all normal ( see figures 15 for a summary of treatment responses ) . this patient presented with rapidly progressive atypical hemolytic uremic syndrome characterized by multiorgan failure , including severe neurologic decline . her renal recovery was remarkable , and she has remained off dialysis with long - term eculizumab treatment . additionally , her creatinine has continued to decrease off dialysis , with recent levels as low as 2.24 mg / dl ( see figure 3 for a summary of creatinine over time ) . while renal failure is generally the most pronounced complication of atypical hemolytic uremic syndrome , neurologic impairment can also occur in severe cases . our patient s rapid and profound neurologic deterioration appears to have resulted from significant thrombotic microangiopathy . despite 11 days in the intensive care unit without meaningful neurologic responses , the patient recovered total neurologic function after only a few doses of eculizumab . twenty - six days after the first dose of eculizumab , a magnetic resonance imaging brain scan was performed upon request by the neurologist . the findings were consistent with a subacute right parietal lobe infarction with areas of hemorrhagic conversion . there was also a suspected tiny subacute infarct within the right centrum semiovale ( figure 6 ) . despite these findings , which suggest extensive thrombotic microangiopathic brain injury , she has recovered neurologically and remains stable . the dosing schema used in this patient is similar to that used in the clinical trials of eculizumab in atypical hemolytic uremic syndrome . maintenance therapy , starting on week 5 , was planned to be 1200 mg every two weeks.46 due to nausea at that dose , the seventh dose was delayed by one day because the patient refused it , but then agreed to a reduced dose of 600 mg weekly . with antiemetic premedications , there was no nausea . nausea has been reported with eculizumab in up to 16% of cases.7 when the seventh dose was due , laboratory investigations suggested relapsing hemolysis , with haptoglobin < 6 mg / dl , indicating her continued need for the drug . as shown in figure 4 , the haptoglobin increased again after the seventh dose , and normalized to 71 mg / dl three days after the eighth dose . the annual incidence of shiga toxin - positive hemolytic uremic syndrome is two cases per 100,000 . the annual incidence of hemolytic uremic syndrome in adults in our patient s age group , ie , 5059 years , is approximately 0.5 cases per 100,000.8,9 shiga toxin - positive hemolytic uremic syndrome primarily affects children . in children under five years 90% are caused by shiga - like toxin - producing e. coli or shiga toxin - producing shigella dysenteriae . this form of hemolytic uremic syndrome , classified as typical or enteropathic hemolytic uremic syndrome , involves diarrhea followed by acute renal failure . streptococcus pneumoniae also causes a severe form of hemolytic uremic syndrome unrelated to shiga or shiga - like toxin - producing organisms.8,9 a minority of hemolytic uremic syndrome cases , generally unrelated to shiga / shiga - like toxin or s. pneumoniae , are termed atypical hemolytic uremic syndrome . atypical hemolytic uremic syndrome accounts for approximately 10% of pediatric hemolytic uremic syndrome cases and the majority of adult cases.810 the prevalence and incidence of atypical hemolytic uremic syndrome , a rare condition , is not well established.11 the estimated prevalence of atypical hemolytic uremic syndrome in european children under 18 years is approximately 3.3 per million.12 the prevalence in adults may be lower . the estimated overall annual incidence of atypical hemolytic uremic syndrome in the us is one in 500,000.13 the pathogenesis of atypical hemolytic uremic syndrome involves genetic or acquired alternative complement pathway regulator deficiencies.810 in atypical hemolytic uremic syndrome , reduced c3 levels may sometimes be observed , further suggesting uncontrolled complement activation and consumption . because the alternative complement pathway convertase requires cleavage of c3 and not c4 , low serum c3 and normal c4 suggest selective alternative pathway activation.9,14 in typical hemolytic uremic syndrome , renal function is preserved in 75% of cases , whereas the majority of patients with atypical hemolytic uremic syndrome develop end - stage renal disease or die.8,9,15,16 in the acute phase of hemolytic uremic syndrome , mortality is 3%5% . in contrast , atypical hemolytic uremic syndrome has a significantly worse prognosis , with a mortality of up to 25% in the acute phase.8,9,14 under 20% of atypical hemolytic uremic syndrome cases are familial . the rest are considered to be sporadic , and are triggered by a variety of pathologies , including human immunodeficiency virus , chemotherapeutic agents , immunotherapeutic agents , antiplatelet agents , cancer , transplant , and pregnancy . in the post - transplant setting , de novo hemolytic uremic syndrome has been associated with calcineurin inhibitors ( cyclosporine [ 5%15% ] , tacrolimus [ 1% ] ) , and humoral rejection . approximately 50% of sporadic cases of atypical hemolytic uremic syndrome show no obvious cause and are considered idiopathic.8,9 prodromic diarrhea has often been used to distinguish typical from atypical hemolytic uremic syndrome . our case of atypical hemolytic uremic syndrome demonstrates how prodromic diarrhea alone can not be used to distinguish these two conditions . approximately 20%30% of atypical hemolytic uremic syndrome cases involve prodromic diarrhea.14 although our patient s stool studies did not reveal any shiga or shiga - like toxin - producing organisms , these organisms are not always recoverable from the stool of patients with hemolytic uremic syndrome . such organisms may trigger hemolytic uremic syndrome in patients with pre - existing complement dysregulation.10 in our patient , thrombotic microangiopathy resulting in ischemic bowel could have caused her diarrhea and hematochezia . in the absence of shiga toxin in stool , assessing a patient s complement system function is appropriate . 14 our patient was on etanercept and an estrogen patch , both of which are potential endothelial offenders and precipitants of atypical hemolytic uremic syndrome / complement dysregulation . eculizumab , a high - affinity humanized monoclonal anti - c5 antibody , blocks terminal complement activity by binding to and blocking cleavage of the terminal complement protein , c5 , into its proinflammatory , prothrombotic , and lytic products : c5a and the cytotoxic membrane - attack complex , c5b-9 . upstream components of complement , most notably c3b , are left intact.3,17,18 in several case reports , eculizumab has been reported to be safe and effective in atypical hemolytic uremic syndrome , a condition of chronic systemic uncontrolled complement activation.3,1727 the doses used in the current clinical trials of eculizumab in atypical hemolytic uremic syndrome are higher than the doses used in paroxysmal nocturnal hemoglobinuria.47 this seems reasonable , given the risk of multiorgan failure as a result of thrombotic microangiopathy in atypical hemolytic uremic syndrome . although no prospective controlled trials have been conducted , plasma exchange has historically been used to manage patients with atypical hemolytic uremic syndrome.28 plasma exchange generally must be used with high frequency to stabilize the condition,24 and often loses efficacy . 16 additionally , plasma intervention has been associated with a 26% chance of major complications.29 furthermore , compared with eculizumab , plasma exchange does not specifically treat the underlying defect in complement pathway regulation.28 thus , eculizumab may prove to be more efficacious in the long term , and further investigation is warranted . complete and sustained terminal complement inhibition is considered necessary for adequate control of atypical hemolytic uremic syndrome . the optimal duration and frequency of therapy remains to be determined and varies among patients . for example , nurnberger et al described an adult with relapsed atypical hemolytic uremic syndrome , despite a second kidney transplant , who maintained remission after only one dose of eculizumab 600 mg.17 unfortunately , 21 months later , this patient suffered allograft loss after failing eculizumab retreatment for recurrence of atypical hemolytic uremic syndrome.14 in contrast , gruppo et al described an infant with atypical hemolytic uremic syndrome refractory to plasma therapy , who required maintenance eculizumab to sustain remission . remission occurred 10 days after the first dose , and lasted for at least four months on maintenance eculizumab 600 mg every two weeks.25 mache et al discussed eculizumab dosing for an adolescent with plasmapheresis - refractory atypical hemolytic uremic syndrome . an initial 600 mg was administered , and platelets normalized in three days , haptoglobin normalized in five days , and renal function improved . atypical hemolytic uremic syndrome relapsed two weeks after recovery of complement - mediated hemolytic activity . treatment with 600 mg was repeated three times in six days , with only minor , transient improvement in renal function , and hemodialysis was ultimately required.1 chatelet et al described the consequences of delaying the ninth dose of eculizumab in a renal transplant patient with recurrent atypical hemolytic uremic syndrome . the patient was on a maintenance dose of 1200 mg every 14 days after receiving 900 mg weekly for four doses . they reported that a six - day delay in administering the ninth dose resulted in recurrent hemolysis and deterioration of transplant function , necessitating resumption of eculizumab . the authors resumed eculizumab as permanent therapy for their patient , hypothesizing that the delayed dose caused complement breakthrough and relapse of thrombotic microangiopathy.20 importantly , after 17 months of eculizumab therapy , acute thrombotic microangiopathy and hemolysis were controlled , renal function was maintained , and the need for blood transfusion decreased.2 around 60% of atypical hemolytic uremic syndrome cases , both familial and sporadic , have been linked to genetic mutations in complement regulatory proteins in the alternative complement pathway or autoantibodies to complement factor h ( cfh).8,9 consequently , we ordered genetic and antibody tests for our patient and the results are still pending . information on genetic abnormalities of the complement system as it relates to atypical hemolytic uremic syndrome can be obtained from two comprehensive papers by norris and remuzzi.8,9 genetic testing is important in the care of patients with atypical hemolytic uremic syndrome because prognosis can vary depending on the gene mutations involved . for example , cfh mutations , with cardiac complications in 20% of cases , are associated with a worse long - term survival . cfh mutations are associated with a 10-year survival rate of only 40%50% compared with cases of anti - cfh antibodies , cfi mutations , and c3 mutations , which have a 10-year survival rate of around 80%90% . furthermore , genetic testing provides the clinician with prognostic and predictive information about the disease course , treatment response , and long - term transplant outcome.8,9 healthy carriers of cfh , mcp , or cfi mutations have an approximately 50% chance of developing atypical hemolytic uremic syndrome . this suggests that complement gene mutations are predisposing rather than causative , and expression of atypical hemolytic uremic syndrome is dependent upon other genetic or environmental factors.1,8,9 genetic testing is relevant in unaffected family members because mutation carriers can be monitored during conditions triggering complement activation , such as infections , drug exposures ( including oral contraceptives ) , and pregnancy . drugs and pregnancy indirectly trigger complement activation by causing endothelial insult.8,9 genetic testing is important for patients with atypical hemolytic uremic syndrome being considered for renal transplant . while outcomes of renal transplant vary depending on the genetic mutations , overall , there is a 50% rate of atypical hemolytic uremic syndrome recurrence in the allograft.8,9 mutations of cfh , cfi , cfb and c3 are associated with post - transplant recurrence . this is because the mutations give rise to abnormalities in circulating proteins which mostly come from the liver , and thus can exist even after kidney transplant.8,9 in total , 80%90% of patients with cfh and cfi mutations had allograft relapse following transplant . consequently , simultaneous liver - kidney transplant has been recommended for these patients.8,9 however , zuber et al described pre - emptive plasma therapy and eculizumab as promising treatments which may reduce the need for simultaneous liver - kidney transplant.14 for patients with c3 mutations undergoing kidney transplant , there is a 40%50% risk of recurrence in the allograft . however , due to significant extrahepatic synthesis of c3 , combined kidney - liver transplant is not recommended . furthermore , for patients with cfb mutations , isolated kidney transplant and combined kidney - liver transplant is not recommended , because those patients are at increased surgical risk due to severe vascular disease.8,9 the annual incidence of shiga toxin - positive hemolytic uremic syndrome is two cases per 100,000 . the annual incidence of hemolytic uremic syndrome in adults in our patient s age group , ie , 5059 years , is approximately 0.5 cases per 100,000.8,9 shiga toxin - positive hemolytic uremic syndrome primarily affects children . in children under five years 90% are caused by shiga - like toxin - producing e. coli or shiga toxin - producing shigella dysenteriae . this form of hemolytic uremic syndrome , classified as typical or enteropathic hemolytic uremic syndrome , involves diarrhea followed by acute renal failure . streptococcus pneumoniae also causes a severe form of hemolytic uremic syndrome unrelated to shiga or shiga - like toxin - producing organisms.8,9 a minority of hemolytic uremic syndrome cases , generally unrelated to shiga / shiga - like toxin or s. pneumoniae , are termed atypical hemolytic uremic syndrome . atypical hemolytic uremic syndrome accounts for approximately 10% of pediatric hemolytic uremic syndrome cases and the majority of adult cases.810 the prevalence and incidence of atypical hemolytic uremic syndrome , a rare condition , is not well established.11 the estimated prevalence of atypical hemolytic uremic syndrome in european children under 18 years is approximately 3.3 per million.12 the prevalence in adults may be lower . the estimated overall annual incidence of atypical hemolytic uremic syndrome in the us is one in 500,000.13 the pathogenesis of atypical hemolytic uremic syndrome involves genetic or acquired alternative complement pathway regulator deficiencies.810 in atypical hemolytic uremic syndrome , reduced c3 levels may sometimes be observed , further suggesting uncontrolled complement activation and consumption . because the alternative complement pathway convertase requires cleavage of c3 and not c4 , low serum c3 and normal c4 suggest selective alternative pathway activation.9,14 in typical hemolytic uremic syndrome , renal function is preserved in 75% of cases , whereas the majority of patients with atypical hemolytic uremic syndrome develop end - stage renal disease or die.8,9,15,16 in the acute phase of hemolytic uremic syndrome , mortality is 3%5% . in contrast , atypical hemolytic uremic syndrome has a significantly worse prognosis , with a mortality of up to 25% in the acute phase.8,9,14 under 20% of atypical hemolytic uremic syndrome cases are familial . the rest are considered to be sporadic , and are triggered by a variety of pathologies , including human immunodeficiency virus , chemotherapeutic agents , immunotherapeutic agents , antiplatelet agents , cancer , transplant , and pregnancy . in the post - transplant setting , de novo hemolytic uremic syndrome has been associated with calcineurin inhibitors ( cyclosporine [ 5%15% ] , tacrolimus [ 1% ] ) , and humoral rejection . approximately 50% of sporadic cases of atypical hemolytic uremic syndrome show no obvious cause and are considered idiopathic.8,9 prodromic diarrhea has often been used to distinguish typical from atypical hemolytic uremic syndrome . our case of atypical hemolytic uremic syndrome demonstrates how prodromic diarrhea alone can not be used to distinguish these two conditions . approximately 20%30% of atypical hemolytic uremic syndrome cases involve prodromic diarrhea.14 although our patient s stool studies did not reveal any shiga or shiga - like toxin - producing organisms , these organisms are not always recoverable from the stool of patients with hemolytic uremic syndrome . such organisms may trigger hemolytic uremic syndrome in patients with pre - existing complement dysregulation.10 in our patient , thrombotic microangiopathy resulting in ischemic bowel could have caused her diarrhea and hematochezia . in the absence of shiga toxin in stool , assessing a patient s complement system function is appropriate . 14 our patient was on etanercept and an estrogen patch , both of which are potential endothelial offenders and precipitants of atypical hemolytic uremic syndrome / complement dysregulation . eculizumab , a high - affinity humanized monoclonal anti - c5 antibody , blocks terminal complement activity by binding to and blocking cleavage of the terminal complement protein , c5 , into its proinflammatory , prothrombotic , and lytic products : c5a and the cytotoxic membrane - attack complex , c5b-9 . upstream components of complement , most notably c3b , are left intact.3,17,18 in several case reports , eculizumab has been reported to be safe and effective in atypical hemolytic uremic syndrome , a condition of chronic systemic uncontrolled complement activation.3,1727 the doses used in the current clinical trials of eculizumab in atypical hemolytic uremic syndrome are higher than the doses used in paroxysmal nocturnal hemoglobinuria.47 this seems reasonable , given the risk of multiorgan failure as a result of thrombotic microangiopathy in atypical hemolytic uremic syndrome . although no prospective controlled trials have been conducted , plasma exchange has historically been used to manage patients with atypical hemolytic uremic syndrome.28 plasma exchange generally must be used with high frequency to stabilize the condition,24 and often loses efficacy . 16 additionally , plasma intervention has been associated with a 26% chance of major complications.29 furthermore , compared with eculizumab , plasma exchange does not specifically treat the underlying defect in complement pathway regulation.28 thus , eculizumab may prove to be more efficacious in the long term , and further investigation is warranted . complete and sustained terminal complement inhibition is considered necessary for adequate control of atypical hemolytic uremic syndrome . the optimal duration and frequency of therapy remains to be determined and varies among patients . for example , nurnberger et al described an adult with relapsed atypical hemolytic uremic syndrome , despite a second kidney transplant , who maintained remission after only one dose of eculizumab 600 mg.17 unfortunately , 21 months later , this patient suffered allograft loss after failing eculizumab retreatment for recurrence of atypical hemolytic uremic syndrome.14 in contrast , gruppo et al described an infant with atypical hemolytic uremic syndrome refractory to plasma therapy , who required maintenance eculizumab to sustain remission . remission occurred 10 days after the first dose , and lasted for at least four months on maintenance eculizumab 600 mg every two weeks.25 mache et al discussed eculizumab dosing for an adolescent with plasmapheresis - refractory atypical hemolytic uremic syndrome . an initial 600 mg was administered , and platelets normalized in three days , haptoglobin normalized in five days , and renal function improved . atypical hemolytic uremic syndrome relapsed two weeks after recovery of complement - mediated hemolytic activity . treatment with 600 mg was repeated three times in six days , with only minor , transient improvement in renal function , and hemodialysis was ultimately required.1 chatelet et al described the consequences of delaying the ninth dose of eculizumab in a renal transplant patient with recurrent atypical hemolytic uremic syndrome . the patient was on a maintenance dose of 1200 mg every 14 days after receiving 900 mg weekly for four doses . they reported that a six - day delay in administering the ninth dose resulted in recurrent hemolysis and deterioration of transplant function , necessitating resumption of eculizumab . the authors resumed eculizumab as permanent therapy for their patient , hypothesizing that the delayed dose caused complement breakthrough and relapse of thrombotic microangiopathy.20 importantly , after 17 months of eculizumab therapy , acute thrombotic microangiopathy and hemolysis were controlled , renal function was maintained , and the need for blood transfusion decreased.2 around 60% of atypical hemolytic uremic syndrome cases , both familial and sporadic , have been linked to genetic mutations in complement regulatory proteins in the alternative complement pathway or autoantibodies to complement factor h ( cfh).8,9 consequently , we ordered genetic and antibody tests for our patient and the results are still pending . information on genetic abnormalities of the complement system as it relates to atypical hemolytic uremic syndrome can be obtained from two comprehensive papers by norris and remuzzi.8,9 genetic testing is important in the care of patients with atypical hemolytic uremic syndrome because prognosis can vary depending on the gene mutations involved . for example , cfh mutations , with cardiac complications in 20% of cases , are associated with a worse long - term survival . cfh mutations are associated with a 10-year survival rate of only 40%50% compared with cases of anti - cfh antibodies , cfi mutations , and c3 mutations , which have a 10-year survival rate of around 80%90% . furthermore , genetic testing provides the clinician with prognostic and predictive information about the disease course , treatment response , and long - term transplant outcome.8,9 healthy carriers of cfh , mcp , or cfi mutations have an approximately 50% chance of developing atypical hemolytic uremic syndrome . this suggests that complement gene mutations are predisposing rather than causative , and expression of atypical hemolytic uremic syndrome is dependent upon other genetic or environmental factors.1,8,9 genetic testing is relevant in unaffected family members because mutation carriers can be monitored during conditions triggering complement activation , such as infections , drug exposures ( including oral contraceptives ) , and pregnancy . drugs and pregnancy indirectly trigger complement activation by causing endothelial insult.8,9 genetic testing is important for patients with atypical hemolytic uremic syndrome being considered for renal transplant . while outcomes of renal transplant vary depending on the genetic mutations , overall , there is a 50% rate of atypical hemolytic uremic syndrome recurrence in the allograft.8,9 mutations of cfh , cfi , cfb and c3 are associated with post - transplant recurrence . this is because the mutations give rise to abnormalities in circulating proteins which mostly come from the liver , and thus can exist even after kidney transplant.8,9 in total , 80%90% of patients with cfh and cfi mutations had allograft relapse following transplant . consequently , simultaneous liver - kidney transplant has been recommended for these patients.8,9 however , zuber et al described pre - emptive plasma therapy and eculizumab as promising treatments which may reduce the need for simultaneous liver - kidney transplant.14 for patients with c3 mutations undergoing kidney transplant , there is a 40%50% risk of recurrence in the allograft . however , due to significant extrahepatic synthesis of c3 , combined kidney - liver transplant is not recommended . furthermore , for patients with cfb mutations , isolated kidney transplant and combined kidney - liver transplant is not recommended , because those patients are at increased surgical risk due to severe vascular disease.8,9 eculizumab can safely reverse neurologic impairment and eliminate the need for dialysis in severe atypical hemolytic uremic syndrome . because clinical trials are underway , the optimal duration of treatment with eculizumab remains to be determined . although genetic abnormalities in complement regulatory factors or anti - cfh antibodies have been identified in approximately 60% of cases , genetic and antibody testing is not required for diagnosis of atypical hemolytic uremic syndrome.8,9 because 20%30% of atypical hemolytic uremic syndrome cases present with diarrhea , this presenting symptom alone can not be used to distinguish atypical and enteropathic hemolytic uremic syndrome.14

this case report describes how eculizumab reversed neurologic impairment and improved renal damage in severe atypical hemolytic uremic syndrome . a 50-year - old female , after presenting with diarrhea and abdominal pain , developed pancolitis , acute renal failure , and thrombocytopenia . the patient underwent total abdominal colectomy . pathology confirmed ischemic colitis with scattered mesenteric microthrombi . due to mental and respiratory decline , she remained intubated . continuous venovenous hemodialysis was initiated . renal failure , neurologic changes , hemolysis , thrombotic microangiopathy , and low complement levels all suggested atypical hemolytic uremic syndrome . eculizumab 900 mg was administered intravenously on hospital day 6 and continued weekly for four doses followed by maintenance therapy . she recovered neurologically and renally after the third dose , and hematologically by the sixth dose . her recovery has been sustained on long - term eculizumab treatment . in severe atypical hemolytic uremic syndrome , eculizumab safely reverses neurologic impairment and eliminates the need for dialysis . the optimal duration of treatment with eculizumab remains to be determined .

Introduction Case report Discussion Atypical versus typical hemolytic uremic syndrome Eculizumab in atypical hemolytic uremic syndrome Genetic abnormalities associated with atypical hemolytic uremic syndrome Conclusion

dysfunctional alternative complement pathway activation reduces host cell protection and results in inappropriate complement activation of platelets and endothelial cells.1,2 atypical hemolytic uremic syndrome can cause multiorgan failure and severe neurologic changes . we present a case of severe atypical hemolytic uremic syndrome with significant neurologic damage amidst multiorgan failure , including severe renal failure . terminal complement inhibition with eculizumab safely reversed the neurologic changes and eliminated the need for dialysis . while eculizumab is not yet approved by the us food and drug administration for atypical hemolytic uremic syndrome , this case report is one of several illustrating its safety and efficacy . a fifty - year - old female with a history of rheumatoid arthritis was transferred to our intensive care unit with sepsis , pancolitis , acute renal failure , and thrombocytopenia . three days prior to transfer , she had presented with malaise , bloody diarrhea , and abdominal pain . at our facility , she remained on broad - spectrum antimicrobials , including fluconazole , ciprofloxacin , metronidazole , and pipercillin - tazobactum . on the day of transfer , she underwent total abdominal colectomy with end ileostomy . this was later confirmed by the pathology report , which described diffuse ischemic colitis with scattered fibrin microthrombi along the mesentery , consistent with microangiopathy . with mental and respiratory decline , she remained intubated . her clinical picture , with severe renal failure , profound neurologic changes , severe hemolysis , thrombotic microangiopathy , and low complement levels , were all suggestive of atypical hemolytic uremic syndrome . because atypical hemolytic uremic syndrome is characterized by chronic uncontrolled systemic complement activation,2 the anti - c5 monoclonal antibody , eculizumab , was administered intravenously at a dose of 900 mg on hospital day 6 . because treatment with eculizumab increases the risk of infection with neisseria meningitidis , the meningococcal vaccine was given prior to the first dose , and 14 days of ciprofloxacin was ordered for meningococcal prophylaxis.3 four days after receiving her first dose of eculizumab , the patient s lactate dehydrogenase dropped to 837 u / l and her complement levels rose : c3 rose to 81 ( 83184 ) mg / dl and c4 rose to 16 ( 1759 ) mg / dl , as shown in figures 1 and 2 . three days after her third dose , she was urinating up to 875 ml , and her cognition and memory had recovered to baseline . three days after her sixth dose , her lactate dehydrogenase , haptoglobin , c3 , and c4 were all normal ( see figures 15 for a summary of treatment responses ) . this patient presented with rapidly progressive atypical hemolytic uremic syndrome characterized by multiorgan failure , including severe neurologic decline . her renal recovery was remarkable , and she has remained off dialysis with long - term eculizumab treatment . while renal failure is generally the most pronounced complication of atypical hemolytic uremic syndrome , neurologic impairment can also occur in severe cases . despite these findings , which suggest extensive thrombotic microangiopathic brain injury , she has recovered neurologically and remains stable . the dosing schema used in this patient is similar to that used in the clinical trials of eculizumab in atypical hemolytic uremic syndrome . maintenance therapy , starting on week 5 , was planned to be 1200 mg every two weeks.46 due to nausea at that dose , the seventh dose was delayed by one day because the patient refused it , but then agreed to a reduced dose of 600 mg weekly . nausea has been reported with eculizumab in up to 16% of cases.7 when the seventh dose was due , laboratory investigations suggested relapsing hemolysis , with haptoglobin < 6 mg / dl , indicating her continued need for the drug . as shown in figure 4 , the haptoglobin increased again after the seventh dose , and normalized to 71 mg / dl three days after the eighth dose . this form of hemolytic uremic syndrome , classified as typical or enteropathic hemolytic uremic syndrome , involves diarrhea followed by acute renal failure . streptococcus pneumoniae also causes a severe form of hemolytic uremic syndrome unrelated to shiga or shiga - like toxin - producing organisms.8,9 a minority of hemolytic uremic syndrome cases , generally unrelated to shiga / shiga - like toxin or s. pneumoniae , are termed atypical hemolytic uremic syndrome . atypical hemolytic uremic syndrome accounts for approximately 10% of pediatric hemolytic uremic syndrome cases and the majority of adult cases.810 the prevalence and incidence of atypical hemolytic uremic syndrome , a rare condition , is not well established.11 the estimated prevalence of atypical hemolytic uremic syndrome in european children under 18 years is approximately 3.3 per million.12 the prevalence in adults may be lower . the estimated overall annual incidence of atypical hemolytic uremic syndrome in the us is one in 500,000.13 the pathogenesis of atypical hemolytic uremic syndrome involves genetic or acquired alternative complement pathway regulator deficiencies.810 in atypical hemolytic uremic syndrome , reduced c3 levels may sometimes be observed , further suggesting uncontrolled complement activation and consumption . because the alternative complement pathway convertase requires cleavage of c3 and not c4 , low serum c3 and normal c4 suggest selective alternative pathway activation.9,14 in typical hemolytic uremic syndrome , renal function is preserved in 75% of cases , whereas the majority of patients with atypical hemolytic uremic syndrome develop end - stage renal disease or die.8,9,15,16 in the acute phase of hemolytic uremic syndrome , mortality is 3%5% . in the post - transplant setting , de novo hemolytic uremic syndrome has been associated with calcineurin inhibitors ( cyclosporine [ 5%15% ] , tacrolimus [ 1% ] ) , and humoral rejection . such organisms may trigger hemolytic uremic syndrome in patients with pre - existing complement dysregulation.10 in our patient , thrombotic microangiopathy resulting in ischemic bowel could have caused her diarrhea and hematochezia . upstream components of complement , most notably c3b , are left intact.3,17,18 in several case reports , eculizumab has been reported to be safe and effective in atypical hemolytic uremic syndrome , a condition of chronic systemic uncontrolled complement activation.3,1727 the doses used in the current clinical trials of eculizumab in atypical hemolytic uremic syndrome are higher than the doses used in paroxysmal nocturnal hemoglobinuria.47 this seems reasonable , given the risk of multiorgan failure as a result of thrombotic microangiopathy in atypical hemolytic uremic syndrome . 16 additionally , plasma intervention has been associated with a 26% chance of major complications.29 furthermore , compared with eculizumab , plasma exchange does not specifically treat the underlying defect in complement pathway regulation.28 thus , eculizumab may prove to be more efficacious in the long term , and further investigation is warranted . the optimal duration and frequency of therapy remains to be determined and varies among patients . for example , nurnberger et al described an adult with relapsed atypical hemolytic uremic syndrome , despite a second kidney transplant , who maintained remission after only one dose of eculizumab 600 mg.17 unfortunately , 21 months later , this patient suffered allograft loss after failing eculizumab retreatment for recurrence of atypical hemolytic uremic syndrome.14 in contrast , gruppo et al described an infant with atypical hemolytic uremic syndrome refractory to plasma therapy , who required maintenance eculizumab to sustain remission . remission occurred 10 days after the first dose , and lasted for at least four months on maintenance eculizumab 600 mg every two weeks.25 mache et al discussed eculizumab dosing for an adolescent with plasmapheresis - refractory atypical hemolytic uremic syndrome . an initial 600 mg was administered , and platelets normalized in three days , haptoglobin normalized in five days , and renal function improved . atypical hemolytic uremic syndrome relapsed two weeks after recovery of complement - mediated hemolytic activity . treatment with 600 mg was repeated three times in six days , with only minor , transient improvement in renal function , and hemodialysis was ultimately required.1 chatelet et al described the consequences of delaying the ninth dose of eculizumab in a renal transplant patient with recurrent atypical hemolytic uremic syndrome . the patient was on a maintenance dose of 1200 mg every 14 days after receiving 900 mg weekly for four doses . the authors resumed eculizumab as permanent therapy for their patient , hypothesizing that the delayed dose caused complement breakthrough and relapse of thrombotic microangiopathy.20 importantly , after 17 months of eculizumab therapy , acute thrombotic microangiopathy and hemolysis were controlled , renal function was maintained , and the need for blood transfusion decreased.2 around 60% of atypical hemolytic uremic syndrome cases , both familial and sporadic , have been linked to genetic mutations in complement regulatory proteins in the alternative complement pathway or autoantibodies to complement factor h ( cfh).8,9 consequently , we ordered genetic and antibody tests for our patient and the results are still pending . furthermore , genetic testing provides the clinician with prognostic and predictive information about the disease course , treatment response , and long - term transplant outcome.8,9 healthy carriers of cfh , mcp , or cfi mutations have an approximately 50% chance of developing atypical hemolytic uremic syndrome . this suggests that complement gene mutations are predisposing rather than causative , and expression of atypical hemolytic uremic syndrome is dependent upon other genetic or environmental factors.1,8,9 genetic testing is relevant in unaffected family members because mutation carriers can be monitored during conditions triggering complement activation , such as infections , drug exposures ( including oral contraceptives ) , and pregnancy . drugs and pregnancy indirectly trigger complement activation by causing endothelial insult.8,9 genetic testing is important for patients with atypical hemolytic uremic syndrome being considered for renal transplant . while outcomes of renal transplant vary depending on the genetic mutations , overall , there is a 50% rate of atypical hemolytic uremic syndrome recurrence in the allograft.8,9 mutations of cfh , cfi , cfb and c3 are associated with post - transplant recurrence . consequently , simultaneous liver - kidney transplant has been recommended for these patients.8,9 however , zuber et al described pre - emptive plasma therapy and eculizumab as promising treatments which may reduce the need for simultaneous liver - kidney transplant.14 for patients with c3 mutations undergoing kidney transplant , there is a 40%50% risk of recurrence in the allograft . this form of hemolytic uremic syndrome , classified as typical or enteropathic hemolytic uremic syndrome , involves diarrhea followed by acute renal failure . atypical hemolytic uremic syndrome accounts for approximately 10% of pediatric hemolytic uremic syndrome cases and the majority of adult cases.810 the prevalence and incidence of atypical hemolytic uremic syndrome , a rare condition , is not well established.11 the estimated prevalence of atypical hemolytic uremic syndrome in european children under 18 years is approximately 3.3 per million.12 the prevalence in adults may be lower . the estimated overall annual incidence of atypical hemolytic uremic syndrome in the us is one in 500,000.13 the pathogenesis of atypical hemolytic uremic syndrome involves genetic or acquired alternative complement pathway regulator deficiencies.810 in atypical hemolytic uremic syndrome , reduced c3 levels may sometimes be observed , further suggesting uncontrolled complement activation and consumption . because the alternative complement pathway convertase requires cleavage of c3 and not c4 , low serum c3 and normal c4 suggest selective alternative pathway activation.9,14 in typical hemolytic uremic syndrome , renal function is preserved in 75% of cases , whereas the majority of patients with atypical hemolytic uremic syndrome develop end - stage renal disease or die.8,9,15,16 in the acute phase of hemolytic uremic syndrome , mortality is 3%5% . in the post - transplant setting , de novo hemolytic uremic syndrome has been associated with calcineurin inhibitors ( cyclosporine [ 5%15% ] , tacrolimus [ 1% ] ) , and humoral rejection . such organisms may trigger hemolytic uremic syndrome in patients with pre - existing complement dysregulation.10 in our patient , thrombotic microangiopathy resulting in ischemic bowel could have caused her diarrhea and hematochezia . 14 our patient was on etanercept and an estrogen patch , both of which are potential endothelial offenders and precipitants of atypical hemolytic uremic syndrome / complement dysregulation . upstream components of complement , most notably c3b , are left intact.3,17,18 in several case reports , eculizumab has been reported to be safe and effective in atypical hemolytic uremic syndrome , a condition of chronic systemic uncontrolled complement activation.3,1727 the doses used in the current clinical trials of eculizumab in atypical hemolytic uremic syndrome are higher than the doses used in paroxysmal nocturnal hemoglobinuria.47 this seems reasonable , given the risk of multiorgan failure as a result of thrombotic microangiopathy in atypical hemolytic uremic syndrome . although no prospective controlled trials have been conducted , plasma exchange has historically been used to manage patients with atypical hemolytic uremic syndrome.28 plasma exchange generally must be used with high frequency to stabilize the condition,24 and often loses efficacy . 16 additionally , plasma intervention has been associated with a 26% chance of major complications.29 furthermore , compared with eculizumab , plasma exchange does not specifically treat the underlying defect in complement pathway regulation.28 thus , eculizumab may prove to be more efficacious in the long term , and further investigation is warranted . complete and sustained terminal complement inhibition is considered necessary for adequate control of atypical hemolytic uremic syndrome . the optimal duration and frequency of therapy remains to be determined and varies among patients . for example , nurnberger et al described an adult with relapsed atypical hemolytic uremic syndrome , despite a second kidney transplant , who maintained remission after only one dose of eculizumab 600 mg.17 unfortunately , 21 months later , this patient suffered allograft loss after failing eculizumab retreatment for recurrence of atypical hemolytic uremic syndrome.14 in contrast , gruppo et al described an infant with atypical hemolytic uremic syndrome refractory to plasma therapy , who required maintenance eculizumab to sustain remission . remission occurred 10 days after the first dose , and lasted for at least four months on maintenance eculizumab 600 mg every two weeks.25 mache et al discussed eculizumab dosing for an adolescent with plasmapheresis - refractory atypical hemolytic uremic syndrome . atypical hemolytic uremic syndrome relapsed two weeks after recovery of complement - mediated hemolytic activity . treatment with 600 mg was repeated three times in six days , with only minor , transient improvement in renal function , and hemodialysis was ultimately required.1 chatelet et al described the consequences of delaying the ninth dose of eculizumab in a renal transplant patient with recurrent atypical hemolytic uremic syndrome . the patient was on a maintenance dose of 1200 mg every 14 days after receiving 900 mg weekly for four doses . the authors resumed eculizumab as permanent therapy for their patient , hypothesizing that the delayed dose caused complement breakthrough and relapse of thrombotic microangiopathy.20 importantly , after 17 months of eculizumab therapy , acute thrombotic microangiopathy and hemolysis were controlled , renal function was maintained , and the need for blood transfusion decreased.2 around 60% of atypical hemolytic uremic syndrome cases , both familial and sporadic , have been linked to genetic mutations in complement regulatory proteins in the alternative complement pathway or autoantibodies to complement factor h ( cfh).8,9 consequently , we ordered genetic and antibody tests for our patient and the results are still pending . furthermore , genetic testing provides the clinician with prognostic and predictive information about the disease course , treatment response , and long - term transplant outcome.8,9 healthy carriers of cfh , mcp , or cfi mutations have an approximately 50% chance of developing atypical hemolytic uremic syndrome . this suggests that complement gene mutations are predisposing rather than causative , and expression of atypical hemolytic uremic syndrome is dependent upon other genetic or environmental factors.1,8,9 genetic testing is relevant in unaffected family members because mutation carriers can be monitored during conditions triggering complement activation , such as infections , drug exposures ( including oral contraceptives ) , and pregnancy . drugs and pregnancy indirectly trigger complement activation by causing endothelial insult.8,9 genetic testing is important for patients with atypical hemolytic uremic syndrome being considered for renal transplant . while outcomes of renal transplant vary depending on the genetic mutations , overall , there is a 50% rate of atypical hemolytic uremic syndrome recurrence in the allograft.8,9 mutations of cfh , cfi , cfb and c3 are associated with post - transplant recurrence . furthermore , for patients with cfb mutations , isolated kidney transplant and combined kidney - liver transplant is not recommended , because those patients are at increased surgical risk due to severe vascular disease.8,9 eculizumab can safely reverse neurologic impairment and eliminate the need for dialysis in severe atypical hemolytic uremic syndrome . because clinical trials are underway , the optimal duration of treatment with eculizumab remains to be determined . although genetic abnormalities in complement regulatory factors or anti - cfh antibodies have been identified in approximately 60% of cases , genetic and antibody testing is not required for diagnosis of atypical hemolytic uremic syndrome.8,9 because 20%30% of atypical hemolytic uremic syndrome cases present with diarrhea , this presenting symptom alone can not be used to distinguish atypical and enteropathic hemolytic uremic syndrome.14

all participants were prospectively recruited and underwent a comprehensive eye examination in the department of ophthalmology at the norfolk and norwich university hospital ( norwich , uk ) and had snellen equivalent visual acuity of 6/12 or better and a normal appearance of the cornea by slit - lamp biomicroscopy . all patients with glaucoma ( primary open angle glaucoma [ poag ] or normal tension glaucoma [ ntg ] ) had glaucomatous visual field defects and optic disc characteristics consistent with glaucoma . patients with poag had pretreatment iops that were consistently > 22 mmhg and the diagnosis of ntg was established using the collaborative normal - tension glaucoma study criteria ( cntgs 1998 ) . patients with ocular hypertension ( oh ) had iop measurements consistently > 22 mmhg and no visual field defect identified by standard automated perimetry . patients were defined as glaucoma suspects ( gs ) if they had an optic nerve head appearance and/or visual field defect that were considered suspicious but were not conclusively glaucomatous . patients with congenital glaucoma , pseudoexfoliation syndrome , pigment dispersion syndrome , or secondary glaucoma were excluded . pseudophakic eyes were included if their clinical diagnosis had been established prior to cataract surgery . individuals with healthy eyes that had an iop < 22 mmhg , a normal optic disc appearance and a normal visual field were not included in the study . one hundred and forty four individuals were consecutively recruited into the study ( 272 eyes ) . there were 71 poag eyes , 50 ntg eyes , 48 oh eyes , and 103 ( gs ) eyes . participants underwent goldmann applanation tonometry and their highest recorded iops were identified from their charts . the cct measurements were made using an ultrasonic pachymeter ( optikon 2000 s.p.a , altair , rome , italy ) by an experienced observer ( lp ) . the measurements of cct were performed as described previously ( gunvant et al 2003 ) . five measurements were performed in rapid succession ( taking no longer than 20 seconds ) , and provided each measurement was within 5 m , the mean of 5 readings was calculated and recorded as the cct . a 24 - 2 threshold visual field test with size iii white stimulus using the standard swedish interactive threshold algorithm strategy ( humphrey 24 - 2 sita - s ) was performed using a humphrey field analyzer - ii ( zeiss - humphrey instruments , carl zeiss meditec inc . , dublin , ca , usa ) . evaluation and screening for visual field artefacts was performed ( by dcb ) and a visual field was considered reliable if it had < 20% false negatives , < 10% false positives , and < 20% fixation losses . the visual field test was classed as glaucomatous based on cntgs criteria ( cntgs 1998 ) . images of the optic disc were obtained using a heidelberg retina tomograph ( hrt - i ; heidelberg engineering , heidelberg , germany ) . the details and the working principles of the hrt - i mean images were drawn using a computer mouse , by an experienced observer ( dcb ) , at the inner aspect of the scleral ring , this being aided by viewing a stereo optic disc image ( captured using the using a nidek 3-dx - nm camera , nidek , japan ) . the global and sectoral raw data parameters selected to be studied were those that represented the area , volume or height / depth and shape of the optic cup , neuroretinal rim and retinal nerve fibre layer . a multiple analysis of covariance ( mancova ) , allowing for the covariance induced by inclusion of both eyes of an individual , was used to investigate the overall difference in cct , refraction and age in different diagnostic groups . a post - hoc analysis was performed using an independent samples t - test to investigate the individual factor differences between groups . a kruskal - wallis test was used to investigate the difference in males and females between the groups and for variables that did not have a normal distribution . a mancova , allowing for the covariance due to inclusion of both eyes of an individual and differences in both age and mean spherical refraction of groups , was also used to investigate the differences in the highest previously recorded iop and iop recorded during the study visit for the various diagnostic groups . following allowance for both eye covariance , pearson s correlation coefficients were determined to identify associations between cct , age , refraction , and iop measured at the study visit . in addition , pearson s correlation coefficients ( allowing for the covariance due to inclusion of both eyes of an individual , age , and refraction ) were determined to identify associations between topographical optic disc parameters and cct . since multiple correlations were investigated between the hrt parameters ( n = 12 ) and cct , the p - value was adjusted using a bonferroni correction to allow for the inflation of alpha . when data was available for both eyes of a participant , one eye was selected randomly for inclusion . further , the highest recorded iop was adjusted to account for the variation in cct using the ehlers algorithm ( ehlers et al 1975 ) . pearson s correlation was used ( allowing for the covariance due to inclusion of both eyes of an individual ) to examine the association between the highest recorded iop with mean deviation of visual field and highest recorded adjusted iop with mean deviation of visual field . when data was only available for one eye of an individual it was included automatically . all statistical analyses were performed using spss version 13.0 ( spss inc . , chicago , il , usa ) . participants underwent goldmann applanation tonometry and their highest recorded iops were identified from their charts . the cct measurements were made using an ultrasonic pachymeter ( optikon 2000 s.p.a , altair , rome , italy ) by an experienced observer ( lp ) . the measurements of cct were performed as described previously ( gunvant et al 2003 ) . five measurements were performed in rapid succession ( taking no longer than 20 seconds ) , and provided each measurement was within 5 m , the mean of 5 readings was calculated and recorded as the cct . a 24 - 2 threshold visual field test with size iii white stimulus using the standard swedish interactive threshold algorithm strategy ( humphrey 24 - 2 sita - s ) was performed using a humphrey field analyzer - ii ( zeiss - humphrey instruments , carl zeiss meditec inc . , dublin , ca , usa ) . evaluation and screening for visual field artefacts was performed ( by dcb ) and a visual field was considered reliable if it had < 20% false negatives , < 10% false positives , and < 20% fixation losses . the visual field test was classed as glaucomatous based on cntgs criteria ( cntgs 1998 ) . images of the optic disc were obtained using a heidelberg retina tomograph ( hrt - i ; heidelberg engineering , heidelberg , germany ) . the details and the working principles of the hrt - i mean images were drawn using a computer mouse , by an experienced observer ( dcb ) , at the inner aspect of the scleral ring , this being aided by viewing a stereo optic disc image ( captured using the using a nidek 3-dx - nm camera , nidek , japan ) . the global and sectoral raw data parameters selected to be studied were those that represented the area , volume or height / depth and shape of the optic cup , neuroretinal rim and retinal nerve fibre layer . a multiple analysis of covariance ( mancova ) , allowing for the covariance induced by inclusion of both eyes of an individual , was used to investigate the overall difference in cct , refraction and age in different diagnostic groups . a post - hoc analysis was performed using an independent samples t - test to investigate the individual factor differences between groups . a kruskal - wallis test was used to investigate the difference in males and females between the groups and for variables that did not have a normal distribution . a mancova , allowing for the covariance due to inclusion of both eyes of an individual and differences in both age and mean spherical refraction of groups , was also used to investigate the differences in the highest previously recorded iop and iop recorded during the study visit for the various diagnostic groups . following allowance for both eye covariance , pearson s correlation coefficients were determined to identify associations between cct , age , refraction , and iop measured at the study visit . in addition , pearson s correlation coefficients ( allowing for the covariance due to inclusion of both eyes of an individual , age , and refraction ) were determined to identify associations between topographical optic disc parameters and cct . since multiple correlations were investigated between the hrt parameters ( n = 12 ) and cct , the p - value was adjusted using a bonferroni correction to allow for the inflation of alpha . thus , when data was available for both eyes of a participant , one eye was selected randomly for inclusion . further , the highest recorded iop was adjusted to account for the variation in cct using the ehlers algorithm ( ehlers et al 1975 ) . pearson s correlation was used ( allowing for the covariance due to inclusion of both eyes of an individual ) to examine the association between the highest recorded iop with mean deviation of visual field and highest recorded adjusted iop with mean deviation of visual field . when data was only available for one eye of an individual it was included automatically . all statistical analyses were performed using spss version 13.0 ( spss inc . , chicago , il , usa ) . table 1 gives the details of age , mean spherical refraction and cct measured for the study participants . a mancova showed that overall the cct ( f = 11.4 , p < 0.0001 ; figure 1 ) , mean spherical refraction ( f = 3.18 , p = 0.02 ) and age ( f = 5.72 , p = 0.001 ) were significantly different for the different diagnostic groups . there was a negative correlation between cct and clinically estimated cup to disc ratio ( r = 0.393 , p < 0.0001 ) , with individuals with thicker cct values having smaller cup to disc ratios . in the glaucoma group , cct correlated with disease severity as measured by mean deviation of visual fields , the eyes with thinner cct values tending to have greater visual field loss ( r = 0.258 , p = 0.012 ; figure 2 ) . the parameters area below reference and volume below reference , which are measures of cup area and cup volume , respectively , had significant negative correlations with cct ( correlation coefficients r = 0.223 and r = 0.207 , respectively ) . third moment ( cup shape measure ) had significant negative correlations with cct ( r = 0.179 , r = 0.268 , and r = 0.254 , respectively ) . these results indicated that the eyes with lower than average cct values had larger , deeper optic cups with altered cup - shape - measure . there were positive correlations identified between parameters measuring amount of neuroretinal rim tissue and cct , with high values of cct being associated with greater amounts of neuroretinal rim tissue . for the parameters volume above reference ( a measure of rim volume ) , rim area , mean rnflthickness and rnflcross - section area , the positive correlations were statistically significant ( r = 0.265 , 0.325 , 0.224 , and 0.215 , respectively ) . relationships between cct and the topographical disc parameters were analyzed for the diagnostic groups and the results are summarized in table 2 . there was no statistically significant difference in degree of visual field loss for the eyes with poag compared with those with ntg , even after accounting for any differences in age or refraction and inclusion of two eyes ( mancova f = 1.849 , p = 0.127 ) . since the mean deviation of visual fields was not normally distributed the difference in poag and ntg group was further analysed using a non - parametric equivalent of one - way anova . the mean deviations for the poag and ntg groups did not differ significantly ( p = 1.0 , kruskal - wallis one - way anova ) . furthermore , the iop measured at the study visit was not significantly different between the eyes with either poag or ntg ( mancova f = 1.849 , p = 0.127 ) . for the eyes with poag there was a significant negative correlation between the cup to disc area ratio hrt parameter and cct ( r = 0.370 , p = 0.002 ) indicating that eyes with thinner than average cct values had a greater cup to disc area ratio. consistent with the latter finding was the fact that there was a positive correlation between the third moment ( cup shape measure ) , correlation coefficients being r = 0.338 ( p = 0.005 ) and r = 0.255 ( p = 0.036 ) , respectively . interestingly , for the eyes with ntg none of the topographical optic disc parameters correlated with cct . the glaucoma suspect eyes were defined as such on the basis of : 1 ) suspicious optic disc appearance with normal visual fields ( 79.6% ) ; 2 ) suspicious glaucomatous visual field with normal optic disc appearance ( 4.9% ) ; and 3 ) suspicious optic disc appearance and visual field analysis ( 15.5% ) . for the gs group as a whole , there was a significant negative correlation between the parameters volume below reference ( a measure of cup volume ) , mean cup depth , and cct ( r = 0.297 ; figure 3 and r = 0.323 ; figure 4 , respectively ) . the parameters area below reference ( a measure of cup area ) and third moment ( cup shape measure) were found to have a negative correlation with cct ( correlation coefficients r = 0.213 and r = 0.225 , respectively ) , but these associations were not statistically significant ( p = 0.035 and p = 0.025 , respectively ) . none of the topographical optic disc parameters correlated significantly with cct for the eyes with oh . the only parameter that showed a trend towards an association was rim area which showed a positive correlation with cct ( r = 0.363 , p = 0.014 ) in the eyes with oh . iop did not correlate significantly with either the mean deviation of visual field defect when using the iop that was unadjusted for cct or when using iop corrected using the ehlers algorithm ( ehlers et al 1975 ) ( correlation coefficients r = 0.033 and r = 0.11 , respectively ; p > 0.05 ) . when only one eye from each participant was included in the statistical analyses , the results were essentially the same with respect to correlations between the topographical optic disc parameters and cct ( results not shown ) . the parameters area below reference and volume below reference , which are measures of cup area and cup volume , respectively , had significant negative correlations with cct ( correlation coefficients r = 0.223 and r = 0.207 , respectively ) . third moment ( cup shape measure ) had significant negative correlations with cct ( r = 0.179 , r = 0.268 , and r = 0.254 , respectively ) . these results indicated that the eyes with lower than average cct values had larger , deeper optic cups with altered cup - shape - measure . there were positive correlations identified between parameters measuring amount of neuroretinal rim tissue and cct , with high values of cct being associated with greater amounts of neuroretinal rim tissue . for the parameters volume above reference ( a measure of rim volume ) , rim area , mean rnflthickness and rnflcross - section area , the positive correlations were statistically significant ( r = 0.265 , 0.325 , 0.224 , and 0.215 , respectively ) . relationships between cct and the topographical disc parameters were analyzed for the diagnostic groups and the results are summarized in table 2 . there was no statistically significant difference in degree of visual field loss for the eyes with poag compared with those with ntg , even after accounting for any differences in age or refraction and inclusion of two eyes ( mancova f = 1.849 , p = 0.127 ) . since the mean deviation of visual fields was not normally distributed the difference in poag and ntg group was further analysed using a non - parametric equivalent of one - way anova . the mean deviations for the poag and ntg groups did not differ significantly ( p = 1.0 , kruskal - wallis one - way anova ) . furthermore , the iop measured at the study visit was not significantly different between the eyes with either poag or ntg ( mancova f = 1.849 , p = 0.127 ) . for the eyes with poag there was a significant negative correlation between the cup to disc area ratio hrt parameter and cct ( r = 0.370 , p = 0.002 ) indicating that eyes with thinner than average cct values had a greater cup to disc area ratio. consistent with the latter finding was the fact that there was a positive correlation between the third moment ( cup shape measure ) , correlation coefficients being r = 0.338 ( p = 0.005 ) and r = 0.255 ( p = 0.036 ) , respectively . interestingly , for the eyes with ntg none of the topographical optic disc parameters correlated with cct . the glaucoma suspect eyes were defined as such on the basis of : 1 ) suspicious optic disc appearance with normal visual fields ( 79.6% ) ; 2 ) suspicious glaucomatous visual field with normal optic disc appearance ( 4.9% ) ; and 3 ) suspicious optic disc appearance and visual field analysis ( 15.5% ) . for the gs group as a whole , there was a significant negative correlation between the parameters volume below reference ( a measure of cup volume ) , mean cup depth , and cct ( r = 0.297 ; figure 3 and r = 0.323 ; figure 4 , respectively ) . the parameters area below reference ( a measure of cup area ) and third moment ( cup shape measure) were found to have a negative correlation with cct ( correlation coefficients r = 0.213 and r = 0.225 , respectively ) , but these associations were not statistically significant ( p = 0.035 and p = 0.025 , respectively ) . none of the topographical optic disc parameters correlated significantly with cct for the eyes with oh . the only parameter that showed a trend towards an association was rim area which showed a positive correlation with cct ( r = 0.363 , p = 0.014 ) in the eyes with oh . iop did not correlate significantly with either the mean deviation of visual field defect when using the iop that was unadjusted for cct or when using iop corrected using the ehlers algorithm ( ehlers et al 1975 ) ( correlation coefficients r = 0.033 and r = 0.11 , respectively ; p > 0.05 ) . when only one eye from each participant was included in the statistical analyses , the results were essentially the same with respect to correlations between the topographical optic disc parameters and cct ( results not shown ) . in this cohort of patients , when considering the whole data set , thinner corneas appeared to be associated with larger , deeper optic disc cups and with increased cup - shape - measure , which are topographic signs regarded to be indicative of glaucomatous optic neuropathy . 1 ) the iop is erroneously high in individuals with thick cornea ( hence an early presentation at clinics ) or erroneously low in patients with thinner cornea ( this delaying referral until more advanced disease is evident ) , thus it is a diagnostic / treatment bias issue . alternatively , 2 ) there may be a true physiological biomechanical relationship between cct and the support structures of the optic nerve . a recent study ( pakravan et al 2007 ) reported a relationship between cct and optic disc size . the current study found no such relationship . in the present study , a subgroup analysis showed different relationships between cct and optic disc morphology for the different diagnostic groups , but no link between cct and optic disc size . the oh eyes ( defined as having normal discs on clinical examination ) had thicker cct values and no identifiable relationship between cct and optic disc morphology , this being despite the eyes in this group having a mean iop on average 6 mmhg higher than the other eyes studied . in contrast , when only considering eyes with established high tension glaucoma ( the poag group ) , the eyes with thinner corneas had optic disc changes indicative of more severe optic neuropathy ( reduced neuroretinal rim area and volume and a greater cup to disc area ratio ) and an associated greater amount of visual field loss in comparison with the poag eyes with thicker corneas . the relationship between cct and optic disc topography did not appear to hold true for the eyes with ntg , where no statistically significant associations were identified . the gs eyes are an interesting group because they represent a group of eyes that can be considered at - risk eyes , although a proportion of these eyes will of course remain stable . in these eyes there was a negative correlation between cct and estimates of cup volume and depth , the gs eyes with thinner corneas having optic disc cups of greater volume and depth . it would appear , therefore , that the eyes with thinner corneas had more changes suspicious of glaucoma in comparison with those having thicker corneas . for a similar group of glaucoma suspects , medeiros and colleagues ( 2003a , 2003b ) have provided further evidence for an increased risk of developing glaucoma in the presence of a thin cornea . patients with oh and normal automated white - on - white visual fields , but early glaucoma detected by either frequency doubling technology perimetric defects ( medeiros et al 2003a ) or short wavelength automated perimetric defects ( medeiros et al 2003b ) , had thinner corneas than those with no such defects . burgoyne and colleagues ( 1995a ) have reported that changes in optic disc compliance and surface position could be detected by digitized image analysis in early experimental glaucoma in the monkey eye . these findings were unlikely to be due to axon loss alone , because they did not occur in eyes with transected nerves ( a model of axon loss for normal iop ) . their results suggested that iop - related damage to the load - bearing connective tissues of the optic nerve head might occur early in the course of glaucoma . quigley and colleagues ( 1991 ) and hernandez ( 1992 ) have reported that there are alterations in the elastin of the optic nerve head in both human and experimental glaucoma and have suggested that differences in elastin function may have a part to play in susceptibility to glaucomatous injury . it remains unknown as to whether such elastin alterations relate to cct differences . as suggested by a previous study ( herndon et al 2004 ) , the results of the present study could be influenced in part by error in iop measurements ( ie , the patients with thinner cct values may have presented late since initially measured iop will have tended to be lower than the it should be noted that , however , in the uk it is mandatory for an ophthalmic evaluation to include optic disc evaluation and thus community glaucoma screening is not limited to iop measurement . furthermore none of the patients were referred on the basis of cct . in the present study we performed an additional analysis to examine the difference between corrected and uncorrected iop on the mean deviation of visual field obtained at presentation to the study . there was no association identified when either the uncorrected iop or the corrected iop ( ehlers algorithm ) ( ehlers et al 1975 ) was used . a prior study examined the accuracy of the presently available tonometric correction factors and reported that they are potentially inaccurate with a tendency to overestimate the effect of cct on iop ( gunvant et al 2005 ) . there is no universally acceptable and correct algorithm that is available as a cct - tonometric correction factor . thus it is difficult to obtain a true estimate of the effect of cct on iop and hence difficult to substantiate or negate any bias issue due to improper measurement of iop . neither can undebatable proof of any physiological biomechanical relationship between cct and the support structures of the optic nerve be determined by the present study . however , the results of the present study suggest that in eyes with glaucoma , thinner corneas are associated with morphological changes of the optic nerve head ( increased cup depth and volume ) that might reflect an increased susceptibility to glaucomatous optic neuropathy . much remains unknown and many further studies are required to unravel the mysteries of the pathogenesis of glaucomatous optic neuropathy and any relationships that there may be with cct . use of alternative tonometers , such as the dynamic contour tonometer ( kaufmann et al 2004 ) or the ocular response analyzer ( luce 2005 ) , which have been reported to be independent of cct , may prove useful in future research . the present study has a specific , important limitation in that it is a clinic - based study and not a population - based study . thus , although the result of the present study provides information on associations in a selected group of patients , a possible selection bias may account for these associations and a larger population - based study is required to confirm the findings .

purposeto identify relationships between central corneal thickness ( cct ) and optic disc topography , as determined by scanning laser ophthalmoscopy ( slo ) , for patients seen in a specialist glaucoma service.methods272 eyes of 144 patients with primary open angle glaucoma ( poag ; n = 71 ) , normal tension glaucoma ( ntg ; n = 50 ) , ocular hypertension ( oh ; n = 48 ) and those considered to be suspicious for glaucoma ( gs ; n = 103 ) underwent ultrasonic pachymetry and optic disc topography by slo . correlations between cct and slo parameter values were identified . a bonferroni correction for multiple comparisons was performed and a p value of < 0.0042 was considered significant.resultsmean cct values were 533 m ( poag ) , 530 m ( ntg ) , 550 m ( gs ) , and 565 m ( oh ) . as a group the gs and oh eyes had significantly thicker cct values than eyes with poag . in addition , the ntg eyes had significantly thinner cct values than gs and oh eyes . overall multiple slo parameters correlated with cct even after accounting for co - variance with age , refraction and inclusion of both eyes . sub - group analysis indicated that optic disc rim area positively correlated with cct ( r = 0.378 ) and cup to disc area ratio negatively correlated with cct ( r = 0.370 ) in the poag group . in the gs group the parameter area below reference ( a measure of cup volume ) and mean cup depth had negative correlations with cct ( r = 0.297 and 0.323 ) indicating that eyes with thinner than average corneal thickness measurements had larger and deeper cups.conclusionthinner corneas appear to be associated with larger and deeper optic disc cups in the eyes of patients seen in a specialist glaucoma service .

Methods Evaluation of participants for the study Optic disc topography Statistical analysis Results Central corneal thickness and topographical optic disc parameters Subgroup analyses Analysis of the eyes with open angle glaucoma (POAG and NTG) Analysis of the eyes with suspicions of glaucoma (GS) Analysis of the eyes with OH IOP and mean deviation of visual field Single eye analysis Discussion

all participants were prospectively recruited and underwent a comprehensive eye examination in the department of ophthalmology at the norfolk and norwich university hospital ( norwich , uk ) and had snellen equivalent visual acuity of 6/12 or better and a normal appearance of the cornea by slit - lamp biomicroscopy . all patients with glaucoma ( primary open angle glaucoma [ poag ] or normal tension glaucoma [ ntg ] ) had glaucomatous visual field defects and optic disc characteristics consistent with glaucoma . patients with poag had pretreatment iops that were consistently > 22 mmhg and the diagnosis of ntg was established using the collaborative normal - tension glaucoma study criteria ( cntgs 1998 ) . patients with ocular hypertension ( oh ) had iop measurements consistently > 22 mmhg and no visual field defect identified by standard automated perimetry . patients were defined as glaucoma suspects ( gs ) if they had an optic nerve head appearance and/or visual field defect that were considered suspicious but were not conclusively glaucomatous . individuals with healthy eyes that had an iop < 22 mmhg , a normal optic disc appearance and a normal visual field were not included in the study . there were 71 poag eyes , 50 ntg eyes , 48 oh eyes , and 103 ( gs ) eyes . five measurements were performed in rapid succession ( taking no longer than 20 seconds ) , and provided each measurement was within 5 m , the mean of 5 readings was calculated and recorded as the cct . evaluation and screening for visual field artefacts was performed ( by dcb ) and a visual field was considered reliable if it had < 20% false negatives , < 10% false positives , and < 20% fixation losses . images of the optic disc were obtained using a heidelberg retina tomograph ( hrt - i ; heidelberg engineering , heidelberg , germany ) . the details and the working principles of the hrt - i mean images were drawn using a computer mouse , by an experienced observer ( dcb ) , at the inner aspect of the scleral ring , this being aided by viewing a stereo optic disc image ( captured using the using a nidek 3-dx - nm camera , nidek , japan ) . a multiple analysis of covariance ( mancova ) , allowing for the covariance induced by inclusion of both eyes of an individual , was used to investigate the overall difference in cct , refraction and age in different diagnostic groups . a mancova , allowing for the covariance due to inclusion of both eyes of an individual and differences in both age and mean spherical refraction of groups , was also used to investigate the differences in the highest previously recorded iop and iop recorded during the study visit for the various diagnostic groups . following allowance for both eye covariance , pearson s correlation coefficients were determined to identify associations between cct , age , refraction , and iop measured at the study visit . in addition , pearson s correlation coefficients ( allowing for the covariance due to inclusion of both eyes of an individual , age , and refraction ) were determined to identify associations between topographical optic disc parameters and cct . since multiple correlations were investigated between the hrt parameters ( n = 12 ) and cct , the p - value was adjusted using a bonferroni correction to allow for the inflation of alpha . when data was available for both eyes of a participant , one eye was selected randomly for inclusion . pearson s correlation was used ( allowing for the covariance due to inclusion of both eyes of an individual ) to examine the association between the highest recorded iop with mean deviation of visual field and highest recorded adjusted iop with mean deviation of visual field . five measurements were performed in rapid succession ( taking no longer than 20 seconds ) , and provided each measurement was within 5 m , the mean of 5 readings was calculated and recorded as the cct . evaluation and screening for visual field artefacts was performed ( by dcb ) and a visual field was considered reliable if it had < 20% false negatives , < 10% false positives , and < 20% fixation losses . the details and the working principles of the hrt - i mean images were drawn using a computer mouse , by an experienced observer ( dcb ) , at the inner aspect of the scleral ring , this being aided by viewing a stereo optic disc image ( captured using the using a nidek 3-dx - nm camera , nidek , japan ) . the global and sectoral raw data parameters selected to be studied were those that represented the area , volume or height / depth and shape of the optic cup , neuroretinal rim and retinal nerve fibre layer . a multiple analysis of covariance ( mancova ) , allowing for the covariance induced by inclusion of both eyes of an individual , was used to investigate the overall difference in cct , refraction and age in different diagnostic groups . a mancova , allowing for the covariance due to inclusion of both eyes of an individual and differences in both age and mean spherical refraction of groups , was also used to investigate the differences in the highest previously recorded iop and iop recorded during the study visit for the various diagnostic groups . following allowance for both eye covariance , pearson s correlation coefficients were determined to identify associations between cct , age , refraction , and iop measured at the study visit . in addition , pearson s correlation coefficients ( allowing for the covariance due to inclusion of both eyes of an individual , age , and refraction ) were determined to identify associations between topographical optic disc parameters and cct . since multiple correlations were investigated between the hrt parameters ( n = 12 ) and cct , the p - value was adjusted using a bonferroni correction to allow for the inflation of alpha . thus , when data was available for both eyes of a participant , one eye was selected randomly for inclusion . pearson s correlation was used ( allowing for the covariance due to inclusion of both eyes of an individual ) to examine the association between the highest recorded iop with mean deviation of visual field and highest recorded adjusted iop with mean deviation of visual field . table 1 gives the details of age , mean spherical refraction and cct measured for the study participants . a mancova showed that overall the cct ( f = 11.4 , p < 0.0001 ; figure 1 ) , mean spherical refraction ( f = 3.18 , p = 0.02 ) and age ( f = 5.72 , p = 0.001 ) were significantly different for the different diagnostic groups . there was a negative correlation between cct and clinically estimated cup to disc ratio ( r = 0.393 , p < 0.0001 ) , with individuals with thicker cct values having smaller cup to disc ratios . in the glaucoma group , cct correlated with disease severity as measured by mean deviation of visual fields , the eyes with thinner cct values tending to have greater visual field loss ( r = 0.258 , p = 0.012 ; figure 2 ) . the parameters area below reference and volume below reference , which are measures of cup area and cup volume , respectively , had significant negative correlations with cct ( correlation coefficients r = 0.223 and r = 0.207 , respectively ) . third moment ( cup shape measure ) had significant negative correlations with cct ( r = 0.179 , r = 0.268 , and r = 0.254 , respectively ) . these results indicated that the eyes with lower than average cct values had larger , deeper optic cups with altered cup - shape - measure . there were positive correlations identified between parameters measuring amount of neuroretinal rim tissue and cct , with high values of cct being associated with greater amounts of neuroretinal rim tissue . for the parameters volume above reference ( a measure of rim volume ) , rim area , mean rnflthickness and rnflcross - section area , the positive correlations were statistically significant ( r = 0.265 , 0.325 , 0.224 , and 0.215 , respectively ) . relationships between cct and the topographical disc parameters were analyzed for the diagnostic groups and the results are summarized in table 2 . there was no statistically significant difference in degree of visual field loss for the eyes with poag compared with those with ntg , even after accounting for any differences in age or refraction and inclusion of two eyes ( mancova f = 1.849 , p = 0.127 ) . furthermore , the iop measured at the study visit was not significantly different between the eyes with either poag or ntg ( mancova f = 1.849 , p = 0.127 ) . for the eyes with poag there was a significant negative correlation between the cup to disc area ratio hrt parameter and cct ( r = 0.370 , p = 0.002 ) indicating that eyes with thinner than average cct values had a greater cup to disc area ratio. consistent with the latter finding was the fact that there was a positive correlation between the third moment ( cup shape measure ) , correlation coefficients being r = 0.338 ( p = 0.005 ) and r = 0.255 ( p = 0.036 ) , respectively . interestingly , for the eyes with ntg none of the topographical optic disc parameters correlated with cct . for the gs group as a whole , there was a significant negative correlation between the parameters volume below reference ( a measure of cup volume ) , mean cup depth , and cct ( r = 0.297 ; figure 3 and r = 0.323 ; figure 4 , respectively ) . the parameters area below reference ( a measure of cup area ) and third moment ( cup shape measure) were found to have a negative correlation with cct ( correlation coefficients r = 0.213 and r = 0.225 , respectively ) , but these associations were not statistically significant ( p = 0.035 and p = 0.025 , respectively ) . none of the topographical optic disc parameters correlated significantly with cct for the eyes with oh . the only parameter that showed a trend towards an association was rim area which showed a positive correlation with cct ( r = 0.363 , p = 0.014 ) in the eyes with oh . iop did not correlate significantly with either the mean deviation of visual field defect when using the iop that was unadjusted for cct or when using iop corrected using the ehlers algorithm ( ehlers et al 1975 ) ( correlation coefficients r = 0.033 and r = 0.11 , respectively ; p > 0.05 ) . when only one eye from each participant was included in the statistical analyses , the results were essentially the same with respect to correlations between the topographical optic disc parameters and cct ( results not shown ) . the parameters area below reference and volume below reference , which are measures of cup area and cup volume , respectively , had significant negative correlations with cct ( correlation coefficients r = 0.223 and r = 0.207 , respectively ) . third moment ( cup shape measure ) had significant negative correlations with cct ( r = 0.179 , r = 0.268 , and r = 0.254 , respectively ) . these results indicated that the eyes with lower than average cct values had larger , deeper optic cups with altered cup - shape - measure . for the parameters volume above reference ( a measure of rim volume ) , rim area , mean rnflthickness and rnflcross - section area , the positive correlations were statistically significant ( r = 0.265 , 0.325 , 0.224 , and 0.215 , respectively ) . relationships between cct and the topographical disc parameters were analyzed for the diagnostic groups and the results are summarized in table 2 . there was no statistically significant difference in degree of visual field loss for the eyes with poag compared with those with ntg , even after accounting for any differences in age or refraction and inclusion of two eyes ( mancova f = 1.849 , p = 0.127 ) . furthermore , the iop measured at the study visit was not significantly different between the eyes with either poag or ntg ( mancova f = 1.849 , p = 0.127 ) . for the eyes with poag there was a significant negative correlation between the cup to disc area ratio hrt parameter and cct ( r = 0.370 , p = 0.002 ) indicating that eyes with thinner than average cct values had a greater cup to disc area ratio. consistent with the latter finding was the fact that there was a positive correlation between the third moment ( cup shape measure ) , correlation coefficients being r = 0.338 ( p = 0.005 ) and r = 0.255 ( p = 0.036 ) , respectively . interestingly , for the eyes with ntg none of the topographical optic disc parameters correlated with cct . the glaucoma suspect eyes were defined as such on the basis of : 1 ) suspicious optic disc appearance with normal visual fields ( 79.6% ) ; 2 ) suspicious glaucomatous visual field with normal optic disc appearance ( 4.9% ) ; and 3 ) suspicious optic disc appearance and visual field analysis ( 15.5% ) . for the gs group as a whole , there was a significant negative correlation between the parameters volume below reference ( a measure of cup volume ) , mean cup depth , and cct ( r = 0.297 ; figure 3 and r = 0.323 ; figure 4 , respectively ) . the parameters area below reference ( a measure of cup area ) and third moment ( cup shape measure) were found to have a negative correlation with cct ( correlation coefficients r = 0.213 and r = 0.225 , respectively ) , but these associations were not statistically significant ( p = 0.035 and p = 0.025 , respectively ) . none of the topographical optic disc parameters correlated significantly with cct for the eyes with oh . the only parameter that showed a trend towards an association was rim area which showed a positive correlation with cct ( r = 0.363 , p = 0.014 ) in the eyes with oh . iop did not correlate significantly with either the mean deviation of visual field defect when using the iop that was unadjusted for cct or when using iop corrected using the ehlers algorithm ( ehlers et al 1975 ) ( correlation coefficients r = 0.033 and r = 0.11 , respectively ; p > 0.05 ) . when only one eye from each participant was included in the statistical analyses , the results were essentially the same with respect to correlations between the topographical optic disc parameters and cct ( results not shown ) . in this cohort of patients , when considering the whole data set , thinner corneas appeared to be associated with larger , deeper optic disc cups and with increased cup - shape - measure , which are topographic signs regarded to be indicative of glaucomatous optic neuropathy . 1 ) the iop is erroneously high in individuals with thick cornea ( hence an early presentation at clinics ) or erroneously low in patients with thinner cornea ( this delaying referral until more advanced disease is evident ) , thus it is a diagnostic / treatment bias issue . alternatively , 2 ) there may be a true physiological biomechanical relationship between cct and the support structures of the optic nerve . a recent study ( pakravan et al 2007 ) reported a relationship between cct and optic disc size . in the present study , a subgroup analysis showed different relationships between cct and optic disc morphology for the different diagnostic groups , but no link between cct and optic disc size . the oh eyes ( defined as having normal discs on clinical examination ) had thicker cct values and no identifiable relationship between cct and optic disc morphology , this being despite the eyes in this group having a mean iop on average 6 mmhg higher than the other eyes studied . in contrast , when only considering eyes with established high tension glaucoma ( the poag group ) , the eyes with thinner corneas had optic disc changes indicative of more severe optic neuropathy ( reduced neuroretinal rim area and volume and a greater cup to disc area ratio ) and an associated greater amount of visual field loss in comparison with the poag eyes with thicker corneas . the relationship between cct and optic disc topography did not appear to hold true for the eyes with ntg , where no statistically significant associations were identified . the gs eyes are an interesting group because they represent a group of eyes that can be considered at - risk eyes , although a proportion of these eyes will of course remain stable . in these eyes there was a negative correlation between cct and estimates of cup volume and depth , the gs eyes with thinner corneas having optic disc cups of greater volume and depth . it would appear , therefore , that the eyes with thinner corneas had more changes suspicious of glaucoma in comparison with those having thicker corneas . patients with oh and normal automated white - on - white visual fields , but early glaucoma detected by either frequency doubling technology perimetric defects ( medeiros et al 2003a ) or short wavelength automated perimetric defects ( medeiros et al 2003b ) , had thinner corneas than those with no such defects . burgoyne and colleagues ( 1995a ) have reported that changes in optic disc compliance and surface position could be detected by digitized image analysis in early experimental glaucoma in the monkey eye . these findings were unlikely to be due to axon loss alone , because they did not occur in eyes with transected nerves ( a model of axon loss for normal iop ) . quigley and colleagues ( 1991 ) and hernandez ( 1992 ) have reported that there are alterations in the elastin of the optic nerve head in both human and experimental glaucoma and have suggested that differences in elastin function may have a part to play in susceptibility to glaucomatous injury . as suggested by a previous study ( herndon et al 2004 ) , the results of the present study could be influenced in part by error in iop measurements ( ie , the patients with thinner cct values may have presented late since initially measured iop will have tended to be lower than the it should be noted that , however , in the uk it is mandatory for an ophthalmic evaluation to include optic disc evaluation and thus community glaucoma screening is not limited to iop measurement . in the present study we performed an additional analysis to examine the difference between corrected and uncorrected iop on the mean deviation of visual field obtained at presentation to the study . neither can undebatable proof of any physiological biomechanical relationship between cct and the support structures of the optic nerve be determined by the present study . however , the results of the present study suggest that in eyes with glaucoma , thinner corneas are associated with morphological changes of the optic nerve head ( increased cup depth and volume ) that might reflect an increased susceptibility to glaucomatous optic neuropathy . use of alternative tonometers , such as the dynamic contour tonometer ( kaufmann et al 2004 ) or the ocular response analyzer ( luce 2005 ) , which have been reported to be independent of cct , may prove useful in future research . thus , although the result of the present study provides information on associations in a selected group of patients , a possible selection bias may account for these associations and a larger population - based study is required to confirm the findings .

california house dust contains some of the highest concentrations of polybrominated diphenyl ether ( pbde ) flame retardants ( frs ) in the world due to a state - wide furniture flammability standard ( technical bulletin 117 ) . pbdes have been associated with thyroid and other endocrine system disruption and adverse neurological development ( see supporting information ( si ) ) . pbdes in california homes and residents often exceed risk - based levels for children , raising concerns about exposures to the many other frs that have not yet been well - characterized . pbde manufacturer , introduced firemaster 550 to replace the pentabde commercial mixture in response to prospective bans in europe and several u.s . states . little is known about the chemical composition , uses , exposure levels and health effects of this mixture or of other brominated , chlorinated , and organophosphate chemicals used as frs . because additive frs shed from consumer products , they are found in house dust . measuring dust concentrations over time can identify exposure trends that result from changes in product formulations . house dust is the primary route of exposure for pbdes , contributing 82% , on average , of a u.s . although diet may also contribute , dust appears to be particularly important in areas , like california , with high concentrations in dust . dust is a direct exposure pathway through incidental ingestion , inhalation of resuspended particles , and dermal absorption , and it is a proxy for exposure from product use . commercial pentabde and octabde mixtures were phased - out in 2004 in the u.s . producers and importers ( chemtura , albermarle , and icl industrial products ) committed to end production , import and sales by the end of 2013 . as pbdes were phased out due to health concerns , other brominated frs ( bfrs ) and organophosphate flame retardants ( opfrs ) were introduced as replacements . chemtura , formerly great lakes chemical corporation , replaced pentabde in polyurethane foam with firemaster 550 , a mixture of 2-ethylhexyl-2,3,4,5-tetrabromobenzoate ( eh - tbb ) , bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate ( beh - tebp ) , triphenyl phosphate ( tphp ) , and a yet - to - be - fully characterized triaryl phosphate isopropylated mixture . concerns are emerging about beh - tebp s environmental persistence and toxicity , since beh - tebp is the brominated version of bis(2-ethylhexyl)phthalate ( dehp ) that adversely affects reproductive development . the u.s . epa recently announced plans to conduct risk assessments for beh - tebp and eh - tbb . several opfrs are used as pbde replacements . in the late 1970s , tris(2,3-dibromopropyl ) phosphate ( tdbpp or brominated tris ) exposure data are limited , although the toxic breakdown product , 2,3-dibromo-1-propanol , was detected in u.s . homes . the chlorinated analog , tris(1,3-dichloro-2-propyl ) phosphate ( tdcipp ) , also a carcinogen , has been found in u.s . house dust and baby products . house dust were recently associated with altered thyroid ( free t4 ) and prolactin hormone levels in men . world have been well established ; however , little is known about levels of other frs . we expect that frs used in polyurethane foam , including pentabde replacements , may be elevated due to the furniture flammability standard . exposure patterns for frs in other applications , such as electronics , are not known because of limited data , including for bde 209 . to provide data on a wider range of frs and on changing exposure patterns , this study measured a broad array of fr chemicals in repeat dust samples collected from 16 california homes . dust collected in california homes in 2006 and in the same homes in 2011 was analyzed for a broad suite of bfrs and opfrs ( n = 49 ) . we also measured 13 legacy chemicals : persistent organochlorines ( ocs ) banned long ago ( e.g. , ddt ) . correlation and cluster analysis of simultaneous fr measurements were used to shed light on mixtures and potential sources . measurement at two time periods allows for the investigation of changes in residential levels , which likely reflect patterns of use . this work contributes to the ongoing characterization of evolving exposures to fr chemicals in homes . dust samples were collected in 16 northern california homes in 2006 and again in the same homes with the same participants in 2011 . these homes were a subset of 50 homes in two san francisco bay area communities further described in brody et al . and rudel et al . samples were collected by trained field staff using a eureka mighty - mite vacuum cleaner fitted with a specially designed ptfe teflon crevice tool attachment modified to collect dust into a cellulose extraction thimble ( 19 90 mm ) . samples were collected by slowly dragging the crevice tool for approximately 30 min over surfaces in the living areas of the home . samples were sieved to < 150 m prior to long - term storage ( 16 c 10 c ) and extraction . residents were surveyed about the presence of furniture , carpets , and electronics , particularly if any items were introduced to the home since the 2006 sample collection . analytes were selected based on previous research , current understanding of potential replacements for pbdes , health concerns , and analytical capability . based on production volumes , hbcyd and tbbpa the health effects of chlorinated and brominated opfrs are of concern and recent work suggests they are found at levels similar to pbdes . nonhalogenated frs are expected to be used in various fr mixtures and may be pervasive given their many other uses in the home . compounds were named following the newly proposed nomenclature presented by bergman et al , with the older name give in parentheses . loq , limit of quantification ; indicates insufficient number of detects to calculate summary statistics . due to the comprehensive list of target analytes and differences in physical - chemical properties , two different sample preparation methods were used in four extracts per sample ( two fractions per method ) for chemical analysis . one sample preparation method , which was used to measure the bulk of bfrs , ocs , and opfrs , involved extraction using hex - ac ( 3:1 , v : v ) and fractionation on florisil . the obtained fractions f1 and f2 were subjected to analysis by gc - ecni / ms and gc - ei / ms ( see si table si1 ) . a second sample preparation method , involving similar extraction and fractionation on silica , the fraction containing pbdes was subjected to gc - ecni / ms and the fraction containing hbcyds and tbbpa was subjected to lc - ms / ms analysis . six procedural blanks were analyzed in the same batches as the samples and concentrations were blank - corrected by subtracting the mean blank values ( in pg ) from the raw analyte values . method limits of quantification ( loq ) were calculated as 3 standard deviation of blank values and divided by the amount of dust used for analysis ( typically 50 mg ) . for compounds not detected in the blanks , the loq was calculated based on the signal - to - noise ratio 10/1 . since loqs are compound - specific variables , they spanned a large range of concentrations . certified reference material srm 2585 ( organics in indoor dust ) was used to test the accuracy . nondetectable concentrations were left at zero for summary statistics , which results in lower values than if other replacement methods were used . concentration ratios ( 2011/2006 concentrations ) were calculated to evaluate changes between the two sampling periods . ratios above 1 indicate higher concentrations in 2011 and ratios below 1 indicate higher 2006 concentrations . spearman rank correlations were used to evaluate associations between absolute concentration differences between rounds ( 20112006 concentrations ) and total number of reported new fr - relevant items ( e.g. , electronics , carpets ) in 2011 . kendall s tau rank correlation estimates were calculated to investigate relationships between analytes within each sampling round and for each analyte across rounds . these estimates were used in cluster analysis to elucidate common mixtures and potential sources . overall , 55 compounds were detected and 41 were found in at least 50% of the 32 samples ( table 1 ) . detected chemicals were 13 pbde congeners , 3 components of firemaster 550 , 15 other bfrs , 4 halogenated opfrs , 7 nonhalogenated opfrs , and 2 dechlorane - plus isomers . table 2 summarizes information on usage and health concerns of these frs grouped by common formulations ( related to exposure patterns ) and chemical structure ( often related to use and toxicity ) . 1 million pounds per year are typically designated by the epa as high production volume chemicals , a voluntary reporting program ( data from 2006 ) . epa action plans have been developed for 10 chemicals considered high priority for risk management . the european union s system of registration , evaluation , authorization , and restriction of chemical substances ( reach ) identifies substances of very high concern ( svhc ) , which are public health hazards proposed for regulation under reach . congeners bde 28 , bde 47 , bde 66 , bde 85 , bde 99 , bde 100 , bde 153 , and bde 154 . the highest concentrations , greater than 0.1 mg / g or 0.01% , were for two chlorinated opfrs , including tcep , which is listed as a carcinogen under california s proposition 65 , and tcipp , and one nonhalogenated opfr ( tboep ) . over the five years between the sample collection periods , firemaster 550 components increased , while pentabde levels decreased . legacy pollutants like ddt also decreased , suggesting that the pbde reduction may be due to decreased loading and/or possibly to differences in sample collection between 2006 and 2011 . figure 1 shows ratios of 2011/2006 concentrations ; ratios > 1 suggest increasing concentrations with time . chemicals with median ratios above 1 were higher in 2011 samples compared with 2006 samples . we found all targeted pbde congeners in at least 50% of samples , with the components of pentabde ( bde 47 and bde 99 ) and decabde ( bde 209 ) mixtures in 100% of samples . median concentrations for all pbde congeners decreased from 2006 to 2011 ( table 1 ) ; however , not all of the means decreased ( data not shown ) , with exceptions likely driven by two homes with substantial increases in the congeners of pentabde mixture ( si figure si3 ) . . ratios of 2011/2006 concentrations are used to evaluate relative concentrations from the two sampling periods . median concentration ratios were less than 1 for all congeners ( figure 1 ) , suggesting a decrease in concentrations between 2006 and 2011 , which could reflect decreased use . however , since we saw decreases for legacy ocs , which should generally have minimal changes between 2006 and 2011 , the pbde reduction may reflect some unidentified but systematic difference in sample collection ( see legacy chemicals below ) . substantial decreases ( up to 20-fold ) in concentrations of pentabde were observed in three homes where participants reported remodeling or acquiring new furniture and/or rugs / carpet between 2006 and 2011 . in fact , there was a significant statistical association between concentration reductions and participant - reported new furniture , electronics , and flooring ( p < 0.05 ) , suggesting that pentabde is no longer present in new household items . reductions are likely the result of phase - outs ( 2004 ) and bans ( 2006 in ca ) of pentabde and octabde . substantial decrease ( 14-fold ) in bde 209 was observed in a home where the participant did not report changes in electronics and furnishings ; possibly some relevant changes were not reported . ng / g in both sampling rounds , which is consistent with previous research showing higher pentabde concentrations in california than elsewhere due to the unique furniture flammability standard . in comparison to other studies within california , the median concentration of bde 47 in 2006 is similar ( within 30% ) ; whereas median concentrations of bde 99 and 100 in 2006 ( 2,200 ng / g and 520 ng / g , respectively ) were lower ( up to 2 ) than other california studies , which used slightly different vacuum sampling techniques . si figures si4 and si5 show that pbde congeners measured in each sampling round correlate / cluster together in the three commercial formulations ( pentabde , octabde , and decabde ) . octabde levels correlate between sampling rounds ( along diagonal in si figure si4 ) , suggesting relatively stable concentrations in the homes over time ; however , pentabde and decabde levels were not significantly correlated over time , likely due to a few homes with substantial changes . chemtura introduced firemaster 550 in 2004 as a replacement for pentabde in polyurethane foam . besides tphp , the other constituents of firemaster 550 were only recently identified as two brominated compounds : eh - tbb and beh - tebp . subsequently , chemtura developed additional products , with undisclosed composition , including firemaster 600 , firemaster 800 , and emerald innovation , with claims of increased efficiency . firemaster 550 is genotoxic and tphp was associated with altered prolactin levels and decreased sperm concentration in men . to our knowledge , carcinogenicity , reproductive and development studies have not been conducted on the brominated components of firemaster 550 . we detected eh - tbb , beh - tebp , and tphp in all but one sample . concentrations of eh - tbb and beh - tebp increased across rounds ( median ratio > 1 ; figure 1 ) , except in one home where beh - tebp was found at 1,935 ng / g in the 2006 sample and not detected ( < 2 ng / g ) in 2011 ( si figure si3 ) . the generally increasing trend for eh - tbb and beh - tebp suggests that firemaster 550 is being used as a pentabde replacement . we compared our 2006 results to two sets of dust samples collected in the boston area ( 50 vacuum bag samples collected between 2002 and 2007 and 20 field technician collected dust samples collected in 2006 ) and vacuum bag dust collected in vancouver , canada in 20072008 . the 2006 eh - tbb and beh - tebp levels in our study were similar to , if not slightly lower than , levels in boston . our 2006 eh - tbb levels were lower than levels in vancouver whereas the 2011 levels are comparable . in contrast , the levels of beh - tebp at both time periods in our study were higher than those in vancouver . eh - tbb and beh - tebp were significantly positively correlated within each sampling round ( si figure si4 ; tau = 0.40.5 ; p < 0.05 ) , which is expected since they are both in firemaster 550 . we compared the observed ratio of eh - tbb / beh - tebp in our samples with the ratio of the commercial mixture and boston - area samples to evaluate if firemaster 550 is the sole source and if eh - tbb and beh - tebp have different fates once applied to a product . we observed a mean eh - tbb / beh - tebp ratio of 0.6 ( 0.043.1 ) in the 2006 samples and 1.5 ( 0.811 ) in the 2011 samples . these ratios are lower than the reported ratio in firemaster 550 ( 4 ) and in boston dust ( mean 4.4 ; range 0.550 ) . this suggests other sources of beh - tebp in california or a different fate of the chemicals . tphp , also present in firemaster 550 , was not significantly correlated with either eh - tbb or beh - tebp in either sampling round , although tphp concentrations increased in homes with substantial increases in eh - tbb and beh - tebp . this suggests that , in addition to firemaster 550 , there are other sources of tphp , for example , as a fr in other formulations or applications or as a plasticizer . hbcyd , the third most used bfr , is used mostly in polystyrene foams in building materials and consumer products . it is being considered for addition to the list of persistent organic pollutants ( pops ) under the stockholm convention , which would substantially limit its production and use . in 2010 , epa released an action plan for hbcyd citing its wide use , presence in humans , bioaccumulation potential , persistence , toxicity to aquatic organisms and concerns about reproductive , neurological and developmental effects in humans . the action plan was followed by a proposed significant new use rule ( snur ) for hbcyd in textiles , where it is often used to meet furniture flammability standards . we detected all hbcyd isomers ( - , - , and -hbcyd ) in all samples , and they were significantly correlated ( tau = 0.40.8 ; p < 0.05 ) within each sampling round . total hbcyd ( sum of three isomers ) concentrations were similar across time periods , ranging from 82 to 6800 ng / g ( median 190 ng / g ) in the 2006 samples and 39 to 1800 ng / g ( median 160 ng / g ) in 2011 . it is unclear whether the phase - out of pentabde and octabde mixtures influenced the pattern of hbcyd use . canadian samples , but less than for uk samples . however , our maxima ( 2006 : 6800 ng / g ; 2011 : 1800 ng / g ) were substantially lower than those reported in boston living room dust ( 130 200 ng / g ) and uk samples ( 110 000 ng / g ) . commercial mixtures of hbcyd mainly consist of -hbcyd ( 7589% ) , while - and -hbcyd are found at lower amounts . however , we observed relative abundances of 4550% , 4045% , and approximately 10% for - , - , and -hbcyd , respectively . this is likely the result of thermal rearrangement at high temperatures in production and processing of hbcyd - added materials or photolysis . this raises cautions about using only source composition information and not evaluating fate and transport of chemicals in products to evaluate potential exposures . tetrabromobisphenol a ( tbbpa ) , the most commonly used bfr , is employed as a reactive fr in circuit boards , plastics , paper and textiles as a plasticizer , in coatings and adhesives , and as an intermediate in the synthesis of other frs . it has been associated with effects on the immune system , reproductive and development effects , and neurotoxicity ( see si table si4 for details and references ) . tbbpa was detected in nearly all homes in both rounds with concentrations ranging from < 10 to 3400 ng / g in 2006 and from 22 to 2000 we found a significant association between concentration reductions and new electronics suggesting that new electronics contain less tbbpa ( rho = 0.69 ; p = 0.003 ) . concentrations are higher ( 1722 at median ) than reported in european homes and similar to michigan offices . btbpe , in production since the 1970s and now used to replace octabde , and dbdpe , introduced in mid-1980s and available as a replacement for decabde , were detected in nearly 100% of samples . the concentrations of btbpe , which has limited toxicity data ( see si table si4 ) , were similar between 2006 and 2011 . in contrast , concentrations of dbdpe , structurally similar to bde 209 and associated with reproductive and developmental toxicities , were generally higher in 2011 ( table 1 and figure 1 ) , and two homes had substantial ( > 20-fold ) increases . another commonly detected fr was the tbbpa derivative tetrabromobisphenol a - bis(2,3-dibromopropylether ) ( tbbpa - bdbpe ) , which is being studied by the national toxicology program ( ntp ) because of the structural similarity with the carcinogenic tdbpp ( brominated tris ) . levels of tbbpa - bdbpe appear fairly stable over time ( table 1 and figure 1 ) and lower than levels reported in belgium . hexabromobenzene ( hbb ) , an additive fr used in paper , wood , textiles , plastics and electronics , and not used in europe , was detected in 50% of 2006 samples and 31% of 2011 samples . octabromo-1,3,3-trimethyl-1-phenylindane was infrequently detected and one home had substantial ( 10-fold ) reductions over the 5 years . studies on exposures and health effects of these bfrs are limited . chlorinated and brominated opfrs have a long history of use in polyurethane foam and textiles and an equally long history of concerns about health effects , particularly cancer . tdbpp or brominated tris was banned from children s sleepwear in the u.s . in 1977 due to carcinogenicity concerns and detection of its mutagenic metabolite in children . it is reported to be used as a fr in polyurethane and polystyrene foams , acrylic furnishings , polyvinyl and phenolic resins , paints and lacquers , styrene - butadiene rubber , and latexes . we detected tdbpp in 62% of 2006 samples and 38% of 2011 samples . as far as we know , this is the first report of tdbpp in house dust , although we previously detected its mutagenic metabolite , 2,3-dibromo-1-propanol , in about 10% of indoor air samples from cape cod , ma . dust concentrations were much lower in 2011 ( mean 40 ng / g ; maximum 310 ng / g ) compared with 2006 ( mean 1000 ng / g ; maximum 8900 ng / g ) , though this may be due to whatever factor led to lower concentrations of legacy pollutants ( see below ) . we also detected three chlorinated opfrs : tcep , tcipp , and tdcipp ( chlorinated tris ) , which are used in polyurethane foams as replacements for pentabde . tdcipp was voluntarily withdrawn from children s pajamas after metabolites 1,3-dichloro-2-propanone and 1,3-dichloro-2-propanol were found to be mutagenic . the consumer product safety commission ( cpsc ) said tdcipp was a potential hazard to consumers , based on cancer and noncancer end points . the cpsc estimate of children s exposure from treated furniture was 5 higher than the agency s acceptable daily intake , with most of the exposure from inhalation of the chemicals volatilized from treated furniture . our reported concentrations of tdcipp comprise tris(1,3-dichloro-2-propyl ) phosphate , which makes up approximately 9095% of tdcipp , and tris(2,3-dibromopropyl ) phosphate . tcep is slated to be banned from children s products in new york by 2014 , and a bill is currently being considered that would expand the ban to tdcipp . median concentrations of all chlorinated opfrs were above 1,000 ng / g , or 1 g / g , in both sampling rounds , and maxima were > 100 000 ng / g or 0.01% , making these the most abundant frs in this study ( table 1 ) . for example , between 2006 and 2011 , one home with a new roof installed between sampling rounds had 20-fold increase in tcep concentration and another home with substantial remodeling had a 14-fold increase in tdcipp . tcipp means ( 2006 mean 1200 ng / g ; 2011 mean 1700 ng / g ) and medians increased ( table 1 ) , suggesting an increase in use between 2006 and 2011 . people who reported new furniture between sampling rounds showed increases in tcipp concentrations ( rho = 0.6 ; p = 0.02 ) , suggesting that tcipp is a pentabde replacement . based on limited comparison data , concentrations of chlorinated opfrs observed in this study are some of the highest in the world ; only concentrations in japan are consistently higher . generally lower levels have been reported for homes in boston , belgium , spain , sweden , and germany , except for higher tcipp in spain and tdcipp in sweden . the highest concentrations of chlorinated opfrs were found in a study of 41 japanese homes , which reported median concentrations 2- to 25-fold higher than seen in our california samples . the levels in japan are likely a result of a voluntary phase - out of pentapbde in the early 1990s . concentrations of the chlorinated opfrs are not correlated with each other , likely because tcipp has been reported as a replacement for tcep and is often used in the same types of products as tdcipp , which is typically used only when a more efficient fr is needed , since it is more expensive . the highest concentrations were for tboep , used as fr as well as in antifoam agents , floor polish , lacquers , plastics , rubbers , and solvents . it had the highest median concentration , 2-fold higher than the next highest , of any analyte ( 2006 : 12 000 ng / g ; 2011 : 11 000 ng / g ) and the highest concentration of any analyte in 2011 ( 170 000 ng / g ) . in addition to tboep , we detected ehdpp and tmpp ( sum of four isomers)used in hydraulic fluids and pvc in all samples . tboep and tmpp generally decreased , whereas ehdpp concentrations generally increased between sampling events ( figure 1 ) . concentrations of tboep were higher than in dust samples collected in belgian and spanish homes , although lower than japanese homes ( 1 570 000 ng / g ) . concentrations of several nonhalogenated opfrs ( tboep , tep , tnbp , and tmpp ) were correlated across sampling rounds ( tau = 0.370.69 ) , indicating that these compounds have temporal stability . dechlorane - plus ( dp ) , a chlorinated fr , is used in electronics and is an alternative to decabde . it is pervasive in the environment and has high potential for long - range transport . dp , measured as two isomers ( syn and anti ) , was detected in all of the homes , although levels were lower than other frs in this study and may have decreased over time . total dp concentrations were generally lower than those reported in ottawa in 20022003 and 2007 samples , whereas concentrations of individual isomers are comparable to those reported in vancouver in 20072008 . to evaluate whether our dust collection methods produced consistent results between the two sampling rounds , we analyzed samples for several legacy compounds that were banned years ago . these chemicals would not be introduced in new products between sampling rounds , though they could possibly increase or decrease with a change in an old item . despite being banned for many years , legacy compounds were frequently detected . pcbs , chlordane , and ddt were detected in almost all homes , with ddt at the highest concentration ( 2006 median 530 ng / g ; 2011 median 160 ng / g ) . polybrominated biphenyls ( pbbs ) were infrequently detected except for congener bb 153 , which was detected in about half of the homes . concentrations of legacy chemicals were generally significantly correlated across sampling rounds , indicating that the rank order was consistent over 5 years . however , the average concentration ratio ( 2011/2006 ) was 0.8 , which means that 2006 concentrations were generally higher than 2011 concentrations . however , it may also be due to some unidentified but systematic difference in sample collection between the two sampling rounds , which could also influence results for other chemicals . for example , pentabde levels went down between 2006 and 2011 , which may reflect decreasing use or may simply be due to the same factor causing decrease in legacy pollutant concentrations . in light of this , the firemaster 550 increase may be underestimated . two homes had substantial ( 1030 ) decreases in ddt and ddd ; one of these homes had significant renovations between rounds , while no explanation was identified for the other home . we were interested in learning which frs co - occurred , suggesting common sources , so we conducted correlation analysis for analytes within each sampling round ( si figure si4 ) , and also used these correlation estimates in cluster analysis to visualize relationships ( si figure si5 ) . as expected , many compounds known to co - occur in commercial formulations were correlated in both rounds . we saw strong correlations for : pbde congeners comprising the pentabde and octabde mixtures , ddt and its breakdown products , the legacy pesticides cis- and trans - chlordane and trans - nonachlor , pcb 153 and pcb 180 , and the dp isomers . interestingly , the brominated firemaster 550 chemicals , eh - tbb and beh - tebp , were also clustered consistently , but the third firemaster 550 constituent , tphp , did not cluster with them , suggesting other sources . as far as we know , this is the first study to analyze for such a broad range of frs in house dust and to analyze samples collected in the same home at two different time periods . this design allowed us to evaluate time trends in concentrations ; however , rigorous longitudinal analysis was not possible due to the small sample size ( n = 16 pairs ) . since our study began in 2006 , we did not fully capture the effects of the 2004 pbde phase - out , and although many participants reported some changes in their homes over the 5 year period , larger differences in fr concentration might be seen in a longer study . we observed differences in concentrations in many homes that reported acquiring furniture , carpets , and electronics ; however , our ability to link chemical concentrations with characteristics of products and residences was limited , because our questionnaire relied on residents recollections . residents may have introduced additional chemical sources that were not identified by our questionnaire , removed major sources without replacing them with new items , or failed to report on changes that we did ask about . there are probably additional frs used in consumer products that are not included because they have not been disclosed by manufacturers . we found that pbdes ; components of firemaster 550 ; other bfrs , such as hbcyd , tbbpa , btbpe , dbdpe ; and opfrs , including the carcinogenic tcep and tdcipp , were abundant and commonly detected , and we hypothesize that they are likely to be found in nearly all california homes . in our study , the levels of individual frs in dust exceeded 0.01% , with a cumulative level of all frs almost 0.03% in one home . such concentration of frs in dust is expected to lead to 30 g / day fr ingestion in a typical child . the average total load of frs in house dust was approximately 8090 g / g . for six chemicals , dust concentrations exceeded risk - based screening levels for residential soil in at least one of the homes , indicating exposure is potentially of health concern . specifically , concentrations of bde 47 , bde 99 , tcep , tdcipp , bb 153 , and ddt exceed screening levels , with 13 of 16 homes exceeding at least one chemical screening level in either sampling round . exposure pathways for residential soil are similar to house dust . screening levels provided in the si . our previous work showed that elevated pentabde levels in california house dust and serum are likely the result of the state s the present study shows california homes still have higher levels of pentabdes than the rest of the world and that california also has some of the highest concentrations of halogenated opfrs , which are also used in furniture foam . the only location with consistently higher opfr concentrations is japan , where the elevated opfrs levels are likely due to the early phase - out of pentabde almost 20 years ago . we also observed that firemaster 550 concentrations are increasing in california homes , suggesting that firemaster 550 is being used as a replacement for pentabde , which was phased - out in 2004 , shortly before our first sample collection . continued monitoring in california and other locations is warranted because we anticipate levels will continue to increase unless manufacturing practices change . following the phase - out of pbdes due to health concerns , other frs with considerable evidence of toxicity some frs appear to be replaced by less - studied chemicals whose health implications are unknown . chlorinated opfrs , some of the most abundant frs in our study , continue to be used despite evidence of carcinogenicity , listing as carcinogens under california s proposition 65 and iarc , and structural similarity to brominated tris ( tdbpp ) , which was banned in children s sleepwear in 1977 . despite this ban , we detected tdbpp in approximately half of the homes . we detected hbcyd in all homes , even though it has been identified under europe s reach program as a substance of very high concern and the u.s . epa initiated a snur to limit its use citing its bioaccumulation potential , persistence , toxicity to aquatic organisms and concerns about human reproductive , neurological , and developmental effects . publicly available health and toxicity information for the pbde replacements , such as firemaster 550 and btbpe , is very limited . the continued use of frs with established health concerns and introduction of replacement frs with limited data highlights the need to modernize u.s . chemical policies to require more complete disclosure and safety testing of consumer product chemicals prior to sale .

higher house dust levels of pbde flame retardants ( frs ) have been reported in california than other parts of the world , due to the state s furniture flammability standard . however , changing levels of these and other frs have not been evaluated following the 2004 u.s . phase - out of pentabde and octabde . we analyzed dust collected in 16 california homes in 2006 and again in 2011 for 62 frs and organohalogens , which represents the broadest investigation of frs in homes . fifty - five compounds were detected in at least one sample ; 41 in at least 50% of samples . concentrations of chlorinated opfrs , including two ( tcep and tdcipp ) listed as carcinogens under california s proposition 65 , were found up to 0.01% in dust , higher than previously reported in the u.s . in 75% of the homes , we detected tdbpp , or brominated tris , which was banned in children s sleepwear because of carcinogenicity . to our knowledge , this is the first report on tdbpp in house dust . concentrations of firemaster 550 components ( eh - tbb , beh - tebp , and tphp ) were higher in 2011 than 2006 , consistent with its use as a pentabde replacement . results highlight the evolving nature of fr exposures and suggest that manufacturers continue to use hazardous chemicals and replace chemicals of concern with chemicals with uncharacterized toxicity .

Introduction Materials and Methods Results and Discussion

california house dust contains some of the highest concentrations of polybrominated diphenyl ether ( pbde ) flame retardants ( frs ) in the world due to a state - wide furniture flammability standard ( technical bulletin 117 ) . pbdes in california homes and residents often exceed risk - based levels for children , raising concerns about exposures to the many other frs that have not yet been well - characterized . house dust is the primary route of exposure for pbdes , contributing 82% , on average , of a u.s . commercial pentabde and octabde mixtures were phased - out in 2004 in the u.s . as pbdes were phased out due to health concerns , other brominated frs ( bfrs ) and organophosphate flame retardants ( opfrs ) were introduced as replacements . chemtura , formerly great lakes chemical corporation , replaced pentabde in polyurethane foam with firemaster 550 , a mixture of 2-ethylhexyl-2,3,4,5-tetrabromobenzoate ( eh - tbb ) , bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate ( beh - tebp ) , triphenyl phosphate ( tphp ) , and a yet - to - be - fully characterized triaryl phosphate isopropylated mixture . epa recently announced plans to conduct risk assessments for beh - tebp and eh - tbb . in the late 1970s , tris(2,3-dibromopropyl ) phosphate ( tdbpp or brominated tris ) exposure data are limited , although the toxic breakdown product , 2,3-dibromo-1-propanol , was detected in u.s . world have been well established ; however , little is known about levels of other frs . we expect that frs used in polyurethane foam , including pentabde replacements , may be elevated due to the furniture flammability standard . exposure patterns for frs in other applications , such as electronics , are not known because of limited data , including for bde 209 . to provide data on a wider range of frs and on changing exposure patterns , this study measured a broad array of fr chemicals in repeat dust samples collected from 16 california homes . dust collected in california homes in 2006 and in the same homes in 2011 was analyzed for a broad suite of bfrs and opfrs ( n = 49 ) . dust samples were collected in 16 northern california homes in 2006 and again in the same homes with the same participants in 2011 . one sample preparation method , which was used to measure the bulk of bfrs , ocs , and opfrs , involved extraction using hex - ac ( 3:1 , v : v ) and fractionation on florisil . overall , 55 compounds were detected and 41 were found in at least 50% of the 32 samples ( table 1 ) . detected chemicals were 13 pbde congeners , 3 components of firemaster 550 , 15 other bfrs , 4 halogenated opfrs , 7 nonhalogenated opfrs , and 2 dechlorane - plus isomers . the highest concentrations , greater than 0.1 mg / g or 0.01% , were for two chlorinated opfrs , including tcep , which is listed as a carcinogen under california s proposition 65 , and tcipp , and one nonhalogenated opfr ( tboep ) . chemicals with median ratios above 1 were higher in 2011 samples compared with 2006 samples . we found all targeted pbde congeners in at least 50% of samples , with the components of pentabde ( bde 47 and bde 99 ) and decabde ( bde 209 ) mixtures in 100% of samples . median concentrations for all pbde congeners decreased from 2006 to 2011 ( table 1 ) ; however , not all of the means decreased ( data not shown ) , with exceptions likely driven by two homes with substantial increases in the congeners of pentabde mixture ( si figure si3 ) . however , since we saw decreases for legacy ocs , which should generally have minimal changes between 2006 and 2011 , the pbde reduction may reflect some unidentified but systematic difference in sample collection ( see legacy chemicals below ) . substantial decreases ( up to 20-fold ) in concentrations of pentabde were observed in three homes where participants reported remodeling or acquiring new furniture and/or rugs / carpet between 2006 and 2011 . reductions are likely the result of phase - outs ( 2004 ) and bans ( 2006 in ca ) of pentabde and octabde . ng / g in both sampling rounds , which is consistent with previous research showing higher pentabde concentrations in california than elsewhere due to the unique furniture flammability standard . in comparison to other studies within california , the median concentration of bde 47 in 2006 is similar ( within 30% ) ; whereas median concentrations of bde 99 and 100 in 2006 ( 2,200 ng / g and 520 ng / g , respectively ) were lower ( up to 2 ) than other california studies , which used slightly different vacuum sampling techniques . octabde levels correlate between sampling rounds ( along diagonal in si figure si4 ) , suggesting relatively stable concentrations in the homes over time ; however , pentabde and decabde levels were not significantly correlated over time , likely due to a few homes with substantial changes . besides tphp , the other constituents of firemaster 550 were only recently identified as two brominated compounds : eh - tbb and beh - tebp . to our knowledge , carcinogenicity , reproductive and development studies have not been conducted on the brominated components of firemaster 550 . we detected eh - tbb , beh - tebp , and tphp in all but one sample . concentrations of eh - tbb and beh - tebp increased across rounds ( median ratio > 1 ; figure 1 ) , except in one home where beh - tebp was found at 1,935 ng / g in the 2006 sample and not detected ( < 2 ng / g ) in 2011 ( si figure si3 ) . the generally increasing trend for eh - tbb and beh - tebp suggests that firemaster 550 is being used as a pentabde replacement . we compared our 2006 results to two sets of dust samples collected in the boston area ( 50 vacuum bag samples collected between 2002 and 2007 and 20 field technician collected dust samples collected in 2006 ) and vacuum bag dust collected in vancouver , canada in 20072008 . the 2006 eh - tbb and beh - tebp levels in our study were similar to , if not slightly lower than , levels in boston . in contrast , the levels of beh - tebp at both time periods in our study were higher than those in vancouver . eh - tbb and beh - tebp were significantly positively correlated within each sampling round ( si figure si4 ; tau = 0.40.5 ; p < 0.05 ) , which is expected since they are both in firemaster 550 . we compared the observed ratio of eh - tbb / beh - tebp in our samples with the ratio of the commercial mixture and boston - area samples to evaluate if firemaster 550 is the sole source and if eh - tbb and beh - tebp have different fates once applied to a product . we observed a mean eh - tbb / beh - tebp ratio of 0.6 ( 0.043.1 ) in the 2006 samples and 1.5 ( 0.811 ) in the 2011 samples . this suggests other sources of beh - tebp in california or a different fate of the chemicals . tphp , also present in firemaster 550 , was not significantly correlated with either eh - tbb or beh - tebp in either sampling round , although tphp concentrations increased in homes with substantial increases in eh - tbb and beh - tebp . it is unclear whether the phase - out of pentabde and octabde mixtures influenced the pattern of hbcyd use . however , our maxima ( 2006 : 6800 ng / g ; 2011 : 1800 ng / g ) were substantially lower than those reported in boston living room dust ( 130 200 ng / g ) and uk samples ( 110 000 ng / g ) . however , we observed relative abundances of 4550% , 4045% , and approximately 10% for - , - , and -hbcyd , respectively . tetrabromobisphenol a ( tbbpa ) , the most commonly used bfr , is employed as a reactive fr in circuit boards , plastics , paper and textiles as a plasticizer , in coatings and adhesives , and as an intermediate in the synthesis of other frs . tbbpa was detected in nearly all homes in both rounds with concentrations ranging from < 10 to 3400 ng / g in 2006 and from 22 to 2000 we found a significant association between concentration reductions and new electronics suggesting that new electronics contain less tbbpa ( rho = 0.69 ; p = 0.003 ) . btbpe , in production since the 1970s and now used to replace octabde , and dbdpe , introduced in mid-1980s and available as a replacement for decabde , were detected in nearly 100% of samples . the concentrations of btbpe , which has limited toxicity data ( see si table si4 ) , were similar between 2006 and 2011 . in contrast , concentrations of dbdpe , structurally similar to bde 209 and associated with reproductive and developmental toxicities , were generally higher in 2011 ( table 1 and figure 1 ) , and two homes had substantial ( > 20-fold ) increases . another commonly detected fr was the tbbpa derivative tetrabromobisphenol a - bis(2,3-dibromopropylether ) ( tbbpa - bdbpe ) , which is being studied by the national toxicology program ( ntp ) because of the structural similarity with the carcinogenic tdbpp ( brominated tris ) . tdbpp or brominated tris was banned from children s sleepwear in the u.s . we detected tdbpp in 62% of 2006 samples and 38% of 2011 samples . as far as we know , this is the first report of tdbpp in house dust , although we previously detected its mutagenic metabolite , 2,3-dibromo-1-propanol , in about 10% of indoor air samples from cape cod , ma . dust concentrations were much lower in 2011 ( mean 40 ng / g ; maximum 310 ng / g ) compared with 2006 ( mean 1000 ng / g ; maximum 8900 ng / g ) , though this may be due to whatever factor led to lower concentrations of legacy pollutants ( see below ) . we also detected three chlorinated opfrs : tcep , tcipp , and tdcipp ( chlorinated tris ) , which are used in polyurethane foams as replacements for pentabde . the cpsc estimate of children s exposure from treated furniture was 5 higher than the agency s acceptable daily intake , with most of the exposure from inhalation of the chemicals volatilized from treated furniture . median concentrations of all chlorinated opfrs were above 1,000 ng / g , or 1 g / g , in both sampling rounds , and maxima were > 100 000 ng / g or 0.01% , making these the most abundant frs in this study ( table 1 ) . based on limited comparison data , concentrations of chlorinated opfrs observed in this study are some of the highest in the world ; only concentrations in japan are consistently higher . generally lower levels have been reported for homes in boston , belgium , spain , sweden , and germany , except for higher tcipp in spain and tdcipp in sweden . the highest concentrations of chlorinated opfrs were found in a study of 41 japanese homes , which reported median concentrations 2- to 25-fold higher than seen in our california samples . the levels in japan are likely a result of a voluntary phase - out of pentapbde in the early 1990s . concentrations of the chlorinated opfrs are not correlated with each other , likely because tcipp has been reported as a replacement for tcep and is often used in the same types of products as tdcipp , which is typically used only when a more efficient fr is needed , since it is more expensive . concentrations of tboep were higher than in dust samples collected in belgian and spanish homes , although lower than japanese homes ( 1 570 000 ng / g ) . dp , measured as two isomers ( syn and anti ) , was detected in all of the homes , although levels were lower than other frs in this study and may have decreased over time . pcbs , chlordane , and ddt were detected in almost all homes , with ddt at the highest concentration ( 2006 median 530 ng / g ; 2011 median 160 ng / g ) . polybrominated biphenyls ( pbbs ) were infrequently detected except for congener bb 153 , which was detected in about half of the homes . however , the average concentration ratio ( 2011/2006 ) was 0.8 , which means that 2006 concentrations were generally higher than 2011 concentrations . however , it may also be due to some unidentified but systematic difference in sample collection between the two sampling rounds , which could also influence results for other chemicals . for example , pentabde levels went down between 2006 and 2011 , which may reflect decreasing use or may simply be due to the same factor causing decrease in legacy pollutant concentrations . we saw strong correlations for : pbde congeners comprising the pentabde and octabde mixtures , ddt and its breakdown products , the legacy pesticides cis- and trans - chlordane and trans - nonachlor , pcb 153 and pcb 180 , and the dp isomers . interestingly , the brominated firemaster 550 chemicals , eh - tbb and beh - tebp , were also clustered consistently , but the third firemaster 550 constituent , tphp , did not cluster with them , suggesting other sources . as far as we know , this is the first study to analyze for such a broad range of frs in house dust and to analyze samples collected in the same home at two different time periods . since our study began in 2006 , we did not fully capture the effects of the 2004 pbde phase - out , and although many participants reported some changes in their homes over the 5 year period , larger differences in fr concentration might be seen in a longer study . we found that pbdes ; components of firemaster 550 ; other bfrs , such as hbcyd , tbbpa , btbpe , dbdpe ; and opfrs , including the carcinogenic tcep and tdcipp , were abundant and commonly detected , and we hypothesize that they are likely to be found in nearly all california homes . in our study , the levels of individual frs in dust exceeded 0.01% , with a cumulative level of all frs almost 0.03% in one home . such concentration of frs in dust is expected to lead to 30 g / day fr ingestion in a typical child . the average total load of frs in house dust was approximately 8090 g / g . for six chemicals , dust concentrations exceeded risk - based screening levels for residential soil in at least one of the homes , indicating exposure is potentially of health concern . specifically , concentrations of bde 47 , bde 99 , tcep , tdcipp , bb 153 , and ddt exceed screening levels , with 13 of 16 homes exceeding at least one chemical screening level in either sampling round . our previous work showed that elevated pentabde levels in california house dust and serum are likely the result of the state s the present study shows california homes still have higher levels of pentabdes than the rest of the world and that california also has some of the highest concentrations of halogenated opfrs , which are also used in furniture foam . the only location with consistently higher opfr concentrations is japan , where the elevated opfrs levels are likely due to the early phase - out of pentabde almost 20 years ago . we also observed that firemaster 550 concentrations are increasing in california homes , suggesting that firemaster 550 is being used as a replacement for pentabde , which was phased - out in 2004 , shortly before our first sample collection . continued monitoring in california and other locations is warranted because we anticipate levels will continue to increase unless manufacturing practices change . following the phase - out of pbdes due to health concerns , other frs with considerable evidence of toxicity some frs appear to be replaced by less - studied chemicals whose health implications are unknown . chlorinated opfrs , some of the most abundant frs in our study , continue to be used despite evidence of carcinogenicity , listing as carcinogens under california s proposition 65 and iarc , and structural similarity to brominated tris ( tdbpp ) , which was banned in children s sleepwear in 1977 . despite this ban , we detected tdbpp in approximately half of the homes . we detected hbcyd in all homes , even though it has been identified under europe s reach program as a substance of very high concern and the u.s .

systemic sclerosis ( ssc ) is a systemic rheumatic disease characterized by vasculopathy , autoimmunity , and fibrosis . cyclophosphamide ( cyc ) has shown modest efficacy in the treatment of ssc - associated interstitial lung disease ( ild ) but its long - term use is accompanied by significant toxicity . therefore , novel therapeutic approaches are desperately needed . during the last decade , b - cell depletion by rituximab ( rtx ) , a monoclonal antibody that targets b - cells , has emerged as a promising therapy for a wide range of systemic autoimmune diseases . it has been approved for the treatment of rheumatoid arthritis but it has also been tried in systemic lupus erythematosus , systemic vasculitides , and multiple sclerosis , among others . an expanding body of experimental evidence suggests that b - cells play a role in the fibrotic process , raising the question of whether b - cell depletion might be a potential therapeutic approach in ssc [ 58 ] . during the last 2 years , 4 small - scale , open - label studies and a few case reports have addressed this question to some extent , reporting encouraging results . in this paper we provide the experimental evidence supporting the active role of b - cells in fibrosis and summarize all the available clinical evidence regarding the use of rtx in patients with ssc . we used the following key words : systemic sclerosis , scleroderma , rituximab , b - cells , fibrosis , ild , and therapy in various combinations . research in ssc has been problematic due to the low prevalence of the disease and the lack of an animal model that possesses all features of the human disease . nevertheless , the tight skin mouse ( tsk ) and the bleomycin ( blm ) induced mouse model of ssc have been extensively used as animal models of the disease . tsk mice are characterized by extensive skin fibrosis and immunological abnormalities including the presence of autoantibodies to topoisomerase-1 , both reminiscent of those observed in the human disease . a lot of research has been done on the potential role of b - cells in this animal model . it has been reported that tsk b - cells exhibit enhanced cd19 signaling compared to wt b - cells , although the expression of this molecule was similar in tsk and wt b - cells . cd19 is a membrane glycoprotein of the immunoglobulin superfamily and part of the hetero - oligomeric complex comprising the complement receptor type 2 , which positively regulates bcr activation . the authors reported enhanced cd19 tyrosine phosphorylation by 45% compared to wt b - cells . tyrosine phosphorylation of vav and lyn kinase , both of which are important downstream steps of cd19 signaling , was also found to be enhanced by 3.4-fold and 22% respectively , compared to wt b - cells . cytoplasmic ca responses , generated by cd19 ligation , were also significantly augmented in tsk b - cells compared to wt b - cells . the crucial role of cd19 signaling in this animal model is underscored by the fact that cd19 deficiency normalizes the hyper responsive phenotype of tsk b - cells and leads to a significant improvement of skin fibrosis compared to control tsk mice . the above data suggest that b - cell hyperactivity and fibrosis are somehow linked in this animal model and raise the question of whether targeting b - cells might be an effective way of attenuating fibrosis . in another study by asano et al . , the effect of enforced cd19 overexpression on b - cells in the tsk mouse model was assessed . more specifically , cd19 transgenic mice that expressed 20% higher levels of cd19 ( cd19 tg4 ) and 200% higher levels ( cd19 tg1 ) exhibited increased antitopoisomerase-1 levels by 7.9-fold and 20-fold , compared to control tsk mice , respectively . despite this increase in autoab production , cd19 overexpression did not lead to worsening of skin fibrosis compared to control tsk mice . the authors also focused on gaining further insight into the hyperactive phenotype of the tsk b - cell and found that this can relate to a defective cd22 signaling , an important negative bcr response regulator . upon stimulation , cd22 tyrosine phosphorylation was 58% lower in tsk b - cells compared to wt b - cells . the hypophosphorylation of cd22 coincided with the hyperphosphorylation of cd19 , which led the investigators to suggest that defective cd22 signaling leads to increased cd19 signaling which is , at least partially , responsible for the hyperresponsive phenotype of the tsk b - cell . recently it was reported that tsk mice have autoantibodies agaist cd22 which were found to be functional and able to attenuate cd22 activation . since cd22 inhibits b - cell activation , these autoabs may contribute to the overactivated phenotype of tsk b - cells . in other words , tsk b - cells produce an autoab that enhances their own activation , something that may have pathogenic implications . once the link between b - cell hyperactivity and fibrosis had been established in the tsk mouse , the next logical step would be to assess the effect of b - cell depletion in this animal model . rtx administration to newborn tsk mice led to a significant attenuation of skin fibrosis by 43% compared to control tsk mice accompanied by a significant reduction in autoantibody production . when the same treatment was applied in 56-day old tsk mice , which are characterized by established skin fibrosis , skin thickening was not reduced , compared to tsk control mice . these data suggest that b - cells may be more important in the early phase of the disease but less so in established disease . it is noteworthy that b - cells are not present in the skin of tsk mice , indicating that their contribution to skin fibrosis may be indirect . despite the improvement of skin thickening , b - cell depletion had no effect on the lung disease of tsk mice , indicating that different manifestations are probably mediated by different pathogenic mechanisms in this animal model . another study explored the effects of b - cell survival factor ( baff ) inhibition in tsk mice and reported improvement of skin fibrosis and attenuation of autoab production . all these data underline the key role of b - cells in the fibrotic process in this animal model . the role of b - cells in fibrosis has also been explored in the blm - induced mouse model of ssc . besides skin involvement , lung fibrosis is a prominent feature of this animal model , making it suitable for the assessment of a given treatment on ild . the investigators reported that blm administration to cd19 knockout mice led to diminished skin thickening compared to blm - treated wt littermates . more importantly , cd19 deficiency led to attenuation of blm - induced lung fibrosis , indicating that b - cells may be actively involved in the fibrotic process not only in the skin but in the lung as well . recently , the role of cd19 during the development of pulmonary fibrosis in the blm - induced model has been extensively assessed . it was reported that cd19 knockout mice exhibited less lung fibrosis in sharp contrast to mice overexpressing cd19 which showed augmented lung fibrosis compared to wt littermates . interestingly , cd19 expression correlated with the number of b - cells in the bronchoalveolar lavage fluid ; cd19 deficiency inhibited the accumulation of b - cells in the alveolar compartment following blm challenge . the above data indicate that cd19 plays a crucial role in pulmonary fibrosis in this mouse model . research on animal models of scleroderma has provided evidence indicating a potential link between b - cell hyperactivity and fibrosis . however , we should note that ssc is a far more complex disease and therefore these data can not be directly extrapolated to humans . only a few studies have addressed the potential contribution of b - cells in the pathogenesis of ssc . in one such study , it was reported that b - cells from patients with ssc had 20% higher cd19 expression compared to b - cells from healthy subjects . detailed phenotypic characterization of b - cells from patients with ssc revealed that peripheral blood b - cells were increased in patients with ssc compared to healthy subjects . naive b - cells were reported to be increased in patients with ssc whereas memory b - cells and plasmablasts were reduced , compared to healthy subjects . memory b - cells from patients with ssc had increased expression of several activation markers , including cd95 and were prone to spontaneous apoptosis . since patients with ssc express higher levels of cd19 on b - cells and since cd19 is a key molecule in the regulation of signaling thresholds in these cells , something that may relate to break of tolerance and induction of autoimmunity , investigators explored the mechanisms involved in cd19 overexpression in ssc b - cells . tsuchiya et al . explored the potential association of cd19 polymorphisms with ssc and the level of cd19 expression on b - cells . they reported a significant association between the 499 t allele in the promoter region of the cd19 gene with ssc , with an odds ratio of 2.18 ; carriers of this allele exhibited significantly higher cd19 levels on b - cells compared to noncarriers . these data raise the question of whether cd19 upregulation in ssc b - cells is genetically determined . it is not clear why ssc b - cells exhibit such an overactivated phenotype ; b - cell survival factors may be implicated . it has been reported that baff serum levels are higher in patients with ssc compared to healthy controls ( p < 0.001 ) . furthermore , patients with the diffuse form had significantly increased levels compared to patients with the limited form of the disease . nevertheless , this was not a disease - specific finding , since patients with sle or dm had similarly increased levels . patients with ssc , exhibiting increased serum baff levels tended to have more severe skin fibrosis as assessed by the mrss tool ( p < 0.01 ) , worse fvc values ( p < 0.05 ) and higher esr ( p < 0.05 ) . furthermore , decreasing serum baff levels were associated with attenuation of skin fibrosis , whereas increasing levels correlated with clinical worsening . the expression of baff in the skin at the mrna level was found to be increased in early disease . moreover , upregulation of the baff receptor was reported on b - cells from patients with ssc compared to healthy subjects . it is also worth mentioning that b - cells from patients with ssc stimulated with baff produced 38% more il-6 , a cytokine able to stimulate fibroblasts , compared to b - cell from healthy subjects . in a recent study it was found that peripheral blood mononuclear cells ( pbmcs ) from ssc patients produced significantly more april ( a proliferation inducing ligand ) , a b - cell survival factor , compared to pbmcs from healthy subjects ( p < 0.01 ) . these data indicate that upregulation of b - cell survival factors may contribute to b - cell hyperactivity and autoimmunity in ssc . baff and april emerge as two interesting therapeutic targets ; inhibition of these molecules may modulate b - cell function in ssc and potentially lead to clinical benefit . an interesting study , performed by whitfield et al . examined gene expression profile , using microarrays in scleroderma skin compared to normal . the authors found that genes characteristic of b - cells , fibroblasts and endothelial cells were differentially expressed in scleroderma compared to normal skin . interestingly , the same expression pattern was evident in patients with ssc in both clinically involved as well as uninvolved skin , underscoring the systemic nature of the disease . since endothelial cells and fibroblasts are considered key players in ssc , these data point towards a potentially active role of b - cells in skin fibrosis . the potential role of b - cells in ssc - associated ild has been inadequately investigated . it has been reported that b - cells are present in lung biopsies from patients with ssc - associated ild and that plasma cell infiltration of the alveolar walls is an early finding . the above studies have documented the presence of b - cells in both skin and lung of patients with ssc there are 4 small - scale , open - label clinical studies exploring the potential clinical efficacy of rtx in ssc , including one from our research group and a few case reports . in the first study by smith et al . , eight patients with early ( defined by disease duration of < 4 years from the first non - raynaud 's disease manifestation ) diffuse ssc received a single course of rtx ( consisting of 2 infusions , 1000 mg each , at day 1 and 15 ) . patients were evaluated clinically at 24 weeks and subjected to skin biopsies at baseline and 12 weeks . five patients had evidence of mild ild ; patients with severe ild were excluded from the study . improvement of skin thickening was reported as assessed by the mrss tool ( mean sd : 24.8 3.4 versus 14.3 3.5 at baseline versus 24 weeks resp . it is worth mentioning that improvement of skin thickening was verified at the histological level , since both collagen and myofibroblast score were reduced significantly following treatment ( p = 0.014 and p = 0.013 , resp . ) . half of the patients had evidence of b - cell infiltration in their skin ; treatment effectively depleted these cells . lung function tests , systolic pulmonary artery pressure , left ventricular ejection fraction , creatinine clearance , and haq score remained stable throughout the study . two serious events were reported ( one patient underwent coronary artery bypass surgery and another was hospitalized due to low - grade fever that spontaneously subsided ) but were considered to be probably unrelated to study drug . this is the first study that provides clinical as well as histological evidence that rtx treatment may favourably affect skin fibrosis in ssc . it should be noted however , that this study has potential limitations which are the relatively small number of patients recruited and the lack of a control arm . resolution of skin fibrosis is associated with the natural course of the disease , therefore uncontrolled studies are difficult to interpret . nevertheless , the improvement of skin fibrosis reported in this study was quite significant , which makes it rather unlikely to have occurred spontaneously , especially within the limited time frame of the study . another study reported the effects of a single course of rtx treatment in 15 patients with early ( as defined by disease duration of < 18 months from the first non - raynaud 's disease manifestation ) diffuse ssc . , only 7 out of 15 patients had evidence of mild ild , since the existence of moderate or severe ild was an exclusion criterion . , no improvement of skin thickening as assessed by the mrss , tool was reported ( mean sd : 20.6 4.4 , 20.2 5.5 and 21.1 5.2 at baseline , 24 wks , and 48 wks , respectively , p = ns ) . however , histologic improvement was found ; myofibroblast score declined from 49.5 to 36.6 ( p < 0.05 ) . skin infiltrating b - cells were significantly increased in patients with ssc compared to healthy controls who had no b - cells and were eliminated posttreatment . pulmonary function tests remained stable at 24 wks compared to baseline ; it is noteworthy though that fvc values increased by an average of 3.5% at 24 wks compared to pretreatment values , but the 95% ci was wide and thus results were not statistically significant . our research group has performed an open label , randomized controlled , 1-year pilot study , assessing the effect of rtx in ssc . we recruited 14 patients with ssc randomized as follows : 8 patients in the treatment arm and 6 patients in the control arm of the study . all patients had diffuse disease , were anti - scl70 positive , and had evidence of ild . there were no differences in terms of disease duration , baseline mrss and baseline pft 's between the treatment and the control group . patients in the treatment arm received 2 cycles of rtx at baseline and 24 weeks ( each cycle consisting of 4 weekly rtx infusions ( 375 mg / m ) ) . we found a significant improvement of both fvc ( mean sd : 68.13 19.69 versus 75.63 19.73% of predicted values , at baseline versus 1 year resp . , p = 0.0018 ) and dlco ( mean sd : 52.25 20.71 versus 62 23.21% of predicted values , at baseline versus 1 year , resp . , p = 0.017 ) in the treatment group whereas no change was noticed in the control group . the median ( upper and lower quartile values ) percentage of improvement of fvc in the rtx group was 10.25% ( 6.1918.65 ) whereas in the control group fvc deteriorated ( median percentage of deterioration ( upper and lower quartile values ) 5.04% ( 4.1111.6 ) ) . direct comparison of fvc changes recorded at 1 year , revealed that the rtx - treated group improved significantly ( p = 0.002 ) compared to the standard - treatment ( control ) group . the median ( upper and lower quartile values ) percentage of improvement of dlco in the rtx group was 19.46% ( 3.730.8 ) whereas in the control group the median percentage of deterioration was 7.5% ( 1.426.57 ) ( p = 0.023 ) . skin thickening , assessed with the mrss , was similar in the two treatment groups at baseline ( p = 0.50 ) . however at the 1 year evaluation , there was a significant decrease of mrss in the rtx group compared to the baseline score ( mean sd , 13.5 6.84 versus 8.37 6.45 at baseline versus 1 year , resp . no significant change in skin scores was noticed in the control group at 1 year when compared to the baseline mrss ( mean sd , 11.50 2.16 versus 9.66 3.38 at baseline versus 1-year , resp . , the median ( upper and lower quartile values ) percentage of improvement in the rtx - treated group was 39.25% ( 27.3364.95 ) compared to 20.80% ( 10.7839.28 ) in the control group . statistical analysis revealed that differences tended to be but were not statistically significant ( p = 0.06 ) . we found a significant reduction of collagen deposition in the papillary dermis at 24 wk compared to baseline in patients treated with rtx ; histologic improvement correlated with skin b - cell depletion . histological data matched the clinical data , since all patients showing histologic improvement also improved clinically . one serious adverse event was reported ( respiratory tract infection ) ; the patient recovered fully following short - term hospitalization . even though this is the first and only so far , randomized , controlled study assessing the efficacy of rtx in ssc , several limitations exist . firstly , the small number of patients recruited does not provide the study with sufficient statistical power to prove efficacy . additionally , most patients had long - standing disease since no disease duration restriction was applied and was heterogeneous in terms of disease duration , severity , and previous treatments . recently , one more study assessing the clinical efficacy of rtx in ssc was published . nine patients with ssc were recruited and received one course of rtx ( consisting of 2 infusions , 1 gr each ) . all patients had severe cutaneous involvement and had experienced worsening of skin score despite treatment with cyc . a significant reduction of skin thickening was reported with patients showing a median decrease of skin score of 43.3% at 6 months compared to baseline . disease activity and severity index also declined . pfts remained stable throughout the study . a significant decline in il-6 levels following treatment was also reported ; the authors hypothesized that this may have contributed to attenuation of skin fibrosis . three case reports have also appeared in the literature regarding the use of rtx in ssc . the first case describing the beneficial effect of rtx on ssc - associated ild was reported by mcgonagle et al . . however treatment effect waned over time and a second course of rtx was administered . these data are in agreement with ours , showing that consecutive treatment courses may be needed to augment and sustain the effect of rtx on pulmonary function . the beneficial effect of rtx on ssc - associated ild has also been documented in a case report by our group . our patient was treated with 4 consecutive rtx courses every 6 months and completed a followup of 2 years . pfts significantly increased ; fvc and dlco reached values of 35% , and 33% respectively , compared to 30% and 14% of pretreatment values . quantification of ground glass lesions using a computer - aided diagnosis system showed a 14% reduction . skin thickening improved as indicated by a decline in mrss from 20 to 9 . clinical improvement coincided with histologic improvement with reduction of collagen accumulation and myofibroblast score ; skin infiltrating b - cells were eliminated post treatment . the functional status of the patient improved as indicated by a decrease in haq score and an increase in 6-minute walking distance . recently , another case of successful treatment of cyc - resistant ssc - associated ild was reported . finally , rtx seems to favourably affect ild in the context of other systemic rheumatic diseases such as the antisynthetase syndrome as indicated by several reports [ 32 , 33 ] . all the available published data regarding the clinical efficacy of rtx in ssc are summarized in table 1 . so far treatment is based on nonspecific immunosuppression , with agents such as cyc or mycophenolate mofetil , with modest results . rtx has been used with varying degrees of success in most systemic autoimmune diseases and this is why , one may argue that its effect on a severe , incurable systemic autoimmune disease such as ssc , is worthwhile exploring . furthermore , there is a strong rationale for the use of rtx in ssc ; an expanding body of evidence from basic research points to the direction that b - cells may be active players in the fibrotic process . on clinical grounds , until now , 43 patients with diffuse ssc have been treated with rtx ( available published data ) . first of all , this agent seems to be well tolerated in ssc since only few adverse events have been reported . but is rtx clinically effective in ssc ? based on the available clinical evidence , definite conclusions can not be drawn ; however results are encouraging . in 3 out of 4 studies skin thickening significantly improved and , even in the single study where no clinical benefit on skin thickening was found , histologic improvement was documented . the most fearful manifestation of ssc is lung disease which is nowadays the leading cause of mortality . the clinical evidence on the efficacy of rtx in ssc - associated ild is limited , since most patients recruited either did not have ild or had only very mild ild . with the exception of our study , the other 3 studies were not designed to test the potential clinical efficacy of rtx in ssc - associated ild . nevertheless , in these three studies pfts remained stable , even though most patients had early disease and therefore were most likely to exhibit declining pfts during their followup . in our study , a significant improvement of lung function was reported in contrast to other studies where pfts remained stable at 6 months compared to baseline . we should note , however , several differences in the design of the studies that could potentially explain these diverse findings . in the other studies most patients did not have ild in contrast to our study where the presence of ild was an inclusion criterion . furthermore , the higher dose of rtx used ( 4 375 mg / m instead of 2 1000 mg ) , consecutive treatments , and the longer evaluation period could also be potential explanations . the significant improvement in lung function tests observed in our study indicates that rtx may favourably affect ssc - associated ild . long - term treatment with rtx may either improve or stabilize lung function over time in patients with ssc . all 8 patients recruited in the treatment arm group of our study remained on rtx treatment and received two additional courses ; their pfts continued to improve during the second year of followup . if rtx turns out to be an effective treatment for ssc , which could be the potential mechanisms of action ? first , b - cells appear to be actively involved in the fibrotic process , as indicated by data derived from both animal models and humans . elimination of skin infiltrating b - cells by rtx has been documented , albeit not consistently . taking into account that b - cell infiltration is a prominent finding in lung biopsies from patients with ssc - associated ild , depletion of this population may be a potential explanation for the clinical improvement . it would therefore be of great interest to study the effect of rtx on lung disease at a histological level , but this may be inherently difficult and challenging . we have also recently shown that rtx - induced improvement of skin fibrosis associates with a decrease in pdgfr phosphorylation ( which corresponds to activation ) in scleroderma skin . finally , another mechanism may involve indirect effect(s ) of rtx on other cells , such as t cells . both experimental and clinical evidence regarding b - cell depletion in ssc is encouraging and certainly points to the direction that further exploration of its clinical efficacy is warranted . the best way forward would be a multicenter , randomized , double blind , placebo - controlled study . if such a study is performed we propose that it should focus on ild rather than skin disease and that it should be designed in such a way , that patients are treated for at least one year and evaluated thereafter ; in this way , a treatment effect on ild ( if any ) would be more easily depicted . to our knowledge , up until now , no such study has been registered . interestingly , however , a phase ii study assessing the clinical efficacy of rtx in ssc - associated pulmonary arterial hypertension has already been launched ( http://clinicaltrials.gov/show/nct01086540 ) . moreover , a phase i study of medi-551 ( mab against cd19 ) has also been launced ( http://clinicaltrials.gov/show/nct00946699 ) . we believe that a large - scale multicenter , randomized study assessing the potential clinical efficacy of rtx in ssc is highly needed .

systemic sclerosis ( ssc ) is a systemic rheumatic disease with poor prognosis since therapeutic options are limited . recent evidence from animal models suggests that b - cells may be actively involved in the fibrotic process . b - cells from tight skin mice , an animal model of scleroderma , display a hyperresponsive phenotype ; treatment with rituximab ( rtx ) significantly attenuates skin fibrosis in this animal model . in humans , b - cell infiltration is a prominent finding in most lung biopsies obtained from patients with ssc - associated interstitial lung disease . several open label studies have assessed the clinical efficacy of rtx in ssc . in most patients skin fibrosis improved ; lung function either improved or remained stable . definite conclusions regarding the clinical efficacy of rtx in ssc can not be drawn but further exploration with a multicenter , randomized study is warranted .

1. Introduction 2. Methods 3. Results 4. Discussion

systemic sclerosis ( ssc ) is a systemic rheumatic disease characterized by vasculopathy , autoimmunity , and fibrosis . cyclophosphamide ( cyc ) has shown modest efficacy in the treatment of ssc - associated interstitial lung disease ( ild ) but its long - term use is accompanied by significant toxicity . during the last decade , b - cell depletion by rituximab ( rtx ) , a monoclonal antibody that targets b - cells , has emerged as a promising therapy for a wide range of systemic autoimmune diseases . an expanding body of experimental evidence suggests that b - cells play a role in the fibrotic process , raising the question of whether b - cell depletion might be a potential therapeutic approach in ssc [ 58 ] . during the last 2 years , 4 small - scale , open - label studies and a few case reports have addressed this question to some extent , reporting encouraging results . in this paper we provide the experimental evidence supporting the active role of b - cells in fibrosis and summarize all the available clinical evidence regarding the use of rtx in patients with ssc . we used the following key words : systemic sclerosis , scleroderma , rituximab , b - cells , fibrosis , ild , and therapy in various combinations . research in ssc has been problematic due to the low prevalence of the disease and the lack of an animal model that possesses all features of the human disease . nevertheless , the tight skin mouse ( tsk ) and the bleomycin ( blm ) induced mouse model of ssc have been extensively used as animal models of the disease . tsk mice are characterized by extensive skin fibrosis and immunological abnormalities including the presence of autoantibodies to topoisomerase-1 , both reminiscent of those observed in the human disease . a lot of research has been done on the potential role of b - cells in this animal model . it has been reported that tsk b - cells exhibit enhanced cd19 signaling compared to wt b - cells , although the expression of this molecule was similar in tsk and wt b - cells . the authors reported enhanced cd19 tyrosine phosphorylation by 45% compared to wt b - cells . tyrosine phosphorylation of vav and lyn kinase , both of which are important downstream steps of cd19 signaling , was also found to be enhanced by 3.4-fold and 22% respectively , compared to wt b - cells . cytoplasmic ca responses , generated by cd19 ligation , were also significantly augmented in tsk b - cells compared to wt b - cells . the crucial role of cd19 signaling in this animal model is underscored by the fact that cd19 deficiency normalizes the hyper responsive phenotype of tsk b - cells and leads to a significant improvement of skin fibrosis compared to control tsk mice . the above data suggest that b - cell hyperactivity and fibrosis are somehow linked in this animal model and raise the question of whether targeting b - cells might be an effective way of attenuating fibrosis . , the effect of enforced cd19 overexpression on b - cells in the tsk mouse model was assessed . more specifically , cd19 transgenic mice that expressed 20% higher levels of cd19 ( cd19 tg4 ) and 200% higher levels ( cd19 tg1 ) exhibited increased antitopoisomerase-1 levels by 7.9-fold and 20-fold , compared to control tsk mice , respectively . the authors also focused on gaining further insight into the hyperactive phenotype of the tsk b - cell and found that this can relate to a defective cd22 signaling , an important negative bcr response regulator . upon stimulation , cd22 tyrosine phosphorylation was 58% lower in tsk b - cells compared to wt b - cells . the hypophosphorylation of cd22 coincided with the hyperphosphorylation of cd19 , which led the investigators to suggest that defective cd22 signaling leads to increased cd19 signaling which is , at least partially , responsible for the hyperresponsive phenotype of the tsk b - cell . since cd22 inhibits b - cell activation , these autoabs may contribute to the overactivated phenotype of tsk b - cells . in other words , tsk b - cells produce an autoab that enhances their own activation , something that may have pathogenic implications . once the link between b - cell hyperactivity and fibrosis had been established in the tsk mouse , the next logical step would be to assess the effect of b - cell depletion in this animal model . rtx administration to newborn tsk mice led to a significant attenuation of skin fibrosis by 43% compared to control tsk mice accompanied by a significant reduction in autoantibody production . when the same treatment was applied in 56-day old tsk mice , which are characterized by established skin fibrosis , skin thickening was not reduced , compared to tsk control mice . these data suggest that b - cells may be more important in the early phase of the disease but less so in established disease . it is noteworthy that b - cells are not present in the skin of tsk mice , indicating that their contribution to skin fibrosis may be indirect . despite the improvement of skin thickening , b - cell depletion had no effect on the lung disease of tsk mice , indicating that different manifestations are probably mediated by different pathogenic mechanisms in this animal model . another study explored the effects of b - cell survival factor ( baff ) inhibition in tsk mice and reported improvement of skin fibrosis and attenuation of autoab production . all these data underline the key role of b - cells in the fibrotic process in this animal model . the role of b - cells in fibrosis has also been explored in the blm - induced mouse model of ssc . besides skin involvement , lung fibrosis is a prominent feature of this animal model , making it suitable for the assessment of a given treatment on ild . more importantly , cd19 deficiency led to attenuation of blm - induced lung fibrosis , indicating that b - cells may be actively involved in the fibrotic process not only in the skin but in the lung as well . recently , the role of cd19 during the development of pulmonary fibrosis in the blm - induced model has been extensively assessed . interestingly , cd19 expression correlated with the number of b - cells in the bronchoalveolar lavage fluid ; cd19 deficiency inhibited the accumulation of b - cells in the alveolar compartment following blm challenge . the above data indicate that cd19 plays a crucial role in pulmonary fibrosis in this mouse model . research on animal models of scleroderma has provided evidence indicating a potential link between b - cell hyperactivity and fibrosis . however , we should note that ssc is a far more complex disease and therefore these data can not be directly extrapolated to humans . only a few studies have addressed the potential contribution of b - cells in the pathogenesis of ssc . in one such study , it was reported that b - cells from patients with ssc had 20% higher cd19 expression compared to b - cells from healthy subjects . detailed phenotypic characterization of b - cells from patients with ssc revealed that peripheral blood b - cells were increased in patients with ssc compared to healthy subjects . naive b - cells were reported to be increased in patients with ssc whereas memory b - cells and plasmablasts were reduced , compared to healthy subjects . memory b - cells from patients with ssc had increased expression of several activation markers , including cd95 and were prone to spontaneous apoptosis . since patients with ssc express higher levels of cd19 on b - cells and since cd19 is a key molecule in the regulation of signaling thresholds in these cells , something that may relate to break of tolerance and induction of autoimmunity , investigators explored the mechanisms involved in cd19 overexpression in ssc b - cells . explored the potential association of cd19 polymorphisms with ssc and the level of cd19 expression on b - cells . they reported a significant association between the 499 t allele in the promoter region of the cd19 gene with ssc , with an odds ratio of 2.18 ; carriers of this allele exhibited significantly higher cd19 levels on b - cells compared to noncarriers . these data raise the question of whether cd19 upregulation in ssc b - cells is genetically determined . it is not clear why ssc b - cells exhibit such an overactivated phenotype ; b - cell survival factors may be implicated . it has been reported that baff serum levels are higher in patients with ssc compared to healthy controls ( p < 0.001 ) . furthermore , patients with the diffuse form had significantly increased levels compared to patients with the limited form of the disease . nevertheless , this was not a disease - specific finding , since patients with sle or dm had similarly increased levels . patients with ssc , exhibiting increased serum baff levels tended to have more severe skin fibrosis as assessed by the mrss tool ( p < 0.01 ) , worse fvc values ( p < 0.05 ) and higher esr ( p < 0.05 ) . moreover , upregulation of the baff receptor was reported on b - cells from patients with ssc compared to healthy subjects . it is also worth mentioning that b - cells from patients with ssc stimulated with baff produced 38% more il-6 , a cytokine able to stimulate fibroblasts , compared to b - cell from healthy subjects . in a recent study it was found that peripheral blood mononuclear cells ( pbmcs ) from ssc patients produced significantly more april ( a proliferation inducing ligand ) , a b - cell survival factor , compared to pbmcs from healthy subjects ( p < 0.01 ) . these data indicate that upregulation of b - cell survival factors may contribute to b - cell hyperactivity and autoimmunity in ssc . baff and april emerge as two interesting therapeutic targets ; inhibition of these molecules may modulate b - cell function in ssc and potentially lead to clinical benefit . the authors found that genes characteristic of b - cells , fibroblasts and endothelial cells were differentially expressed in scleroderma compared to normal skin . interestingly , the same expression pattern was evident in patients with ssc in both clinically involved as well as uninvolved skin , underscoring the systemic nature of the disease . since endothelial cells and fibroblasts are considered key players in ssc , these data point towards a potentially active role of b - cells in skin fibrosis . the potential role of b - cells in ssc - associated ild has been inadequately investigated . it has been reported that b - cells are present in lung biopsies from patients with ssc - associated ild and that plasma cell infiltration of the alveolar walls is an early finding . the above studies have documented the presence of b - cells in both skin and lung of patients with ssc there are 4 small - scale , open - label clinical studies exploring the potential clinical efficacy of rtx in ssc , including one from our research group and a few case reports . in the first study by smith et al . , eight patients with early ( defined by disease duration of < 4 years from the first non - raynaud 's disease manifestation ) diffuse ssc received a single course of rtx ( consisting of 2 infusions , 1000 mg each , at day 1 and 15 ) . five patients had evidence of mild ild ; patients with severe ild were excluded from the study . half of the patients had evidence of b - cell infiltration in their skin ; treatment effectively depleted these cells . lung function tests , systolic pulmonary artery pressure , left ventricular ejection fraction , creatinine clearance , and haq score remained stable throughout the study . this is the first study that provides clinical as well as histological evidence that rtx treatment may favourably affect skin fibrosis in ssc . nevertheless , the improvement of skin fibrosis reported in this study was quite significant , which makes it rather unlikely to have occurred spontaneously , especially within the limited time frame of the study . another study reported the effects of a single course of rtx treatment in 15 patients with early ( as defined by disease duration of < 18 months from the first non - raynaud 's disease manifestation ) diffuse ssc . skin infiltrating b - cells were significantly increased in patients with ssc compared to healthy controls who had no b - cells and were eliminated posttreatment . pulmonary function tests remained stable at 24 wks compared to baseline ; it is noteworthy though that fvc values increased by an average of 3.5% at 24 wks compared to pretreatment values , but the 95% ci was wide and thus results were not statistically significant . our research group has performed an open label , randomized controlled , 1-year pilot study , assessing the effect of rtx in ssc . we recruited 14 patients with ssc randomized as follows : 8 patients in the treatment arm and 6 patients in the control arm of the study . patients in the treatment arm received 2 cycles of rtx at baseline and 24 weeks ( each cycle consisting of 4 weekly rtx infusions ( 375 mg / m ) ) . , p = 0.017 ) in the treatment group whereas no change was noticed in the control group . skin thickening , assessed with the mrss , was similar in the two treatment groups at baseline ( p = 0.50 ) . however at the 1 year evaluation , there was a significant decrease of mrss in the rtx group compared to the baseline score ( mean sd , 13.5 6.84 versus 8.37 6.45 at baseline versus 1 year , resp . no significant change in skin scores was noticed in the control group at 1 year when compared to the baseline mrss ( mean sd , 11.50 2.16 versus 9.66 3.38 at baseline versus 1-year , resp . , the median ( upper and lower quartile values ) percentage of improvement in the rtx - treated group was 39.25% ( 27.3364.95 ) compared to 20.80% ( 10.7839.28 ) in the control group . we found a significant reduction of collagen deposition in the papillary dermis at 24 wk compared to baseline in patients treated with rtx ; histologic improvement correlated with skin b - cell depletion . histological data matched the clinical data , since all patients showing histologic improvement also improved clinically . even though this is the first and only so far , randomized , controlled study assessing the efficacy of rtx in ssc , several limitations exist . additionally , most patients had long - standing disease since no disease duration restriction was applied and was heterogeneous in terms of disease duration , severity , and previous treatments . recently , one more study assessing the clinical efficacy of rtx in ssc was published . nine patients with ssc were recruited and received one course of rtx ( consisting of 2 infusions , 1 gr each ) . all patients had severe cutaneous involvement and had experienced worsening of skin score despite treatment with cyc . pfts remained stable throughout the study . three case reports have also appeared in the literature regarding the use of rtx in ssc . the first case describing the beneficial effect of rtx on ssc - associated ild was reported by mcgonagle et al . however treatment effect waned over time and a second course of rtx was administered . these data are in agreement with ours , showing that consecutive treatment courses may be needed to augment and sustain the effect of rtx on pulmonary function . the beneficial effect of rtx on ssc - associated ild has also been documented in a case report by our group . clinical improvement coincided with histologic improvement with reduction of collagen accumulation and myofibroblast score ; skin infiltrating b - cells were eliminated post treatment . recently , another case of successful treatment of cyc - resistant ssc - associated ild was reported . finally , rtx seems to favourably affect ild in the context of other systemic rheumatic diseases such as the antisynthetase syndrome as indicated by several reports [ 32 , 33 ] . all the available published data regarding the clinical efficacy of rtx in ssc are summarized in table 1 . rtx has been used with varying degrees of success in most systemic autoimmune diseases and this is why , one may argue that its effect on a severe , incurable systemic autoimmune disease such as ssc , is worthwhile exploring . furthermore , there is a strong rationale for the use of rtx in ssc ; an expanding body of evidence from basic research points to the direction that b - cells may be active players in the fibrotic process . on clinical grounds , until now , 43 patients with diffuse ssc have been treated with rtx ( available published data ) . but is rtx clinically effective in ssc ? based on the available clinical evidence , definite conclusions can not be drawn ; however results are encouraging . the clinical evidence on the efficacy of rtx in ssc - associated ild is limited , since most patients recruited either did not have ild or had only very mild ild . with the exception of our study , the other 3 studies were not designed to test the potential clinical efficacy of rtx in ssc - associated ild . nevertheless , in these three studies pfts remained stable , even though most patients had early disease and therefore were most likely to exhibit declining pfts during their followup . in our study , a significant improvement of lung function was reported in contrast to other studies where pfts remained stable at 6 months compared to baseline . in the other studies most patients did not have ild in contrast to our study where the presence of ild was an inclusion criterion . the significant improvement in lung function tests observed in our study indicates that rtx may favourably affect ssc - associated ild . long - term treatment with rtx may either improve or stabilize lung function over time in patients with ssc . first , b - cells appear to be actively involved in the fibrotic process , as indicated by data derived from both animal models and humans . elimination of skin infiltrating b - cells by rtx has been documented , albeit not consistently . taking into account that b - cell infiltration is a prominent finding in lung biopsies from patients with ssc - associated ild , depletion of this population may be a potential explanation for the clinical improvement . it would therefore be of great interest to study the effect of rtx on lung disease at a histological level , but this may be inherently difficult and challenging . we have also recently shown that rtx - induced improvement of skin fibrosis associates with a decrease in pdgfr phosphorylation ( which corresponds to activation ) in scleroderma skin . both experimental and clinical evidence regarding b - cell depletion in ssc is encouraging and certainly points to the direction that further exploration of its clinical efficacy is warranted . the best way forward would be a multicenter , randomized , double blind , placebo - controlled study . if such a study is performed we propose that it should focus on ild rather than skin disease and that it should be designed in such a way , that patients are treated for at least one year and evaluated thereafter ; in this way , a treatment effect on ild ( if any ) would be more easily depicted . interestingly , however , a phase ii study assessing the clinical efficacy of rtx in ssc - associated pulmonary arterial hypertension has already been launched ( http://clinicaltrials.gov/show/nct01086540 ) . we believe that a large - scale multicenter , randomized study assessing the potential clinical efficacy of rtx in ssc is highly needed .

natural product ( np ) research is a demanding science , requiring an in - depth knowledge of many aspects of organic , analytical , and biological chemistry , including separation science , spectroscopy , biosynthesis , and pharmacology , as well as the biology and taxonomy of the investigated phyla . nonetheless , most contemporary practitioners would agree that the role of this discipline within biomedical science has declined , as evidenced by its present abandonment by most large pharmaceutical companies in their search for new chemical entities to provide new drug discovery leads . this review takes a comprehensive look at the current practice of np research , with the aim of pinpointing potential areas where the practitioners might improve the overall efficiency of this type of work . by focusing on np chemistry as one fundamental aspect of np research , it might be possible to recognize patterns that impact the bigger picture and identify opportunities that otherwise would go unnoticed . this review intends to stimulate discussion and inspire the development of new approaches to yield more rapid results and a greater number of new chemical entities discovered , and thereby promote the future role of np research in interdisciplinary programs . a series of excellent articles , coauthored by g. m. cragg , d. j. newman , and colleagues , has documented the invaluable role of nps in drug discovery . underlying evidence came from an extensive meta - analysis of the primary literature of all drugs , in or completing fda - approved studies within a set time frame and classifying them according to their origin as nps , inspired by nps , analogues of these two classes , or from non - np sources . these analyses have indicated that a high proportion of new drugs approved in western countries in recent decades are , in some manner , connected to nps . as primordial biosynthetic pathways endow nature s library of chemicals with an evolutionary advantage over man - made chemicals , np libraries are keyed to nature s biochemistry and diversity and , thus , continue to be an attractive source for new bioactive agents , for both therapeutic and diagnostic uses . moreover , the chemical diversity in nps is tied intrinsically to the complexity of the metabolome contained in the source material . ultimately , both the discovery and the resupply of bioactive nps depend on the availability of preparative - scale analytical methods having the capability of resolving the complex primary and secondary metabolomic mixtures that are typically isolated from the source organism , yielding a purified np ( np in figure 1 ) , and eventually providing a well - characterized np as a single chemical entity ( sce ; figure 1 ) . it should be noted that , in the practice of np chemistry research , a purified np does not necessarily represent an sce , but may only have been purified to the degree necessary , e.g. , for structure elucidation or identification . a sce may be defined as a substance for which all chemical , physical , and biological characteristics can be attributed to a single molecular structure . accordingly , a np becomes a sce only after its singleton character has been demonstrated ( high - purity np ) . this is in line with practice for sces that are used and regulated as drugs : their purity plays a pivotal role in all pharmacopoeias worldwide . this topic has recently received global public attention when an isosorbide-5-mononitrate preparation containing pyrimethamine as an impurity caused the death of more than 100 patients in pakistan . this tragedy demonstrates the importance of purity as a parameter for the safety of medicines , but also exemplifies that purity should never be ignored and always be part of quality control of drugs and nps . the process involves repeated ( n - times ) preparative- and analytical - scale separation and , depending on the methods and n , results in a np that is linked to varying residual complexity ( rc ) , reflecting both its metabolomic heritage and the purification protocol . subsequent analytical characterization including purity assessment is required to generate a fully quality controlled np ( cnp ) or single chemical entity ( sce , figure 2 ) . the nearly 2000 publications evaluated employ bioassays to address screening of crude nps , bioassay - guided fractionation , biological assessment of purified nps , and detailed pharmacological investigation of , for example , structure activity and structure purity relationships ( sars and pars , respectively ; see text and figure 2 ) . the majority of pure nps represent rare chemicals of extremely limited supply . frequently , particularly in the case of newly reported structures , such compounds are also unique commodities and are only immediately available from a single source , namely , the original investigators , or by re - isolation . practitioners of np chemistry can generally observe additional factors that contribute to the exclusivity of np samples : ( i ) their consumption in the bioassay systems of contemporary np research programs ; ( ii ) a general trend to smaller sample sizes , leading to smaller yields ; ( iii ) the frequently unfavorable consistency of small - scale isolation products , and ( iv ) the practical challenges of handling small samples for distribution , such as precise weighing in the submilligram range . considering both commercial and noncommercial / academic sources and supply chains , most pure np compounds can be traced back ultimately to crude natural materials ( extracts ) that require various purification steps before being considered pure . consequently , pure nps carry a natural signature in the form of a characteristic impurity profile called residual complexity ( rc ) , which originates ultimately from the biosynthetic cocktail(s ) of the producing organism(s ) . as a result of the authors own experience components to the purified np , such as sorbents , laboratory pollutants , residual solvents , or other chemicals , which can evade detection by the analytical methods used . these considerations also affect studies with a biological or pharmacological focus that utilize as tools nps , which might be acquired from outside sources . most such studies generally consider nps as fine chemicals rather than a material derived from nature . exceptions may be compounds obtained by ( semi)synthesis , a process typically only accomplished at an advanced discovery stage and for select nps . even in these instances , as minor congeners potentially can undergo the same reaction , carryover of minor components ( commonly analogues ) through semisynthetic schemes has to be considered . all of these considerations reveal nps as being both highly sought after and hard to obtain entities . they also explain why the np drug discovery process and the biological assessment of nps to date are almost inevitably tied to preparative - scale analytical methods used for np purification . the ability to purify a few milligrams of a rare np from kilograms of a crude extract has been one of the significant skills of scientists trained in np chemistry , pharmacognosy , and analogous disciplines and represents one of the keys to np research . in clinical research , numeric meta - analysis of literature is a well - established tool , allowing recognition of more general trends , and is used frequently to improve clinical practice . while such meta - analyses are rarely done in np research , they can be very helpful tools for gaining new and more generalized insights . one example of such a report is the study by g. a. cordell et al . , revealing that only about 3% of some 20 000 known alkaloids have been evaluated biologically in more than five test systems , whereas 36% of alkaloids that were evaluated in 20 or more bioassays are pharmaceutically relevant . the present contribution is based on the meta - analysis of the recent literature with a focus on parameters that reflect the analysis and purification of bioactive nps ( anapurna ) . the production of pure nps of controlled quality ( cnps ) involves two main aspects : ( a ) the actual purification process used for np isolation , i.e. , the ( semi)preparative - scale analytical method employed ; ( b ) the assessment of the purity [ or residual complexity ( rc ) ] of the isolated np , including the analytical method used for purity assessment . the aim of this review is to describe the status quo regarding both aspects , through a comprehensive assessment of the contemporary literature on bioactive nps . the present report summarizes over a decade of data - mining activity by the authors , which involved manual screening of > 80 000 pages of scientific literature during the periods 19981999 , 20042005 , and 20092010 . to date , data have been extracted from nearly 2000 peer - reviewed articles , forming the foundation of this survey of analysis and purification of bioactive natural products ( anapurna ) . the framework of this study was designed at the survey onset and in a prospective fashion . throughout the study , the literature was examined for a set of predefined parameters , which were recorded using predefined scoring and key systems . furthermore , a set of 15 questions to be answered was developed at the beginning of the study . this review is organized as follows : the methodology section describes the data - mining methodology employed as well as the journal and time coverage of the survey . subsequent sections present the survey results as well as the numerical and statistical measures developed from these data . the next section concentrates on the following aspects : sources of purified nps ; chromatographic methodology used for np isolation ; spectroscopic methods used for np characterization ; and the role of purity and the methods used for the purity analysis of nps . the final section of the review summarizes the findings from the perspective of potential new approaches to the analysis of np complexity and the achievement of novelty . final discussions are devoted to the role of np integrity including purity and linkages between chemical and biological properties of bioactive nps . the integration of these aspects potentially could help in advancing the future role of nps as a viable source of new biologically active agents . the source journals ( n = 13 ) , intervals monitored ( 1998/1999 [ period i ] , 2004/2005 [ period ii ] , 2009/2010 [ period iii ] ) , and coverage of evaluated articles ( ntot = 1823 ) are summarized in table 1 . all journals screened are well - established and peer - reviewed and dedicated to or frequently publish studies on bioactive nps . they are focused on drug discovery and/or pharmacology involving nps and exhibit a wide range of isi impact factors ( ca . journals were assigned to two groups , a and b , according to the depth of reported spectroscopic information ( see methods section and main discussion for details ) . these two columns give the journal volume number and the number of articles that fulfilled the inclusion criteria and were evaluated , respectively . only 21 articles were assessed , although about 80 articles would have fulfilled the inclusion criteria . only this journal was assessed for the period 20002003 ( n = 77 ; not included in ntot ) . in the initial stage , the survey consisted of a large - volume screening of reports from both years of period i , which involved manual screening of ca . 55 000 journal pages from 12 journals . upon compilation and preliminary data evaluation , this led to the selection of six priority journals and the addition of one journal ( journal of asian natural products research ) for continuation of the survey in the subsequent periods ii and iii , which mostly focused on one year of the two - year periods ( see table 1 for details ) . the seven priority journals were selected due to their much higher information density , i.e. , the number of qualifying articles . accordingly , the number of published pages to be screened was reduced to ca . 15 000 and about 12 000 pages in the periods ii and iii . of the journals with a lower prevalence of qualifying reports , one ( journal of pharmacology and experimental therapeutics ) was included with the seven priority journals and assessed for the entire period 20002003 ( n = 77 ; not included in ntot ) plus one year of period ii , in order to provide an example of extended coverage for these journals . for any given journal volume included in the study , all articles were prescreened manually for the following inclusion criteria : they had to report on both bioactivity and chemistry of nps and provide a substantial experimental description , regardless of how well the np - related portion was developed . exclusion criteria were as follows : reports in which bioactivity was clearly a minor aspect of the work ; reports in which np and/or synthetic chemistry was so dominant that the bioactivity portion was insignificant ; and reports with ambiguous experimental descriptions of the analytical parameters . by default however , depending on the journal and its editorial framework , in some instances such as limited coverage of a given volume or year , publications in content - limiting formats ( e.g. , notes ) that fulfilled the other inclusion criteria and had a sufficient level of detail to address the key study parameters were included in the survey . by ensuring that formatting restrictions did not impact the scores , these added publications contributed to the statistical significance of the survey by increasing the total number of articles evaluated . the raw data were collected into tabbed spreadsheets ( microsoft excel 2010 ) and analyzed using mathematical , sorting , and boolean and other logics as well as conditional formatting functions of the software . the parameters extracted from the primary literature , as well as the scoring and key system used to record the information in a standardized spreadsheet format , resulted from a preliminary , randomized screening of ca . 100 articles and were defined before starting the main survey . as a means of prospective guidance for the data - mining process , all of the questions addressed under the observations made section of this review were also formulated at the onset of the study . while the insight gained during the study led to additional hypotheses that were eventually tested with the complete survey data , the scores initially implemented and keys as well as the basic set of questions were maintained constant during the entire study . in addition to the basic header information about each article ( author name , journal volume , page ) , the primary biological activity or target of each report was recorded . furthermore , for each report , the total number of nps and the predominant class of compounds were recorded , and the following data were determined : total number of compounds ; number of compounds that were isolated by purification from natural material ; number of compounds that were synthesized ( full or partial ) ; number of compounds that were gifts from colleagues ; and the number of new structures . the experimental section of each report was evaluated for parameters that reflect the methods used for the purification of the nps and their spectroscopic characterization . as many reported isolation procedures are rather convoluted , the assessment included determination of the longest purification pathway and the highest degree of diversification of the methods employed . the maximum number of isolation steps was counted , excluding extraction and solvent partition procedures . the use of normal - phase silica gel as a primary or secondary purification step , after any partitioning or precipitation steps , was recorded as a binary number . in addition , the diversity of the purification methods was assessed and encoded into binary format as a byte integer , consisting of the following five bits : 0 = undefined or literature reference only ; 2 = precipitation or crystallization ; 2 = paper or thin - layer chromatography ( tlc ) , including centrifugal tlc ; 2 = column liquid chromatography ( lc ) , vacuum lc , and low - pressure lc , and the value 2 was added to encode repetition in the entire scheme ; 2 = medium - pressure and high - pressure lc ( mlpc , hplc ) , and also the value 2 was added to encode repetition in the entire scheme ; 2 = countercurrent chromatography . the reported lc techniques applied numerous different solid - phase packings , which were not individually differentiated in the survey and included primarily the following : normal- and reversed - phase ( rp-8/18 ; cyano ) silica gel ; sephadex lh-20 ( see ref ( 10 ) for a comprehensive review ) ; styrene resins ( see ref ( 11 ) for theory and applications ) . data collected on the spectroscopic characterization of the nps , for which bioactivities were reported , included the number of compounds for which spectroscopic data were reported , the comprehensive nature of the general physical / spectroscopic data in general , and , separately , the nature of the nmr data utilized in particular . for this study , depth is defined by the completeness , detail of interpretation , and comprehensive nature of data . this was assessed and scored as follows : for the general spectroscopic and other analytical data , 1 = highly comprehensive ( x - ray and/or very comprehensive 1d and 2d nmr , ms , physical data ) ; 2 = comprehensive ( 1d and some 2d nmr , ms , physical data ) ; 3 = as for 2 but with apparent gaps ; 4 = mainly or fully lacking ; 5 = literature reference only , or in cases where no spectroscopic data were reported , or referred to as previously described , with reference to other literature . similarly , the depth of nmr data were scored : 1 = highly comprehensive ( 1d and 2d nmr and/or special experiments such as selective pulse experiments , spectral simulation , connection with molecular modeling studies ) ; 2 = comprehensive ; 3 = as for 2 but with gaps ; 4 = mainly or fully lacking ; 5 = literature reference only or in cases where no spectroscopic data were reported . in judging the completeness of physical data , the reports were considered adequate despite not providing uv data if the compounds had no chromophore , and similarly optical rotation was not expected if the molecules were achiral . for the assessment of reports from the most recent time period , iii , the provision of spectroscopic data as supporting information ( si ) was considered as added comprehensiveness that was linked via cross - references in the main text . due to workload and practical considerations , however , the si materials were not screened . finally , each article in its entirety was mined for information about purity assessment of the bioactive nps . the information was encoded into binary format as a byte with five bits , as follows : 0 = undefined or obscure method ; 2 = taken from vendor label ; 2 = single spot on tlc ; 2 = determined by hplc ; 2 = determined by quantitative h nmr ( qhnmr ) through basic integration using the 100% method ; and 2 = determined by qhnmr with calibration or by titration , data given for each compound . all authors were involved in the data - mining process , which involved manual page turning of journal hard copies , judgment of inclusion criteria of each article in the screened volumes , and mining of the aforementioned data from each of the 1823 articles . it is realized that the scoring systems involve an element of subjectivity that may lead to deviations in scores assigned by individual assessors . another potential source of variation was the screening of experimental sections for data about isolation methods , in particular in reports where the purification procedures were lengthy and/or convoluted . while the information was mined with particular care and attention to detail , the extracted data might have deviated slightly in a few instances ; for example , the number of isolation steps might be off by one step from the actual experiments . given the workload of manually screening 80 000 pages of literature information , it was not feasible to perform the entire survey in triplicate and/or by multiple individuals . as no averaging was performed , the data represent the outcome of single assessments . the authors distributed their efforts as assessors across the journals , as this helped by averaging the influence of interindividual subjectivity . moreover , a limited amount of cross - checking between the authors was also undertaken . data from this anapurna study are presented in the following paragraphs and are discussed with respect to the questions ( q ; numbered ) that were formulated initially in the prospective study . as the study evolved over the last 10 + years , additional aspects for which the survey data could provide insight were added and are included . np isolation accompanied by bioactivity measurement is considered to be the specialization of a larger discipline . while many journals occasionally publish articles on the isolation of nps and their bioactivity , only a few journals regularly publish such reports . this study is an in - depth investigation of a handful of journals ( limited for practical reasons ) that publish articles routinely on the bioactivity of nps , rather than a broad study of the general literature . this systematic literature review focuses on a selection of journals that contribute heavily to the specialization of np research , with a particular focus on pharmacognosy and natural products chemistry . independent of the choice of journals , it is likely that the editorial policies of the selected journals influence the data . this reflects the natural flow of disciplines in science , where areas of specialization form their own communities and at the same time contribute to the greater scientific endeavor in a variety of ways . for example , purified nps may be incorporated into human clinical trials , which will be published in a medical journal rather than a natural products journal . seven of the 13 journals ( table 1 ) represent 79% coverage of qualifying reports . this number increases to 88% when including the proportional numbers of qualifying articles in planta medica from periods ii and iii . the articles assessed were almost evenly distributed over the time periods i , ii , and iii , providing 716 , 501 , and 597 surveyed reports , respectively . when ranking the journals by number of qualifying articles , about one - third of them were published in the journal of natural products . following in the ranks are biological / chemical and pharmaceutical bulletin ( combined ) , planta medica , phytochemistry , fitoterapia , and the journal of asian natural products research . the latter was included in the survey for period iii , in order to get a perspective on a publication that reflects the outlet of the very productive nps research community in asia . a graphical overview of the journals by contributed survey articles is provided in figure s1 , supporting information . there were four main sources of the nps : ( i ) isolation and purification of the np by the authors as reported in a scientific publication ; ( ii ) purchase of nps from commercial sources ; ( iii ) receipt of nps from colleagues who have performed the isolation and purification themselves ; and ( iv ) ( semi)synthesis . the proportion of bioactive nps that were described as gifts from colleagues has dropped over the survey time period from 1.6% in period i to 0.7% in period ii and 0.6% in period iii . looking only at the group a journals , gifts were reported for around 12% of all investigated nps ( 0.6% in period i , 2.5% in period ii , 1.1% in period iii ) and , thus , contribute to only a very small proportion of the studies . the overall reduction in shared compounds might be a result of the trend toward smaller isolation yields and their consumption in the bioassays , together reducing the availability of the compounds . these observations are also in line with an observed trend toward collaborative research , which indicates that teams involving nps researchers produce compounds dedicated to biological evaluation . this again may result in the unavailability of the compounds for subsequent studies . recently , some journals have implemented requirements for the inclusion of copies of original spectra as supporting information , which facilitates structural dereplication by other researchers . at the same time , this new mechanism may contribute to the observed reduction of sharing of the actual compounds among researchers . the involvement of synthetic nps has seen a significant decline , by 75% , over the study period : while they contributed a similar proportion of study compounds in period i in all journals ( 11.9% in journal group a , 7.9% in journal group b ) , their overall contribution to all study compounds decreased from a relatively high 9.5% in period i to 6.3% in period ii and 2.8% in period iii in the group a journals and from 11.5% in period i to 6.5% in period ii and 3.1% in period iii when adding both groups a and b together . this shows that the role of ( semi)synthetic chemistry in the surveyed journals has diminished over the observation period . addressing this question eventually required a more elaborate analysis of the data , including differentiation by looking at individual journals and the groups of journals . in this study , it was determined that the vast majority of nps used in bioactivity studies were isolated and purified by the authors from their natural sources by the protocols described in the experimental section included in the publication . the average proportions of purified nps across all journals rose from 78% in period i to 93% in period ii and 95% in period iii . the main reason for both the high proportion and the rise may be that the probability of a major or even breakthrough discovery is lower with a compound that has been extensively investigated due to its unrestricted ( commercial ) availability . however , dividing all journals into two groups , a and b , according to the overall depth of spectroscopic data ( see methodology section , table 1 , and details below ) reveals a different trend : while in group a , ca . 85% of compounds reported in each journal were isolated and characterized , their proportion in group b is only about 55% . considering that gifts and synthetic substances are generally minor sources of nps , this implies that the amount of purchased nps has increased in the group b journal reports . these interpretations are supported by the analysis of all 12 journals from period i : only 3.5% of nps ( 107 ) reported in the group a journals were from commercial sources , but their proportion in the group b journals was 6 times higher , at 22% ( 305 ) . for one group b journal ( journal of pharmacology and experimental therapeutics ) analyzed in period ii conversely , in group b the proportion of isolated / characterized compounds was as low as 36% in reports within an individual journal . these observations regarding the sourcing of the investigated nps are independent of differences in scope and policy of the journals in groups a and b and also of the diverse foci ( e.g. , chemistry or biology orientation ) of individual reports . the high percentage of bioactive nps that are isolated and characterized ( currently about 95% ) also indicates the rarity of purified nps in that most researchers tend to produce these compounds by themselves rather than obtaining them commercially . this observation is important , because one consequence of this practice is that the authors themselves are responsible for establishing not only the identity of the np but its purity as well . in cases where nps are obtained from commercial sources , the isolation process may well be proprietary ; however , the nps will also carry a specification sheet , certificate of analysis , and/or certificate of origin that includes a purity statement conforming to the standards of the manufacturer . the percentage of reports on new chemical entities has been remarkably stable over time . in the group a journals , an average of 30.1% of reported nps were new chemical entities ( 30.2% in period i , 26.5% in period ii , 31.0% in period iii ) , which represents a 4-fold higher incidence than in group b. since the beginning of the survey , the reports in one journal ( fitoterapia ) included in the seven priority journals have shown a significant increase in new nps and today closely match the average of the group a journals ( 28.3% in period iii ) . considering this further , in most cases the principal division of labor in the surveyed reports is the sharing of responsibilities between the np chemist performing the isolation and the biologist completing the bioassay work . this means that at a certain point the isolated np(s ) are handed off from a np laboratory to a biology laboratory . in this case , unless activity - guided chromatographic fractionation is conducted , it seems that the investigations will almost always be chemistry driven , so that the chemist will select the most interesting and accessible nps to isolate . typically , the bioactivity of the crude extract was reported along with the bioactivities of the final isolation product(s ) , while the potency of fractions throughout the separation scheme was reported much less frequently . it is important to monitor the activity of nps ( extracts , fractions , purified compounds ) through at least three purification steps in order to establish the correlation between chemical purity and biological activity . activity relationships ( pars ) are quantitative correlations between chemical ( purity ) and biological ( potency ) parameters , which indicate whether or not the observed biological activity can be attributed to the main component , assigned as active principle . as such considering the role of purity in the literature , as observed in this study , this type of information might currently be under - utilized . notably , pars can also be established at the level of purified compounds ( nps and cnps ; figure 1 ) , e.g. , by comparing the potencies of the same np purified from different sources and/or by different purification protocols . another measure of the importance of the biological component may be how many nps that produce promising hits in the biological assay are investigated further for their biological activity . this interface between np chemistry and biology is crucial to the ongoing success of this specialization , which seeks to harmonize these two aspects of scientific research . today , numerous chromatographic procedures with widely differing characteristics ( selectivity , mechanism , resolution , loading capacity , scale - up behavior ) are available to the np researcher . the chromatographic information extracted from the surveyed literature provides insights into the ongoing use of this diverse toolbox . the binary encoding and scoring of characteristics of the purification methods used in surveying all reports is described in the methodology section and rests on a thorough case - by - case analysis of the experimental section of each report . considering the correlation between the metabolomic complexity of crude extracts and the residual complexity of purified nps ( figure 1 ) , the codes and scores were designed to provide metrics to answer questions about the depth and diversity of isolation procedures as they are used in laboratory practice . while the number of nps per individual report varies considerably , the average number of nps per report has increased slightly over the survey time period as follows : 6.6 7.3 ( sd ) in period i , 6.1 5.8 in period ii , and 7.8 7.7 in period iii . an upward trend is also noticed for the proportion of new nps , which has increased by more than half from 26% in period i to 37% in period ii and 41% in period iii . it is noteworthy that this observed trend applies primarily to journals in which reports include a comprehensive coverage of the spectroscopic data ( group a journals and fitoterapia ; see table 1 and discussion below ) . one significant outcome of this literature analysis is the revelation that the average number of steps taken to isolate and purify a natural product is less than three ( n = 1823 ) . in addition , this number has not changed significantly in the time period covered by the study . in period i , an average of 2.0 isolation steps ( sd 1.7 ) was used to yield a pure np . this increased to 2.4 in period ii ( sd 1.5 ) and 2.7 in period iii ( sd 1.6 ) . taking into account that 22.9% of reports did not employ any isolation steps ( assigned value of 0 ) , the other studies employed an average of three isolation steps . the data fit a gaussian normal distribution reasonably well ( figure s2 , supporting information ) , with a tail toward higher numbers representing the very few studies ( n = 39 , 0.6% ) that employed six to 10 isolation steps . one conclusion from this data is that compounds that can be isolated in three steps or less are the predominantly isolated np . on the other hand , this observation also indicates that compounds present in very small amounts and/or similar to more abundant congeners are currently rarely pursued , likely because they are more arduous to isolate . an interesting example of this is that ginkgolides a , b , c , and j have been reisolated from ginkgo biloba l. ( ginkgoaceae ) hundreds of times , while ginkgolides l and m are described in only one publication . recently , the two new ginkgolide congeners p and q have been isolated in less than 30 mg quantities from 8 kg of g. biloba leaves . this question includes two aspects : the number of steps is addressed here , and the chromatographic methodology in the following section . about half of all reports ( 48.8% ) either did not perform an isolation or employed only one or two steps to produce the bioactive np . isolation efforts included a maximum of three steps in about three - quarters of all reports ( 76.3% ) . publications that described at least four or five isolation steps contributed to 23.7% or 7.2% of studies , respectively , and , in turn , can be considered in - depth isolation studies . there was no clear trend of their prevalence over the survey periods i / ii / iii , with 21.8/18.1/30.8% and 6.8/5.4/9.0% of 4- and 5-step studies , respectively . the effort required to achieve single chemical entity parameters for an isolated np depends on many factors , including ( i ) the concentration of the np in the crude material ( the higher , the easier the purification ) ; ( ii ) the physicochemical characteristic of the compound , in particular solubility ( precipitation ) and tendency to form crystals ( a historically important property of nps ) ; ( iii ) the match between the selectivity characteristics of the chosen purification methods and the np ( some methods appear to work better than others for certain compound classes or types of source materials ; different standard protocols for marine vs microbial vs plant nps ) ; and ( iv ) the nature of the matrix components in the crude np , which may cause difficulties in the purification process ( e.g. , polyphenols or chlorophyll in plants , high - polarity overlap with primary metabolites and other polar substances in the case of marine nps ) . accordingly , a one - step isolation procedure might be sufficient to purify a np that is present at relatively high concentration , i.e. , in the 0.2% range ( relative to dry weight of the biomass ) and above . during this survey , numerous examples of such rapid access to a purified np were noted in the literature . they involve typically solvent partitioning and just one step of normal - phase silica gel column chromatography , sometimes followed by precipitation or crystallization . examples well - known to the authors are vitexin from vitex agnus - castus ( 0.10.2% content ) and xanthorrhizol from curcuma xanthorrhiza ( > 0.2% content ) . as the information about purity in the literature has generally been very scarce ( see below ) , there is very little basis for judgment of the properties of these kinds of materials and its impact on the biological activity . given the long history of nps research , it appears to be likely that more elaborate isolation schemes could produce new insights and novel structural and biological information , in particular when performing research on nps that have previously been ( extensively ) studied . on the basis of the entire data set ( n = 1823 ) , about two - thirds of all studies utilize normal - phase silica gel for the isolation of nps . the proportion of these studies has increased over the observation period from 57% ( i ) to 63% ( ii ) and recently 71% ( iii ) . interestingly , studies that use normal - phase silica gel report isolation of crystalline compounds 25 times more often than studies that do not use this sorbent . comparing studies that use normal - phase silica gel with those that do not , the ratio of the average number of crystalline compounds per study was 2.0 in period i , 2.6 in period ii , and 4.9 in period iii . in the same time interval , the proportion of crystalline isolates has declined from 10.1% to 7.3% and recently 4.9% , respectively . overall , this may attest to the ability of normal - phase silica gel to concentrate and/or remove unwanted constituents and offer one reason for its steady popularity . its widely known disadvantages such as irreversible absorption or degradation of desirable constituents are less frequently conveyed for bonded silica gel derivatives . assessment of the actual impact of these unpredictable properties of silica gel - based stationary lc phases on the outcome of the purification protocol requires dedicated studies . one such example has been reported by pinel et al . , who directly compared normal - phase silica gel and liquid only based lc ( countercurrent separation ) for the purification of xanthanolides from zanthium macrocarpum ( asteraceae ) . 13-fold reduced yield of one particular xanthanolide , xanthatin , when using the solid - phase method . this almost selective removal of a compound from a crude np might inspire future developments and/or validation of silica gel - based purification methods . with regard to the generation of crystalline nps , it is noteworthy that their proportion has dropped from 10.1% to recently 4.9% . this may reflect the trend to smaller starting amounts of biomass and isolation yields based on the capability of modern spectroscopy to obtain structural information from smaller and/or less pure samples . these observations are in line with a conclusion recently made by meyer and imming , underscoring the value of practical skills in compound crystallization for contemporary research programs that involve purification of nps and other drug leads . considering the extremely wide use of normal - phase silica gel , it is not surprising that one- to two - third of studies ( 63.7/35.5/32.5% in the periods i / ii / iii , respectively ) used gravity - driven column chromatography exclusively . while this proportion is declining , the data show that a large proportion of isolation procedures are uniform rather than diverse . likely the most prevalent isolation methodology consists of normal - phase silica gel , ( repeated ) gravity - driven column chromatography , and hplc . this combination was found to also increase in popularity and has most recently been employed by almost one - half of all studies ( 27.4/36.1/45.6% in the periods i / ii / iii , respectively ) . these observations may reflect preferences for fast approaches such as automated flash chromatography and preparative hplc and/or may also be a sign of the increased availability of such equipment . although not specifically tracked and encoded in this survey , a general observation is the very frequent use of c18 reversed - phase silica gel and sephadex lh-20 as stationary phases for lc purification of nps . both materials are significantly more costly than normal - phase silica gel , which might explain their relatively lesser use , but they have the advantage of being reusable . reversed - phase silica gel appears to be the second most widely used stationary phase and like normal - phase silica gel is widely employed in ( semi)automated lc applications such as hplc , mplc , and vacuum and flash lc ( including high - throughput settings ) . in the present meta - analysis , np purification schemes have two primary dimensions : the number of purification steps and the chromatographic methodology used in each step . while together they describe the overall depth of the purification process , a chemically diverse metabolome likely requires a chromatographically diverse purification scheme for the efficient mining of nps . the binary scores given in this study for the diversity of the purification methods ( see methodology ) allowed us to study the relationships between the number of steps and the chromatographic methodology ( see scatter plot , figure s3 , supporting information ) . a general observation from the distribution of the purification diversity scores is that an increase in the number of purification steps does not necessarily indicate an increase in chromatographic diversity . the data exhibit the presence of general trends , as follows : two - step procedures mostly consisted of two lc steps ( often repeated ) or a combination of one lc and one hplc step . three- and four - step procedures frequently applied repeated lc and one level of hplc , although a relatively large number of these purification schemes apply gravity- , vacuum- , or low - pressure - driven lc methods only . emerging from the authors research and interest in countercurrent separation ( cs ; syn . ccc ; see ref ( 18 ) for a review ) , this survey also explored how widespread the use of this methodology is . in all studies and over the entire survey period , countercurrent methods such as hsccc , cpc , and dccc are used only sporadically ( average 0.9% ) . in fact , despite recent developments of countercurrent technology , its proportional use in studies on bioactive nps has actually decreased over the project period , from 1.7% in period i to 0.3% in period iii . even when looking only at in - depth isolation studies ( see above ) , the proportion of countercurrent chromatography use fell from 4.5% in period i to 0.5% most recently . however , the number of reports that employ countercurrent techniques and fractionate nps in - depth , by using at least three ( 58/67/50% in periods i / ii / iii , average 58.3% ) or four isolation steps ( 83/67/50% in periods this implies that countercurrent methodology is applied primarily in more complex isolation schemes rather than as an alternative to other techniques . these observations reflect the need for specialized countercurrent chromatography equipment , which might not be widely available even to well - equipped laboratories . another consideration is that , unlike many ( semi)automated solid - phase lc methods ( e.g. , preparative hplc ) , countercurrent separation techniques require some time to be optimized . while this may be perceived as being disadvantageous , significant progress has been made recently on key aspects such as solvent system selection , instrument design , and operation modes , and there is a wealth of recent reports on efficient np purification protocols that employ countercurrent techniques ( see ref ( 18 ) and references therein ) . following from the observations made in q7 , the uniformity of isolation approaches may also be due to the fact that this systematic literature survey looked at only a limited number of journals . for example , there are dozens of articles published every year featuring the isolation of nps with countercurrent separation with subsequent analysis of bioactivity . similar considerations apply for supercritical fluid separations . that having been said , the use of normal - phase silica gel as a chromatographic method of choice is much more entrenched than can be simply explained by the fact that some alternatives are considered to be specialized techniques . the reported use of normal - phase silica gel has actually increased during the time period of this literature survey . numerous preparative - scale analytical methodologies are used in minor compound purification in the laboratories of np researchers . owing to the complexity , newly developed techniques are often test driven in nps laboratories . examples are the development of countercurrent chromatography , as pioneered by y. ito and co - workers , and the advent of hplc in the 1970s . while a few techniques have established themselves as mainstream , it remains unclear as to what other techniques have to offer and what roles they can play in the future . once a np has been isolated from its metabolomic background ( figure 1 ) , characterization of its chemical structure ( verification , dereplication , or elucidation ) is the next step toward a quality - controlled material ( cnp , figure 1 ) for biological evaluation . the questions posed were as follows : in order to answer these questions , both the physical data in the experimental sections as well as the tables and descriptions in the main text of the articles were assessed , and the extracted information was coded as previously described under methodology . the criteria took into account the widespread availability of spectroscopic equipment ( nmr , ms , uv , ir , less so cd / ord ) . while the depth of spectroscopic analysis per se is scientifically independent , editorial policies and journal format constraints undeniably have an impact on the information finally reported and , very possibly , which experiments are performed . therefore , to apply equal measures in the entire survey , the same coding scheme was applied to all publications and across the survey time period . compounding the scores for the depth of spectroscopic data for all articles ( n = 1908 ) yielded a numerical average of 2.5 on the discrete scale from 1 to 5 ( lower number better thus , on average , the spectroscopic foundation of all reported bioactive nps ( ncpd = 12 570 ) was between comprehensive ( 2 ) and with gaps ( 3 ) . the distribution of the scores ( s4 , supporting information ) shows tailing toward higher scores , as a result of 21.9% of the reports lacking support by spectroscopic characteristics at all ( 13.3% ) or in the same publication ( 8.6% ) . in relation to the nps , spectroscopic data were provided for less than half of all compounds ( 5510 = 44% ) , with only minor differences over the 12-year survey period . when analyzing reports by journal source , the distribution and average depth of spectroscopic information in the 13 surveyed journals were heterogeneous . this is not an unexpected outcome for a number of possible reasons already discussed above . in fact , when evaluating the depth scores of both general spectroscopic and nmr spectroscopic data for the entire survey period , a clear gap was noted between average scores of 2.5 and 3.0 , as can be seen in the tables and graphs in s5 and s6 of the supporting information , respectively . this led to the classification of the journals into the groups a and b ( 2.5 [ five journals ] vs 3.0 [ eight journals ] , respectively ; see also table 1 ) . the five group a journals showed an average score of 2.1 and covered greater than four - fifths ( 10 660 = 84.7% ) of all studied bioactive nps . conversely , reports of less than one - fifth of the compounds ( 1827 = 14.5% ) were in the group b journals , which gave an average score of 3.8 . considering that about one - fifth of the reported nps lack support by spectroscopic characteristics ( see above ) , the distributions of the spectroscopic depth scores in the two journal groups are almost mirror images of each other ( s5 , supporting information ) . analogous observations were made for nmr spectroscopic data , which is usually essential for structure elucidation and compound identification . of the seven priority journals selected for long - term surveillance over the whole 19982010 period , five were group a journals . the total average depth score ( see methodology section ) for the nmr spectroscopic data ( 2.7 ) is almost identical to that of the general spectroscopy ( 2.5 ) . the two sets of spectroscopic depth measures also show parallel behavior over time and have experienced a steady improvement over the three survey periods : from 3.3 to 2.3 for the nmr and 3.1 to 2.0 for the general spectroscopic data . this can be seen clearly from the score distribution plots provided in s7 , supporting information . these results indicated that nmr spectroscopy in general and 2d - nmr , in particular , have become the mainstay of structure elucidation . the observation that in the most recent period , iii , 49% of all nps were reported with nmr spectroscopic information categorized as comprehensive and an additional 18% as highly comprehensive can be interpreted as a sign of strong nmr evidence for the structure of about two - thirds of all bioactive nps . these encouraging observations , however , do not necessarily indicate that dereplication of two - thirds of all nps is straightforward . while the scoring system was not designed to specifically address this question , it is the authors impression that unambiguous dereplication requires ( nmr ) spectroscopic data sets that scored typically as one in this survey . while additional studies will be necessary to draw conclusions about the level of detail that is needed and/or practical for structure dereplication and , thus , full reproducibility , the survey indicates that nmr spectroscopy has been playing an increasingly strong role in this regard . what has changed over time is that the nmr spectra are now included as supporting information in most journals , especially in the case of new compounds , due to space constraints . unfortunately , the depth of the nmr data can not be assessed in many cases simply because the spectra are not available . one way to assess depth of nmr data is level of detail , such as the completeness of the assignments , the coupling pathways , the coupling constants , and the multiplicity assignments . in addition , not only may structural data be lost but valuable information on the purity of an isolated np may be disregarded by consigning nmr data to a table or brief listing . another way to assess the depth of structural information is to consider the number and sophistication of the spectroscopic tests that are reported . for example , 2d nmr techniques generally reveal more structural subtleties than can typically be deduced from 1d h and c experiments only . this brings up an important point of the sophistication of both the technique and the individual who interprets the data . two scenarios present themselves : a rather simple technique in the hands of a skilled researcher can reveal structurally accurate conclusions , while a sophisticated technique may be poorly interpreted and even misinterpreted . all in all , the depth of structural information relies on what constitutes an adequate attempt to assign a structure to a given compound . with nmr prediction and simulation techniques becoming more mainstream ( see refs ( 25 , 26 ) and citations therein ) , it is possible that computational analysis of nmr spectra may be encouraged in the future as supporting or possibly even definitive evidence of a correct spectroscopic interpretation . the role of purity is typically , but not necessarily ( see discussion below ) , assessed last in the np isolation workflow ( figure 1 ) . ideally , the purity of quality - controlled np for biological evaluation is high , making it a single chemical entity . the short answer is that reports occur rather infrequently and at a declining rate . compounding the information for all journals and sorting by survey period , the topic of purity is only addressed ( not necessarily measured ) in 4.68.4% of the reports ( 6.3% total average ) . over time , purity reporting has been on a decline and was found in only 31 of 597 reports evaluated in the most recent period , iii ( 5.2% ) . interestingly , when considering the whole survey period , 4.2% ( 76 ) of reports from the group b journals address purity vs 1.4% ( 26 ) reports in group a. assuming a general awareness of purity as a parameter , it is possible that some studies determined purities without publishing this information , but there was no way of determining the abundance of such cases . in summary , purity analysis was reported as being performed for less than 10 in 1000 compounds , and hplc or more elaborate methodology was used for less than five in 1000 . it is important to differentiate between awareness and actual assessment of purity : an average of 3.6% of all reports included some form of purity analysis , and the rate has been declining over the survey period ( 4.6% to 3.2% to 2.8% in periods i , ii , and iii , respectively ) . ( 6.3% vs 3.6% of all reports ) suggests that about 40% of reported purity information is taken from ( vendor ) labels or derived from undocumented or otherwise obscure methods . in contrast to the declining awareness of purity , the use rate of hplc for purity analysis is flat over the survey period ( 2.22.4% ) : an average of 2.3% of reports , representing about one - third of all reports surveyed , used hplc for this purpose . accordingly , hplc is the most common method of purity measurement when purity is reported . ostensibly , this is done because an hplc method was developed as part of the isolation scheme to either prepare the target np(s ) or assess the purity of fractions . hplc provides the chromatography method , and the detector actually monitors the composition of the column effluent . the uv vis method of compound detection is used widely in hplc and other liquid chromatography methods . it can be a highly sensitive method to detect and quantify a target compound and has the potential to be universally applied to nps that involve a uv vis hplc method at some stage of the purification protocol . on the other hand , this method often has severe limitations in detecting sample impurities . more sophisticated methods of lc detection are available , including ms(ms ) , elsd , and corona charged aerosol detector , and used for this purpose . these methods all are limited to varying degrees in that they have altered sensitivity between different compounds , and thus can not be considered universal detectors and require carefully tuned parameter optimization . relevant to purity assays , but primarily of importance to the metabolomic aspects of np research , it shall be noted that recent developments in lc ( e.g. , uhplc ) and hyphenated technology ( e.g. , lc ms , lc nmr ) have significantly advanced analytical capabilities for the characterization of both complex and purified nps ( see reviews ( 28 , 29 ) and references therein ) . the survey data clearly show that the purity of nps used in bioassays is rarely reported . there may be some feeling that if a np is pure enough to determine its structure by nmr and mass spectrometry , it is sufficiently pure to analyze its bioactivity . as shown in figures 2 and 3 , this may or may not be the case , depending on the balance of np and its rc component and their interactions with the target and the biome of the test system . parameters such as purity , specificity , and ( residual ) complexity are involved in both the chemical and the biological portions of the analyses , and they play equally important roles in the outcome . from the chemical perspective , there can be no question that the purity of a np is of utmost importance in determining its bioactivity . at best , an inactive impurity will dilute the apparent activity that is usually measured in terms of bioactivity per mass of np . more impactful is the possibility that the bioactivity of a minor component could mask the true bioactivity , or lack of it , for the target np . activity relationships as a way of correlating the measured bioactivity and the bioactivity of the target np . a pure np ideally represents a single chemical entity ( sce ) . its interaction with a defined biological target ( t ) establishes a definite structure activity relationship ( sar ) and typifies how the majority of bioactivities of nps are characterized . however , due to variation in purification protocols and their source , nps are inevitably ( chain ) impure profiles , by virtue of residual complexity ( rc ) from the source organism s metabolome . similar considerations apply to the bioassay : whole cell assays in particular entail the entirety of biological targets and processes ( biome ) with which the np sample interacts . interactions between the sce and/or the rc and the biome can lead to a response and need to be considered when interpreting outcomes . depending on the proportions of sce and rc and the interactions with t and the biome , the sars and purity activity relationship ( par ) of a np will interfere , with possibly profound impact on the outcome . it should be noted that the aforementioned 3.6% rate of hplc purity reports only referred to qualifying hplc statements , whereas these reports did not include details of the analytical methods used such as chromatograms , integrals , and calculations . elaborate reports of purity were coded separately and were very rare at a total average of 0.9% . moreover , the rate of detailed purity assessment has declined to levels of 0.200.34% in the most recent two survey periods . despite research progress and increasing interest in the methodology , quantitative [ h ] nmr ( q[h]nmr ; see refs ( 30 , 31 ) for a literature overview ) so far has rarely been used for purity assessment , with only 13 reports , or 0.72% , employing qhnmr . interestingly , the survey did not detect any further use of qhnmr for purity analysis of nps involved in bioactivity studies in the most recent time period , iii . however , virtually all investigators today use h nmr spectroscopy in the structural elucidation of their nps and , therefore , have at their fingertips the data to determine the purities of the nps isolated . considering that qhnmr methodology is well - established , purity evaluation using the 100% method is straightforward from most existing h nmr data sets . calculation and citation of such data are relatively uncomplicated and would add important evidence to the matter of rc highlighted here , as well as to related discussions about bioactive nps . 40% of reported purities are taken from ( vendor ) labels or undocumented methods and an additional ca . 30% from hplc statements , the reported purity values have to be interpreted with caution . in order to put the small numbers of purity - tested nps into perspective , of the total number of bioactive nps ( ncpd = 12 570 ) , an average of seven compounds ( sd 7 ) were included in one report . given 102 reports on actual purity analysis by hplc ( statement ) or better , this potentially affected ca . due to the design of the study , the actual number of analyzed compounds was not recorded in the periods i and ii . during the literature assessment it became clear that this number is much lower , likely by a factor of 5 to 10 . this is in line with two other observations : ( i ) the low ( 0.11.1% ) proportion of compounds in reports with hplc or better purity analysis and ( ii ) that 85% of purity statements contained one single value rather than a range of purities , indicating that only one or very few nps were analyzed and/or individual samples were not differentiated . based on the evaluation of 102 reports , the following can be said about purity statements : the vast majority of reports ( 87% ) state purities of 95% or higher , and almost two - thirds ( 60% ) of which report purities of 98% and above . while a few studies ( 3% ) even report absolute purity ( 100% ) , the same proportion reports purities below 80% . in general , at least three additional dimensions of complexity affect the interpretation of research data on bioactive nps and will be addressed in the following : ( i ) the role and relationship of in vivo ( here including whole cell- and animal - based ) vs in vitro bioassays used to assess bioactivity in the np purification and characterization workflow ( figure 1 ) ; ( ii ) the depth and diversity of the purification workflow ; and ( iii ) the role of purity and rc . in addition , just as rc is almost inevitable when purifying nps , biological test systems are seldom singleton but rather residually complex or even very complex entities ( e.g. , in vivo systems ) . potential further dimensions to consider relate to the connectivity between sce , rc , and bioassay ( figure 2 ) , i.e. , the specificity , both qualitative and quantitative , of the bioassay . finally , both the bioassay and the purified np may behave like the proverbial tip of the iceberg , depending on their individual rc characteristics . as a result , a matrix of scenarios can be conveyed ( figure 3 ) , which reflect the multidimensional interplay of the np / sce , its purity , its rc characteristics , the activities of the sce and the rc component(s ) , and the specificity of the bioassay . figure 3 shows that , depending on the combination of these factors , observations from the lack of bioactivity to the presence of strong activity potentially can be explained for nps that otherwise appear to be identical or at least comparable entities . nps in bioassays that comprise different response elements ( biome ; figure 2 ) can be symbolized by triangles in which proverbial tips of the iceberg represent both the well - defined target ( t ) of the bioassay and the residual complexity ( rc ; figure 2 ) of the np , respectively . as purity decreases and rc increases , four main scenarios , a d , can be distinguished : ( a ) highly pure np , only the sce interacts with t ; ( b ) like a , but the sce interacts with additional biome processes ; ( c ) in a bioassay with reduced specificity , nps containing some impurities exhibit intermediate bioactivities , which can result from all four interactions depicted in figure 2 ; ( d ) in impure nps , the rc component dominates the biological response , even if t is highly defined . in addition to a d , depending on whether only sce or rc or both components are active principles , three series , 13 , of biological potency may be observed . particularly relevant ( marked * ) for nps research are ( a1 ) the ideal case : the purification leads to a near pure sce as bioactive principle ; ( c1 ) false potency : the inactive rc dilutes the bioactivity of the sce such that potency is misjudged ; ( c2 and d2 ) false assignment of bioactive principle : the purification yields the active principle , but it is contained in the rc component and not represented by the ( apparent ) sce . the importance and potential impact of rc on , for example , the efficiency of drug discovery workflows can not be underestimated . the initial discovery of an inverse correlation of anti - tb activity and the purity of ursolic acid ( purity activity relationships ) has led to the routine integration of qhnmr purity assessment in the authors laboratory . a recent report by fitch et al . describes the comprehensive efforts aimed at establishing solid structure activity relationships for the frog alkaloid epiquinamide . their studies involved chiral synthesis of three and pharmacological study ( nicotinic acetyl choline receptor ) of all four stereoisomers of the np to eventually determine that all of them are inactive . the authors state that the misleading activity in the natural product material is concluded to be trace contamination by co - occurring epibatidine , a finding that bodes heavily on the relevance of purity cragg and newman et al . , backed by extensive nps research experience , stated that the potential for the discovery of new chemotypes from plants , comparable to the taxanes and camptothecins , appears to be relatively low . on the other hand , two recent articles by kinghorn et al . provide convincing evidence , also backed by long - term research , for the relevance of higher plants and other terrestrial organisms as sources for new bioactive lead compounds . emphasizing the exceptional role of camptothecin and taxol ( paclitaxel ) , kinghorn et al . provide a thought - provoking interpretation that counters the other apparently discouraging outlook : the possibility that an informative in vitro screen is not a substitute for a relevant in vivo assay . for the discovery of five anticancer leads in the 1970s and 1980s , there was an early involvement of in vivo testing . however , the insights from the present anapurna study add yet another possible interpretation . as the purification of highly active principles that are minor constituents likely requires more effort , it is conceivable that an increase in the fractionation depth ( n , figure 1 ) and/or diversification of the preparative - scale separation methodology is a viable means of improving the purification process and , thus , potentially can contribute to a discovery being made . moreover , as the sce and rc characters of nps are closely linked ( figure 2 ) a correlation that opens multiple possibilities for the interpretation of observed biological activity / potency ( figure 3 ) it can seldom be ruled out that bioactivities originate , in full or in part , from the rc portion of the np ( see scenarios c2 and d2 in figure 3 ) . the assessment of purity and rc ( see discussion above ) and the establishment of pars are potential valuable methods for the nps discovery process . in light of the findings of this study , all these factors represent aspects that could stimulate future research design . it is widely recognized that structure elucidation , unless supported by direct atomic evidence from x ray diffraction analysis , is largely based on indirect spectroscopic evidence , primarily from nmr , ms , ir , uv , and cd / ord methods . as a result , elucidation is an asymptotic process and can be compared with a balancing act between the interpretation of the spectroscopic data and the possible structural variations that can potentially be aligned with it . accordingly , the non - x - ray approach to structure determination includes an element of uncertainly ( residual doubt ) , which depends on the depth of the analysis in terms of the choice of type and number of spectroscopic experiments , but also on how well the chemical space is probed for alternative structures ( e.g. , isomers , compounds with heteroatoms beyond n and o ) that potentially fit the spectroscopic information . there are clear indications in the literature that in - depth studies lead to higher confidence in the assigned structure ( reduced residual doubt ) and frequently lead to reassignment and revision of structures . one such example is the case of hypurticin , a 2h - pyran-2-one , which was recently reassigned to contain a 3,5,6- rather than a 3,4,6-tri - oac side chain . make a convincing case by employing detailed density functional theory and h nmr analysis , including h nmr spectral simulation and the synthesis of an analogue . a similar approach in the authors laboratory also employed h nmr spectral simulation and involved a detailed analysis of the higher order j coupling patterns of the sugar moiety . this led to the identification of the first cyanogenic glycoside with -allose rather than -glucose attached to the cyanogenic methine carbon , which has broad implications for the enzymology of cyanogenesis . during the present survey , the authors frequently encountered articles in which the reported h nmr spectroscopic data , in particular the interpretation of coupling patterns and the deduction of coupling constants ( j ) , were lacking or ambiguous and , thus , would not allow the distinction of diastereoisomers . in many cases , even though other spectroscopic evidence was provided , this did not provide the distinction of the given structure from potential ( stereo)isomeric alternatives . the most frequently encountered examples are epimeric hexopyranoses , which in the h nmr domain require a relatively tedious analysis of their h nmr multiplets between 3.2 and 4.5 ppm to sort out the correct j couplings and chemical shifts . the aforementioned cases of hypurticin and -d - allopyranosyloxy-2-phenylacetonitrile , among many others , demonstrate that taking on the challenge of residual doubt can lead to significant discovery . during the writing of this review , the authors became aware of an excellent book chapter by a. d. wright , which includes a meta - analysis of the literature regarding the isolation of marine nps . the author analyzed 115 reports during 1995 , published in journal of the american chemical society , journal of natural products , the journal of organic chemistry , and tetrahedron and recorded parameters similar to the present survey . one aspect of the study was the differentiation of stationary phases and specific chromatographic methods . isolation methods in marine np chemistry might be different from those used for nps from terrestrial organisms . distinct differences seem to exist with regard to the use of silica gel ( 6% of studies analyzed in ref ( 38 ) compared with 5771% in the above discussion ) or ccc ( 7% of studies analyzed in ref ( 38 ) , 0.31.7% in the present survey , depending on time period ) . while the surveys can not be directly compared , future meta - analyses of the np literature , including the continuation of the present anapurna study , can likely benefit from extended parameter sets that enable addressing additional aspects in the contemporary methods used to purify and analyze bioactive nps . during the extensive literature study performed , the present authors observed that only very few publications include new preparative - scale analytical methods in their np isolation schemes . it seems that innovative chromatographic methods would enhance the systematic exploitation of np diversity . this is exactly the chemical space that is the focus of the application of metabolomics to the study of nps . in effect , metabolomics absolutely requires an organized investigation of the thousands , and possibility ten of thousands , of chemical entities that a single organism may contain . a recent editorial in phytochemical analysis points out that , although many articles include the term metabolomics in their title , the content of the publications concerned tend to reflect only standard methodology and reporting . ultimately , the depth ( see methodology section ) of the spectroscopic data of a quality - controlled np ( figure 1 ) and the thoroughness of their reporting represent important parameters of the integrity in np research . this applies from two angles : from the perspective of a novel structure and its discoverer(s ) , as mentioned above , it effects an ambiguity to the structure elucidation and the amount of residual doubt . np integrity has been coined by the national center for complementary and alternative medicine of the u.s . national institutes of health in relation to research on biologically active agents used in complementary and alternative medicine , particularly including botanicals and other dietary supplements . these guidelines are intended to ensure reproducibility of preclinical , translational , and clinical studies with np agents , which are known to exhibit much larger variation in constitution than other common intervention materials , such as sce - based drugs . from the perspective of reproducibility , research involving ( re)isolation , characterization , and/or other operations with previously published nps performed by the same or other scientists , the integrity of spectroscopic information influences the degree of certainty with which structures can be dereplicated and/or distinguished from close congeners . this makes the depth of spectroscopic information , as assessed in this survey , an element of np integrity and a factor in reproducibility . increasing considerations of purity as a standard or required physicochemical parameter might be ( mis)interpreted as a quest for ever - increasing purities . high purity certainly has its merit , because it allows the np to approach sce status and simplifies interpretation and understanding of a biological outcome ( figures 2 and 3 ) . at the same time , requiring nps to be highly pure often imposes overly proportional or even unrealistic efforts and costs on the research . depending on the biological application and research aim , a certain degree of rc can also be of value , as residually complex nps more closely reflect the natural character of a np . provided that rc and purity of a np are known and documented , even less pure materials can be potentially useful and/or are suitable as unique research tools for biological studies , as long as their greater chemical complexity is considered during the interpretation of the results ( figure 3 ) . examples of relevant biological topics that can benefit from the availability of such materials include additive , synergistic , and antagonistic action and their use as markers in standardization of biological agents such as botanicals . while even very thorough analysis may not solve the challenge of reproducing identical nps with identical rc patterns , biological studies with such highly characterized nps ( cnps ) at least can be compared on a more rational basis . the suitability of np and cnp materials , e.g. , attaining certain potency levels or confirmation of activity at a given target , can usually be assessed only after their purification . accordingly , studies aimed at the biological evaluation of nps can greatly benefit from assessing the status of any np sourced from the pipeline from crude np to sce / cnp ( figure 1 ) and from publically disseminating this information as equally valuable along with the chemical and biological data .

based on a meta - analysis of data mined from almost 2000 publications on bioactive natural products ( nps ) from > 80 000 pages of 13 different journals published in 19981999 , 20042005 , and 20092010 , the aim of this systematic review is to provide both a survey of the status quo and a perspective for analytical methodology used for isolation and purity assessment of bioactive nps . the study provides numerical measures of the common means of sourcing nps , the chromatographic methodology employed for np purification , and the role of spectroscopy and purity assessment in np characterization . a link is proposed between the observed use of various analytical methodologies , the challenges posed by the complexity of metabolomes , and the inescapable residual complexity of purified nps and their biological assessment . the data provide inspiration for the development of innovative methods for np analysis as a means of advancing the role of naturally occurring compounds as a viable source of biologically active agents with relevance for human health and global benefit .

Introduction Methodology Observations Made Summary and Conclusions

nonetheless , most contemporary practitioners would agree that the role of this discipline within biomedical science has declined , as evidenced by its present abandonment by most large pharmaceutical companies in their search for new chemical entities to provide new drug discovery leads . this review takes a comprehensive look at the current practice of np research , with the aim of pinpointing potential areas where the practitioners might improve the overall efficiency of this type of work . this review intends to stimulate discussion and inspire the development of new approaches to yield more rapid results and a greater number of new chemical entities discovered , and thereby promote the future role of np research in interdisciplinary programs . underlying evidence came from an extensive meta - analysis of the primary literature of all drugs , in or completing fda - approved studies within a set time frame and classifying them according to their origin as nps , inspired by nps , analogues of these two classes , or from non - np sources . moreover , the chemical diversity in nps is tied intrinsically to the complexity of the metabolome contained in the source material . ultimately , both the discovery and the resupply of bioactive nps depend on the availability of preparative - scale analytical methods having the capability of resolving the complex primary and secondary metabolomic mixtures that are typically isolated from the source organism , yielding a purified np ( np in figure 1 ) , and eventually providing a well - characterized np as a single chemical entity ( sce ; figure 1 ) . the nearly 2000 publications evaluated employ bioassays to address screening of crude nps , bioassay - guided fractionation , biological assessment of purified nps , and detailed pharmacological investigation of , for example , structure activity and structure purity relationships ( sars and pars , respectively ; see text and figure 2 ) . they also explain why the np drug discovery process and the biological assessment of nps to date are almost inevitably tied to preparative - scale analytical methods used for np purification . the ability to purify a few milligrams of a rare np from kilograms of a crude extract has been one of the significant skills of scientists trained in np chemistry , pharmacognosy , and analogous disciplines and represents one of the keys to np research . in clinical research , numeric meta - analysis of literature is a well - established tool , allowing recognition of more general trends , and is used frequently to improve clinical practice . the present contribution is based on the meta - analysis of the recent literature with a focus on parameters that reflect the analysis and purification of bioactive nps ( anapurna ) . , the ( semi)preparative - scale analytical method employed ; ( b ) the assessment of the purity [ or residual complexity ( rc ) ] of the isolated np , including the analytical method used for purity assessment . the aim of this review is to describe the status quo regarding both aspects , through a comprehensive assessment of the contemporary literature on bioactive nps . the present report summarizes over a decade of data - mining activity by the authors , which involved manual screening of > 80 000 pages of scientific literature during the periods 19981999 , 20042005 , and 20092010 . to date , data have been extracted from nearly 2000 peer - reviewed articles , forming the foundation of this survey of analysis and purification of bioactive natural products ( anapurna ) . this review is organized as follows : the methodology section describes the data - mining methodology employed as well as the journal and time coverage of the survey . the next section concentrates on the following aspects : sources of purified nps ; chromatographic methodology used for np isolation ; spectroscopic methods used for np characterization ; and the role of purity and the methods used for the purity analysis of nps . the final section of the review summarizes the findings from the perspective of potential new approaches to the analysis of np complexity and the achievement of novelty . final discussions are devoted to the role of np integrity including purity and linkages between chemical and biological properties of bioactive nps . the integration of these aspects potentially could help in advancing the future role of nps as a viable source of new biologically active agents . upon compilation and preliminary data evaluation , this led to the selection of six priority journals and the addition of one journal ( journal of asian natural products research ) for continuation of the survey in the subsequent periods ii and iii , which mostly focused on one year of the two - year periods ( see table 1 for details ) . of the journals with a lower prevalence of qualifying reports , one ( journal of pharmacology and experimental therapeutics ) was included with the seven priority journals and assessed for the entire period 20002003 ( n = 77 ; not included in ntot ) plus one year of period ii , in order to provide an example of extended coverage for these journals . for any given journal volume included in the study , all articles were prescreened manually for the following inclusion criteria : they had to report on both bioactivity and chemistry of nps and provide a substantial experimental description , regardless of how well the np - related portion was developed . as a means of prospective guidance for the data - mining process , all of the questions addressed under the observations made section of this review were also formulated at the onset of the study . furthermore , for each report , the total number of nps and the predominant class of compounds were recorded , and the following data were determined : total number of compounds ; number of compounds that were isolated by purification from natural material ; number of compounds that were synthesized ( full or partial ) ; number of compounds that were gifts from colleagues ; and the number of new structures . the experimental section of each report was evaluated for parameters that reflect the methods used for the purification of the nps and their spectroscopic characterization . as many reported isolation procedures are rather convoluted , the assessment included determination of the longest purification pathway and the highest degree of diversification of the methods employed . in addition , the diversity of the purification methods was assessed and encoded into binary format as a byte integer , consisting of the following five bits : 0 = undefined or literature reference only ; 2 = precipitation or crystallization ; 2 = paper or thin - layer chromatography ( tlc ) , including centrifugal tlc ; 2 = column liquid chromatography ( lc ) , vacuum lc , and low - pressure lc , and the value 2 was added to encode repetition in the entire scheme ; 2 = medium - pressure and high - pressure lc ( mlpc , hplc ) , and also the value 2 was added to encode repetition in the entire scheme ; 2 = countercurrent chromatography . data collected on the spectroscopic characterization of the nps , for which bioactivities were reported , included the number of compounds for which spectroscopic data were reported , the comprehensive nature of the general physical / spectroscopic data in general , and , separately , the nature of the nmr data utilized in particular . for this study , depth is defined by the completeness , detail of interpretation , and comprehensive nature of data . for the assessment of reports from the most recent time period , iii , the provision of spectroscopic data as supporting information ( si ) was considered as added comprehensiveness that was linked via cross - references in the main text . finally , each article in its entirety was mined for information about purity assessment of the bioactive nps . all authors were involved in the data - mining process , which involved manual page turning of journal hard copies , judgment of inclusion criteria of each article in the screened volumes , and mining of the aforementioned data from each of the 1823 articles . given the workload of manually screening 80 000 pages of literature information , it was not feasible to perform the entire survey in triplicate and/or by multiple individuals . for example , purified nps may be incorporated into human clinical trials , which will be published in a medical journal rather than a natural products journal . following in the ranks are biological / chemical and pharmaceutical bulletin ( combined ) , planta medica , phytochemistry , fitoterapia , and the journal of asian natural products research . there were four main sources of the nps : ( i ) isolation and purification of the np by the authors as reported in a scientific publication ; ( ii ) purchase of nps from commercial sources ; ( iii ) receipt of nps from colleagues who have performed the isolation and purification themselves ; and ( iv ) ( semi)synthesis . addressing this question eventually required a more elaborate analysis of the data , including differentiation by looking at individual journals and the groups of journals . the high percentage of bioactive nps that are isolated and characterized ( currently about 95% ) also indicates the rarity of purified nps in that most researchers tend to produce these compounds by themselves rather than obtaining them commercially . notably , pars can also be established at the level of purified compounds ( nps and cnps ; figure 1 ) , e.g. the binary encoding and scoring of characteristics of the purification methods used in surveying all reports is described in the methodology section and rests on a thorough case - by - case analysis of the experimental section of each report . considering the correlation between the metabolomic complexity of crude extracts and the residual complexity of purified nps ( figure 1 ) , the codes and scores were designed to provide metrics to answer questions about the depth and diversity of isolation procedures as they are used in laboratory practice . this question includes two aspects : the number of steps is addressed here , and the chromatographic methodology in the following section . the effort required to achieve single chemical entity parameters for an isolated np depends on many factors , including ( i ) the concentration of the np in the crude material ( the higher , the easier the purification ) ; ( ii ) the physicochemical characteristic of the compound , in particular solubility ( precipitation ) and tendency to form crystals ( a historically important property of nps ) ; ( iii ) the match between the selectivity characteristics of the chosen purification methods and the np ( some methods appear to work better than others for certain compound classes or types of source materials ; different standard protocols for marine vs microbial vs plant nps ) ; and ( iv ) the nature of the matrix components in the crude np , which may cause difficulties in the purification process ( e.g. comparing studies that use normal - phase silica gel with those that do not , the ratio of the average number of crystalline compounds per study was 2.0 in period i , 2.6 in period ii , and 4.9 in period iii . in the present meta - analysis , np purification schemes have two primary dimensions : the number of purification steps and the chromatographic methodology used in each step . the binary scores given in this study for the diversity of the purification methods ( see methodology ) allowed us to study the relationships between the number of steps and the chromatographic methodology ( see scatter plot , figure s3 , supporting information ) . that having been said , the use of normal - phase silica gel as a chromatographic method of choice is much more entrenched than can be simply explained by the fact that some alternatives are considered to be specialized techniques . examples are the development of countercurrent chromatography , as pioneered by y. ito and co - workers , and the advent of hplc in the 1970s . the questions posed were as follows : in order to answer these questions , both the physical data in the experimental sections as well as the tables and descriptions in the main text of the articles were assessed , and the extracted information was coded as previously described under methodology . compounding the scores for the depth of spectroscopic data for all articles ( n = 1908 ) yielded a numerical average of 2.5 on the discrete scale from 1 to 5 ( lower number better thus , on average , the spectroscopic foundation of all reported bioactive nps ( ncpd = 12 570 ) was between comprehensive ( 2 ) and with gaps ( 3 ) . the observation that in the most recent period , iii , 49% of all nps were reported with nmr spectroscopic information categorized as comprehensive and an additional 18% as highly comprehensive can be interpreted as a sign of strong nmr evidence for the structure of about two - thirds of all bioactive nps . one way to assess depth of nmr data is level of detail , such as the completeness of the assignments , the coupling pathways , the coupling constants , and the multiplicity assignments . this brings up an important point of the sophistication of both the technique and the individual who interprets the data . it is important to differentiate between awareness and actual assessment of purity : an average of 3.6% of all reports included some form of purity analysis , and the rate has been declining over the survey period ( 4.6% to 3.2% to 2.8% in periods i , ii , and iii , respectively ) . as shown in figures 2 and 3 , this may or may not be the case , depending on the balance of np and its rc component and their interactions with the target and the biome of the test system . parameters such as purity , specificity , and ( residual ) complexity are involved in both the chemical and the biological portions of the analyses , and they play equally important roles in the outcome . activity relationships as a way of correlating the measured bioactivity and the bioactivity of the target np . however , due to variation in purification protocols and their source , nps are inevitably ( chain ) impure profiles , by virtue of residual complexity ( rc ) from the source organism s metabolome . depending on the proportions of sce and rc and the interactions with t and the biome , the sars and purity activity relationship ( par ) of a np will interfere , with possibly profound impact on the outcome . interestingly , the survey did not detect any further use of qhnmr for purity analysis of nps involved in bioactivity studies in the most recent time period , iii . however , virtually all investigators today use h nmr spectroscopy in the structural elucidation of their nps and , therefore , have at their fingertips the data to determine the purities of the nps isolated . in order to put the small numbers of purity - tested nps into perspective , of the total number of bioactive nps ( ncpd = 12 570 ) , an average of seven compounds ( sd 7 ) were included in one report . due to the design of the study , the actual number of analyzed compounds was not recorded in the periods i and ii . based on the evaluation of 102 reports , the following can be said about purity statements : the vast majority of reports ( 87% ) state purities of 95% or higher , and almost two - thirds ( 60% ) of which report purities of 98% and above . in general , at least three additional dimensions of complexity affect the interpretation of research data on bioactive nps and will be addressed in the following : ( i ) the role and relationship of in vivo ( here including whole cell- and animal - based ) vs in vitro bioassays used to assess bioactivity in the np purification and characterization workflow ( figure 1 ) ; ( ii ) the depth and diversity of the purification workflow ; and ( iii ) the role of purity and rc . as a result , a matrix of scenarios can be conveyed ( figure 3 ) , which reflect the multidimensional interplay of the np / sce , its purity , its rc characteristics , the activities of the sce and the rc component(s ) , and the specificity of the bioassay . nps in bioassays that comprise different response elements ( biome ; figure 2 ) can be symbolized by triangles in which proverbial tips of the iceberg represent both the well - defined target ( t ) of the bioassay and the residual complexity ( rc ; figure 2 ) of the np , respectively . particularly relevant ( marked * ) for nps research are ( a1 ) the ideal case : the purification leads to a near pure sce as bioactive principle ; ( c1 ) false potency : the inactive rc dilutes the bioactivity of the sce such that potency is misjudged ; ( c2 and d2 ) false assignment of bioactive principle : the purification yields the active principle , but it is contained in the rc component and not represented by the ( apparent ) sce . the initial discovery of an inverse correlation of anti - tb activity and the purity of ursolic acid ( purity activity relationships ) has led to the routine integration of qhnmr purity assessment in the authors laboratory . the assessment of purity and rc ( see discussion above ) and the establishment of pars are potential valuable methods for the nps discovery process . as a result , elucidation is an asymptotic process and can be compared with a balancing act between the interpretation of the spectroscopic data and the possible structural variations that can potentially be aligned with it . during the writing of this review , the authors became aware of an excellent book chapter by a. d. wright , which includes a meta - analysis of the literature regarding the isolation of marine nps . the author analyzed 115 reports during 1995 , published in journal of the american chemical society , journal of natural products , the journal of organic chemistry , and tetrahedron and recorded parameters similar to the present survey . while the surveys can not be directly compared , future meta - analyses of the np literature , including the continuation of the present anapurna study , can likely benefit from extended parameter sets that enable addressing additional aspects in the contemporary methods used to purify and analyze bioactive nps . ultimately , the depth ( see methodology section ) of the spectroscopic data of a quality - controlled np ( figure 1 ) and the thoroughness of their reporting represent important parameters of the integrity in np research .

fall - related injuries in the elderly population are a rapidly growing public health concern that lead to a wide range of financial , physical , and emotional complications . one out of three elderly adults fall each year in the us , resulting in more than 1.5 million fragility fractures annually.1,2 due to the aging baby boomer generation , these numbers will continue to grow . the impact of these injuries will also continue to be an economic burden , with the cost for fall - related injuries projected to exceed us$30 billion by 2020.3 fragility fractures often leave patients in great emotional distress and unable to perform activities of daily living.4,5 the impact of these fractures is dramatic enough that a survey of older women indicated that 80% of them preferred death to a hip fracture that would result in admission to a nursing home.5 mortality for patients with fragility fractures is also significantly increased compared to expected mortality rates for this demographic population.6 a multicenter canadian study found that hip fracture patients had a 4.2 times greater risk of death during the first year after fracture than would be expected for hip fracture patients.7 mortality 5 years after hip or vertebral fracture is also ~20% greater than would be expected for elderly individuals without fracture.4 identifying functional deficits that may put elderly patients at risk for falls is crucial to help prevent the morbidity and mortality that can occur after a fragility fracture . impaired balance , lumbopelvic control , and handgrip strength all have shown strong correlations with fall risk.813 rehabilitation studies demonstrate that both improvements in postural sway and increases in strength measures are possible in geriatric patients after a fragility fracture.1416 in addition , multiple systematic reviews have shown that targeted interventions , mainly exercise and balance exercises , can reduce fall risk in elderly patients , thus preventing future complications.1720 because of the high morbidity and mortality associated with falls in the elderly , the american and british geriatric societies recommend that all the elderly individuals who present for medical attention because of a fall , report recurrent falls in the past year , or report difficulties in walking or balance should have a multifactorial fall risk assessment performed to determine their future fall risk.21 this assessment should include a detailed evaluation of gait , balance , and lower extremity joint function . given the great morbidity and mortality complications with fall - related injuries in the elderly , along with the potential for functional improvement after treatment and rehabilitation , there is a need to develop inexpensive , objective , and portable technologies to help evaluate people for fall risk quickly and accurately . the affordable health care act emphasizes improvement of quality and efficiency of health care , public health , access to innovative medical therapies , and prevention of chronic disease . as a result , the orthopedic surgery field faces new socioeconomic pressures to develop performance - based criteria and evidence - based clinical assessment tools to evaluate outcomes of treatment for musculoskeletal injuries . three clinical assessment tools were identified and utilized to assess stability ( wii balance board [ wbb ] , ipod touch level belt ) , postural sway ( wii balance board , ipod touch level belt ) , and handgrip strength ( saehan handheld dynamometer ) . inclusion criteria required that each tool be commercially available , easily portable , reasonably inexpensive , and operational with little training or expertise . the wbb ( nintendo , redmond , wa , usa ) was selected to evaluate a patient s balance and stability . recent studies have already demonstrated that the wbb has the ability to detect subtle changes in balance and center of pressure with good test retest reliability when compared to the gold standard , a research - grade force platform.22,23 the ipod touch level belt ( perfect practice , columbus , oh , usa ) was selected to evaluate a patient s lumbopelvic control . the ipod touch level belt is an ipod touch application available in the apple itunes store and has been used to measure postural sway in baseball players during functional tasks.24 the built - in accelerometer of the ipod touch ( apple , cupertino , ca , usa ) has already been shown to reliably and validly assess gait and posture in the elderly population.25 accelerometry has been used in other studies to measure trunk stability in the elderly population and has been suggested as a possible screening tool for future fall risk.26 the handheld dynamometer was selected to evaluate a patient s handgrip strength . handheld dynamometers have been used to measure handgrip in many studies to provide quick and accurate strength quantification.9,16,2729 in addition , decreased isometric handgrip strength has been identified as a risk factor for future falls in a 1-year prospective study.10 the goal of this study was to identify inexpensive , noninvasive , portable tools that can be used to assess potential deficits including balance , handgrip strength , and lumbopelvic control that may put elderly patients at risk for falls . we hypothesize that our tools will be able to quickly and easily identify balance , strength , and stability deficits in fragility fracture patients in a variety of clinical settings . prior to study participation , all subjects signed informed consent and agreed to participate in the study as approved by the the ohio state university s biomedical sciences institutional review board . the participants were recruited via fliers from three orthopedic outpatient clinics and the general community . the two groups evaluated were patients with a history of one or more fragility fractures and controls without a history of fragility fractures . a fragility fracture was defined as a fracture occurring from a fall from a standing height or less , without major trauma such as a motor vehicle accident . the fracture must have occurred after the patient was 50 years of age.30 further inclusion criteria required that all subjects in the study be 50 years old or greater , able to walk 40 feet unassisted , able to follow simple verbal and written directions , and have an absence of peripheral neuropathies or mobility disorders . each participant completed a written questionnaire about age , sex , weight , current ambulatory status , fragility fracture details , and activity limitations . included in the patient questionnaire was a section from the 36-item short form health survey ( sf-36 ) , which evaluates daily activity restrictions based on the subject s current state of health . participants were then asked to perform a series of functional assessment activities over the course of 20 minutes utilizing the clinical assessment tools in a prognostic case - control study . stability was evaluated using the nintendo wii video game platform and the wbb . in conjunction with the wbb , two mini - games ( torso twists and single leg stand ) from the wii fitness program were used to assess postural sway and stability in participants . all hardware and software is commercially available and was not altered from its purchased state . participants were asked to remove their shoes and stand on the wbb facing a member of the research team . for each trial , the researcher navigated the on - screen menu and selected the activity to be performed . in addition , the researcher also performed the activity at the same time as the participant to ensure that the correct movements and pace were maintained throughout the duration of the exercise . as shown in figure 1 , the researcher was able to see the wii instructions on the tv screen , but the screen was blocked from the participant s view . the first activity selected from the wii fitness program was the torso twist activity , which requires patients to raise their arms parallel to the ground and rotate it 90 to one side , then to the other , alternating sides for a total of three twists to each side . at the end of the trial , the maximum horizontal and vertical scores were 50 for each , and the maximum total score was 100 , which was a sum of the horizontal and vertical components . the vertical score correlated to the amount of shift in the anterior posterior ( ap ) direction , while the horizontal score correlated to shift in the medial lateral ( ml ) direction . the second activity selected from the wii fitness program was the single leg stand activity which measures balance as the participant performs a series of movements while standing on one leg . the recommended motion from the wii fitness program was simplified by only asking patients to stand with your hands on your hips and raise your leg off the ground . participants were told that if they felt unstable , they could briefly grab the safety bars that were placed on either side of them or tap their raised foot down , but to reset to the one - leg stand position as soon as possible . the trial lasted 15 seconds on each leg . at the end of each trial , again , the maximum right and left scores were 50 for each , and the maximum total score was 100 , which was a sum of the right and left components . as with the torso twist activity , higher scores indicated decreased postural sway throughout the duration of the task . in addition , hand and foot faults were measured as a secondary internal measure of balance and sway . participants were asked to hold their forearm parallel to the ground with their elbow flexed at 90 and pointed laterally . participants were asked to grip as hard as they can for 3 seconds , then release . encouragement was provided to each participant throughout the task and they were told when to release . the apparatus consists of an elastic belt to which an ipod touch is attached by velcro ( velcro usa , manchester , nh , usa ) . the belt is placed across both of the participant s posterior superior iliac spines with the ipod touch centered in the middle of the back . the application uses an accelerometer - based sensor to measure ap and ml tilt as the participant walks . the participant is instructed to walk normally for 40 m to the end of the hall and stop . the data obtained during the middle half of the walking stage were used in the study in order to capture the normal gait phase of walking and not acceleration or deceleration phases of a walk . torso twist and single leg stand balance scores reported by the wbb as well as faults during the single leg stand were averaged over the three trials . for handgrip strength , the best score out of three trials for each hand was used for statistical analysis . the ipod touch level belt records the ap and ml tilt in degrees during the walking trials 60 times per second . for both the ap and ml tilt data , the standard deviation of the tilt data during the middle half of the walking stage was calculated . two - sample student s t - tests were conducted to compare balance , handgrip strength , and lumbopelvic tilt between fragility fracture patients and controls . normality assumptions of the t - tests were investigated , and sensitivity analyses using nonparametric methods were conducted to confirm the results . prior to study participation , all subjects signed informed consent and agreed to participate in the study as approved by the the ohio state university s biomedical sciences institutional review board . the participants were recruited via fliers from three orthopedic outpatient clinics and the general community . the two groups evaluated were patients with a history of one or more fragility fractures and controls without a history of fragility fractures . a fragility fracture was defined as a fracture occurring from a fall from a standing height or less , without major trauma such as a motor vehicle accident . the fracture must have occurred after the patient was 50 years of age.30 further inclusion criteria required that all subjects in the study be 50 years old or greater , able to walk 40 feet unassisted , able to follow simple verbal and written directions , and have an absence of peripheral neuropathies or mobility disorders . each participant completed a written questionnaire about age , sex , weight , current ambulatory status , fragility fracture details , and activity limitations . included in the patient questionnaire was a section from the 36-item short form health survey ( sf-36 ) , which evaluates daily activity restrictions based on the subject s current state of health . participants were then asked to perform a series of functional assessment activities over the course of 20 minutes utilizing the clinical assessment tools in a prognostic case - control study . stability was evaluated using the nintendo wii video game platform and the wbb . in conjunction with the wbb , two mini - games ( torso twists and single leg stand ) from the wii fitness program were used to assess postural sway and stability in participants . all hardware and software is commercially available and was not altered from its purchased state . participants were asked to remove their shoes and stand on the wbb facing a member of the research team . for each trial , the researcher navigated the on - screen menu and selected the activity to be performed . in addition , the researcher also performed the activity at the same time as the participant to ensure that the correct movements and pace were maintained throughout the duration of the exercise . as shown in figure 1 , the researcher was able to see the wii instructions on the tv screen , but the screen was blocked from the participant s view . the first activity selected from the wii fitness program was the torso twist activity , which requires patients to raise their arms parallel to the ground and rotate it 90 to one side , then to the other , alternating sides for a total of three twists to each side . at the end of the trial , the maximum horizontal and vertical scores were 50 for each , and the maximum total score was 100 , which was a sum of the horizontal and vertical components . the vertical score correlated to the amount of shift in the anterior posterior ( ap ) direction , while the horizontal score correlated to shift in the medial the second activity selected from the wii fitness program was the single leg stand activity which measures balance as the participant performs a series of movements while standing on one leg . the recommended motion from the wii fitness program was simplified by only asking patients to stand with your hands on your hips and raise your leg off the ground . participants were told that if they felt unstable , they could briefly grab the safety bars that were placed on either side of them or tap their raised foot down , but to reset to the one - leg stand position as soon as possible . the trial lasted 15 seconds on each leg . at the end of each trial , again , the maximum right and left scores were 50 for each , and the maximum total score was 100 , which was a sum of the right and left components . as with the torso twist activity , higher scores indicated decreased postural sway throughout the duration of the task . in addition , hand and foot faults were measured as a secondary internal measure of balance and sway . participants were asked to hold their forearm parallel to the ground with their elbow flexed at 90 and pointed laterally . participants were asked to grip as hard as they can for 3 seconds , then release . encouragement was provided to each participant throughout the task and they were told when to release . the apparatus consists of an elastic belt to which an ipod touch is attached by velcro ( velcro usa , manchester , nh , usa ) . the belt is placed across both of the participant s posterior superior iliac spines with the ipod touch centered in the middle of the back . the application uses an accelerometer - based sensor to measure ap and ml tilt as the participant walks . the participant is instructed to walk normally for 40 m to the end of the hall and stop . the data obtained during the middle half of the walking stage were used in the study in order to capture the normal gait phase of walking and not acceleration or deceleration phases of a walk . stability was evaluated using the nintendo wii video game platform and the wbb . in conjunction with the wbb , two mini - games ( torso twists and single leg stand ) from the wii fitness program were used to assess postural sway and stability in participants . all hardware and software is commercially available and was not altered from its purchased state . participants were asked to remove their shoes and stand on the wbb facing a member of the research team . for each trial , the researcher navigated the on - screen menu and selected the activity to be performed . in addition , the researcher also performed the activity at the same time as the participant to ensure that the correct movements and pace were maintained throughout the duration of the exercise . as shown in figure 1 , the researcher was able to see the wii instructions on the tv screen , but the screen was blocked from the participant s view . the first activity selected from the wii fitness program was the torso twist activity , which requires patients to raise their arms parallel to the ground and rotate it 90 to one side , then to the other , alternating sides for a total of three twists to each side . at the end of the trial , the maximum horizontal and vertical scores were 50 for each , and the maximum total score was 100 , which was a sum of the horizontal and vertical components . the vertical score correlated to the amount of shift in the anterior posterior ( ap ) direction , while the horizontal score correlated to shift in the medial the second activity selected from the wii fitness program was the single leg stand activity which measures balance as the participant performs a series of movements while standing on one leg . the recommended motion from the wii fitness program was simplified by only asking patients to stand with your hands on your hips and raise your leg off the ground . participants were told that if they felt unstable , they could briefly grab the safety bars that were placed on either side of them or tap their raised foot down , but to reset to the one - leg stand position as soon as possible . the trial lasted 15 seconds on each leg . at the end of each trial , again , the maximum right and left scores were 50 for each , and the maximum total score was 100 , which was a sum of the right and left components . as with the torso twist activity , higher scores indicated decreased postural sway throughout the duration of the task . in addition , hand and foot faults were measured as a secondary internal measure of balance and sway . participants were asked to hold their forearm parallel to the ground with their elbow flexed at 90 and pointed laterally . participants were asked to grip as hard as they can for 3 seconds , then release . encouragement was provided to each participant throughout the task and they were told when to release . the apparatus consists of an elastic belt to which an ipod touch is attached by velcro ( velcro usa , manchester , nh , usa ) . the belt is placed across both of the participant s posterior superior iliac spines with the ipod touch centered in the middle of the back . the application uses an accelerometer - based sensor to measure ap and ml tilt as the participant walks . the participant is instructed to walk normally for 40 m to the end of the hall and stop . the screen displayed during the walking trial is shown in figure 2 . the data obtained during the middle half of the walking stage were used in the study in order to capture the normal gait phase of walking and not acceleration or deceleration phases of a walk . torso twist and single leg stand balance scores reported by the wbb as well as faults during the single leg stand were averaged over the three trials . for handgrip strength , the best score out of three trials for each hand was used for statistical analysis . the ipod touch level belt records the ap and ml tilt in degrees during the walking trials 60 times per second . for both the ap and ml tilt data , the standard deviation of the tilt data during the middle half of the walking stage was calculated . two - sample student s t - tests were conducted to compare balance , handgrip strength , and lumbopelvic tilt between fragility fracture patients and controls . normality assumptions of the t - tests were investigated , and sensitivity analyses using nonparametric methods were conducted to confirm the results . the participants comprised 20 older adults with a history of fragility fracture and 21 older adults with no history of fragility fracture . the distribution of fragility fracture sites in the fracture group is summarized in table 2 . no significant differences were observed in sex , age , or weight when comparing fracture patients to control patients . the participants average activity level was significantly lower in the fracture group when compared to the control group ( p=0.026 ) . results from the two - sample t - tests showed that participants with a history of fragility fracture had lower scores on the vertical component of the wbb torso twist task ( p=0.042 ) and increased ml sway during a 40 m walk ( p=0.026 ) when compared to controls . fracture patients also had higher left leg and total balance scores as well as less faults on the left leg than control patients during the single leg stand task on the wbb ( p=0.020 , 0.010 , 0.003 , respectively ) . there was no statistical difference in horizontal or total balance score during the wbb torso twist , right leg scores during the single leg stand task , right leg or total faults during the single leg stand task , dominant or nondominant handgrip strength , or ap sway during a 40 m walk when comparing the two groups . comparisons of wbb scores and ipod touch level belt results between the fracture patients and controls can be seen in figures 3 and 4 , respectively . the purpose of this study was to identify inexpensive , noninvasive , portable tools that can be used to assess potential deficits that may put elderly patients at risk for falls . current techniques used to measure gait and balance often require highly specialized , expensive , and cumbersome equipment . these factors make it difficult to identify balance deficits in elderly patients conveniently and efficiently.28 the torso twist evaluation on the wbb is able to assess a patient s real - time balance during a reaching exercise , very similar to situations that occur when actual falls take place . in addition , the ipod touch level belt is able to measure real - time sway during normal walking . handgrip dynamometry uses a simulation of gripping handrails and has been shown to be highly predictive of functional limitations and disability later in life.28,31 these clinical assessment tools are able to assess patients in situations very closely mimicking normal daily activities , thus allowing the results from this study to be highly clinically relevant . the portability and accessibility of these tools in a clinical orthopedic setting allow for patient data collection in convenient ways that a biomechanics laboratory can not provide . in addition , commonly used balance evaluations , such as the berg balance scale and the timed up and go test , do exist ; however , these evaluation tools are limited in their ability to detect subtle changes and often can not distinguish between fallers and nonfallers.22,29,32 in contrast , the clinical assessment tools used in this study were able to detect differences in postural sway between fragility fracture patients and controls . all of the clinical assessment tools were easily transported into nearly any small - sized exam room and could be set up within 10 minutes . this study took place at multiple locations and demonstrates the potential to use this technology as part of multi center studies . all tools were commercially available and unmodified from their factory state , allowing any clinical site to easily adopt and utilize our testing protocol . a previous pilot study conducted with nine advanced practice providers demonstrated that all nine participants could be trained in under an hour to effectively and confidently use the clinical assessment tools to evaluate patients for fall risk in a clinical setting.33 this study demonstrates that these clinical assessment tools identified are portable , affordable , and efficient to use in clinical settings . after sustaining an initial fragility fracture , patients are 23 times more likely to sustain a second future fracture than the general population.34,35 thus , being proactive in regard to evaluating patients for fall risk and initiating fall prevention measures at earlier ages is crucial in this high - risk population . clinical assessment tools provide opportunities for both fall risk assessment and patient - specific rehabilitation . for example , these tools can be used to identify a patient s specific weaknesses in stability and strength , from which an individualized rehabilitation plan can be developed to address each patient s specific functional needs . clinical assessment tools can continue to be involved in the rehabilitation process , as well in the assessment of a patient s recovery process . in our analysis of participant demographics , the daily activity level of the fracture patients was significantly decreased when compared to controls . given that fragility fractures have been shown to limit patients ability to complete activities of daily living , this finding seemed logical.4,5 coinciding with our initial hypothesis , the clinical assessment tools were able to detect both increased ml sway and lower scores on the wbb torso twist task in fracture patients when compared to controls . while dominant and nondominant handgrip strength for fracture patients were approximately 3 kg lower than the controls , the differences were not statistically significant ( p=0.334 , 0.268 ) . unexpectedly , the fracture patients had significantly higher scores on the left leg and total components of the wbb single leg stand task than the controls , indicating decreased postural sway . the right leg component for the fracture patients , although not significant , was still higher than the scores for the controls ( p=0.085 ) . paralleling these findings , the fracture patients recorded significantly less faults during the left leg task and less total faults when compared to the controls ( p=0.003 , 0.085 ) . one possible explanation for this difference is that the fracture patients concentrate harder on staying balanced because they are more concerned about falling than the control patients . healthy individuals may also naturally have a more random pattern of sway as a strategy to adapt to changes in the environment , and individuals with injuries lose this compensatory mechanism and demonstrate decreased sway during balance tasks . a similar phenomenon was also reported in a study measuring postural coordination in athletes who have sustained an anterior cruciate ligament injury.36 interestingly , when looking at a more functional dynamic task of gait compared to single leg balance , these same postural sway deficits actually reversed and fragility fracture patients had decreased postural sway control ( increased ml sway ) . gait requires a different level of concentration with a controlled perturbation in ground contact with each step . this is compelling evidence that variability of movement during different tasks may reveal functional deficits in fragility fracture patients that may put them at risk for future fracture and falls . the fragility fracture population has the potential for baseline balance strength and stability deficits that put them at risk for future falls . this study revealed that this population differs from healthy control subjects in functions measured by the clinical assessment tools , and these tools identified some deficits associated with a high risk for falling . while the risk factors identified in this pathologic population were not their baseline measures associated with falling risk , these tests are promising to be used in future longitudinal study to predict the risk for future falls . additional information about the subjects such as socioeconomic status , prefracture activity levels , and motivation levels could improve risk stratification in our fragility fracture group . however , one of the main goals of this study was to identify tools that could assess fragility fracture patients in a clinical setting both quickly and efficiently . in addition , portable technology inherently has some limitations , such as the possible need for calibration with each use and the simplification of internal equipment . both the wii and handheld dynamometer tests are well validated in the literature.10,14,23,28,37 the ipod touch level belt has also been validated ; however , it is relatively a newer technology and more validation studies are emerging each year related to the technology.24 the analysis for the ipod touch level belt was performed by the techniques detailed by the inventors , and this emerging technology is being used in high level sport and rehabilitation settings.24 similar accelerometer - based testing using the ipod touch has demonstrated high validity and reliability during gait and posture testing in adults of all ages.25 the differences in handgrip strength may not have reached significance due to the smaller sample size , and future work will investigate this further . the fragility fracture population has the potential for baseline balance strength and stability deficits that put them at risk for future falls . this study revealed that this population differs from healthy control subjects in functions measured by the clinical assessment tools , and these tools identified some deficits associated with a high risk for falling . while the risk factors identified in this pathologic population were not their baseline measures associated with falling risk , these tests are promising to be used in future longitudinal study to predict the risk for future falls . additional information about the subjects such as socioeconomic status , prefracture activity levels , and motivation levels could improve risk stratification in our fragility fracture group . however , one of the main goals of this study was to identify tools that could assess fragility fracture patients in a clinical setting both quickly and efficiently . in addition , portable technology inherently has some limitations , such as the possible need for calibration with each use and the simplification of internal equipment . both the wii and handheld dynamometer tests are well validated in the literature.10,14,23,28,37 the ipod touch level belt has also been validated ; however , it is relatively a newer technology and more validation studies are emerging each year related to the technology.24 the analysis for the ipod touch level belt was performed by the techniques detailed by the inventors , and this emerging technology is being used in high level sport and rehabilitation settings.24 similar accelerometer - based testing using the ipod touch has demonstrated high validity and reliability during gait and posture testing in adults of all ages.25 the differences in handgrip strength may not have reached significance due to the smaller sample size , and future work will investigate this further . the clinical assessment tools utilized in this study are relatively cheap , portable , and were able to detect differences in postural sway between fragility fracture patients and controls . these tools can provide real - time , objective functional data inexpensively , efficiently , and in nearly any clinical setting . in the orthopedic trauma setting , fractures in the elderly population lead to profound morbidity , mortality , and social and financial consequences for the patients and the orthopedic community . utilization of clinical assessment tools to identify patients at risk for falls and future fractures may help clinicians develop targeted intervention strategies to help prevent future falls and fragility fractures . prevention of even a small percentage of fragility fractures could have substantial medical and economic benefits for society .

purposeto identify inexpensive , noninvasive , portable , clinical assessment tools that can be used to assess functional performance measures that may put older patients at risk for falls such as balance , handgrip strength , and lumbopelvic control.patients and methodstwenty fragility fracture patients and 21 healthy control subjects were evaluated using clinical assessment tools ( nintendo wii balance board [ wbb ] , a handheld dynamometer , and an application for the apple ipod touch , the level belt ) that measure functional performance during activity of daily living tasks . the main outcome measurements were balance ( wbb ) , handgrip strength ( handheld dynamometer ) , and lumbopelvic control ( ipod touch level belt ) , which were compared between fragility fracture patients and healthy controls.resultsfragility fracture patients had lower scores on the vertical component of the wbb torso twist task ( p=0.042 ) and greater medial lateral lumbopelvic sway during a 40 m walk ( p=0.026 ) when compared to healthy controls . unexpectedly , the fracture patients had significantly higher scores on the left leg ( p=0.020 ) and total components ( p=0.010 ) of the wbb single leg stand task as well as less faults during the left single leg stand task ( p=0.003).conclusionthe clinical assessment tools utilized in this study are relatively inexpensive and portable tools of performance measures capable of detecting differences in postural sway between fragility fracture patients and controls .

Introduction Patients and methods Subjects Procedure Stability Handgrip strength Lumbopelvic control Statistical analysis Results Discussion Study limitations Conclusion

the impact of these injuries will also continue to be an economic burden , with the cost for fall - related injuries projected to exceed us$30 billion by 2020.3 fragility fractures often leave patients in great emotional distress and unable to perform activities of daily living.4,5 the impact of these fractures is dramatic enough that a survey of older women indicated that 80% of them preferred death to a hip fracture that would result in admission to a nursing home.5 mortality for patients with fragility fractures is also significantly increased compared to expected mortality rates for this demographic population.6 a multicenter canadian study found that hip fracture patients had a 4.2 times greater risk of death during the first year after fracture than would be expected for hip fracture patients.7 mortality 5 years after hip or vertebral fracture is also ~20% greater than would be expected for elderly individuals without fracture.4 identifying functional deficits that may put elderly patients at risk for falls is crucial to help prevent the morbidity and mortality that can occur after a fragility fracture . impaired balance , lumbopelvic control , and handgrip strength all have shown strong correlations with fall risk.813 rehabilitation studies demonstrate that both improvements in postural sway and increases in strength measures are possible in geriatric patients after a fragility fracture.1416 in addition , multiple systematic reviews have shown that targeted interventions , mainly exercise and balance exercises , can reduce fall risk in elderly patients , thus preventing future complications.1720 because of the high morbidity and mortality associated with falls in the elderly , the american and british geriatric societies recommend that all the elderly individuals who present for medical attention because of a fall , report recurrent falls in the past year , or report difficulties in walking or balance should have a multifactorial fall risk assessment performed to determine their future fall risk.21 this assessment should include a detailed evaluation of gait , balance , and lower extremity joint function . given the great morbidity and mortality complications with fall - related injuries in the elderly , along with the potential for functional improvement after treatment and rehabilitation , there is a need to develop inexpensive , objective , and portable technologies to help evaluate people for fall risk quickly and accurately . as a result , the orthopedic surgery field faces new socioeconomic pressures to develop performance - based criteria and evidence - based clinical assessment tools to evaluate outcomes of treatment for musculoskeletal injuries . three clinical assessment tools were identified and utilized to assess stability ( wii balance board [ wbb ] , ipod touch level belt ) , postural sway ( wii balance board , ipod touch level belt ) , and handgrip strength ( saehan handheld dynamometer ) . inclusion criteria required that each tool be commercially available , easily portable , reasonably inexpensive , and operational with little training or expertise . recent studies have already demonstrated that the wbb has the ability to detect subtle changes in balance and center of pressure with good test retest reliability when compared to the gold standard , a research - grade force platform.22,23 the ipod touch level belt ( perfect practice , columbus , oh , usa ) was selected to evaluate a patient s lumbopelvic control . the ipod touch level belt is an ipod touch application available in the apple itunes store and has been used to measure postural sway in baseball players during functional tasks.24 the built - in accelerometer of the ipod touch ( apple , cupertino , ca , usa ) has already been shown to reliably and validly assess gait and posture in the elderly population.25 accelerometry has been used in other studies to measure trunk stability in the elderly population and has been suggested as a possible screening tool for future fall risk.26 the handheld dynamometer was selected to evaluate a patient s handgrip strength . handheld dynamometers have been used to measure handgrip in many studies to provide quick and accurate strength quantification.9,16,2729 in addition , decreased isometric handgrip strength has been identified as a risk factor for future falls in a 1-year prospective study.10 the goal of this study was to identify inexpensive , noninvasive , portable tools that can be used to assess potential deficits including balance , handgrip strength , and lumbopelvic control that may put elderly patients at risk for falls . we hypothesize that our tools will be able to quickly and easily identify balance , strength , and stability deficits in fragility fracture patients in a variety of clinical settings . each participant completed a written questionnaire about age , sex , weight , current ambulatory status , fragility fracture details , and activity limitations . included in the patient questionnaire was a section from the 36-item short form health survey ( sf-36 ) , which evaluates daily activity restrictions based on the subject s current state of health . participants were then asked to perform a series of functional assessment activities over the course of 20 minutes utilizing the clinical assessment tools in a prognostic case - control study . stability was evaluated using the nintendo wii video game platform and the wbb . in conjunction with the wbb , two mini - games ( torso twists and single leg stand ) from the wii fitness program were used to assess postural sway and stability in participants . participants were asked to remove their shoes and stand on the wbb facing a member of the research team . as shown in figure 1 , the researcher was able to see the wii instructions on the tv screen , but the screen was blocked from the participant s view . the first activity selected from the wii fitness program was the torso twist activity , which requires patients to raise their arms parallel to the ground and rotate it 90 to one side , then to the other , alternating sides for a total of three twists to each side . at the end of the trial , the maximum horizontal and vertical scores were 50 for each , and the maximum total score was 100 , which was a sum of the horizontal and vertical components . the vertical score correlated to the amount of shift in the anterior posterior ( ap ) direction , while the horizontal score correlated to shift in the medial lateral ( ml ) direction . at the end of each trial , again , the maximum right and left scores were 50 for each , and the maximum total score was 100 , which was a sum of the right and left components . as with the torso twist activity , higher scores indicated decreased postural sway throughout the duration of the task . torso twist and single leg stand balance scores reported by the wbb as well as faults during the single leg stand were averaged over the three trials . for handgrip strength , the best score out of three trials for each hand was used for statistical analysis . the ipod touch level belt records the ap and ml tilt in degrees during the walking trials 60 times per second . for both the ap and ml tilt data , the standard deviation of the tilt data during the middle half of the walking stage was calculated . two - sample student s t - tests were conducted to compare balance , handgrip strength , and lumbopelvic tilt between fragility fracture patients and controls . normality assumptions of the t - tests were investigated , and sensitivity analyses using nonparametric methods were conducted to confirm the results . a fragility fracture was defined as a fracture occurring from a fall from a standing height or less , without major trauma such as a motor vehicle accident . included in the patient questionnaire was a section from the 36-item short form health survey ( sf-36 ) , which evaluates daily activity restrictions based on the subject s current state of health . stability was evaluated using the nintendo wii video game platform and the wbb . in conjunction with the wbb , two mini - games ( torso twists and single leg stand ) from the wii fitness program were used to assess postural sway and stability in participants . participants were asked to remove their shoes and stand on the wbb facing a member of the research team . as shown in figure 1 , the researcher was able to see the wii instructions on the tv screen , but the screen was blocked from the participant s view . the first activity selected from the wii fitness program was the torso twist activity , which requires patients to raise their arms parallel to the ground and rotate it 90 to one side , then to the other , alternating sides for a total of three twists to each side . at the end of the trial , the maximum horizontal and vertical scores were 50 for each , and the maximum total score was 100 , which was a sum of the horizontal and vertical components . the vertical score correlated to the amount of shift in the anterior posterior ( ap ) direction , while the horizontal score correlated to shift in the medial the second activity selected from the wii fitness program was the single leg stand activity which measures balance as the participant performs a series of movements while standing on one leg . at the end of each trial , again , the maximum right and left scores were 50 for each , and the maximum total score was 100 , which was a sum of the right and left components . as with the torso twist activity , higher scores indicated decreased postural sway throughout the duration of the task . stability was evaluated using the nintendo wii video game platform and the wbb . in conjunction with the wbb , two mini - games ( torso twists and single leg stand ) from the wii fitness program were used to assess postural sway and stability in participants . participants were asked to remove their shoes and stand on the wbb facing a member of the research team . at the end of the trial , the maximum horizontal and vertical scores were 50 for each , and the maximum total score was 100 , which was a sum of the horizontal and vertical components . the vertical score correlated to the amount of shift in the anterior posterior ( ap ) direction , while the horizontal score correlated to shift in the medial the second activity selected from the wii fitness program was the single leg stand activity which measures balance as the participant performs a series of movements while standing on one leg . at the end of each trial , again , the maximum right and left scores were 50 for each , and the maximum total score was 100 , which was a sum of the right and left components . as with the torso twist activity , higher scores indicated decreased postural sway throughout the duration of the task . the participant is instructed to walk normally for 40 m to the end of the hall and stop . the data obtained during the middle half of the walking stage were used in the study in order to capture the normal gait phase of walking and not acceleration or deceleration phases of a walk . torso twist and single leg stand balance scores reported by the wbb as well as faults during the single leg stand were averaged over the three trials . for handgrip strength , the best score out of three trials for each hand was used for statistical analysis . the ipod touch level belt records the ap and ml tilt in degrees during the walking trials 60 times per second . for both the ap and ml tilt data , the standard deviation of the tilt data during the middle half of the walking stage was calculated . two - sample student s t - tests were conducted to compare balance , handgrip strength , and lumbopelvic tilt between fragility fracture patients and controls . the participants comprised 20 older adults with a history of fragility fracture and 21 older adults with no history of fragility fracture . the participants average activity level was significantly lower in the fracture group when compared to the control group ( p=0.026 ) . results from the two - sample t - tests showed that participants with a history of fragility fracture had lower scores on the vertical component of the wbb torso twist task ( p=0.042 ) and increased ml sway during a 40 m walk ( p=0.026 ) when compared to controls . fracture patients also had higher left leg and total balance scores as well as less faults on the left leg than control patients during the single leg stand task on the wbb ( p=0.020 , 0.010 , 0.003 , respectively ) . there was no statistical difference in horizontal or total balance score during the wbb torso twist , right leg scores during the single leg stand task , right leg or total faults during the single leg stand task , dominant or nondominant handgrip strength , or ap sway during a 40 m walk when comparing the two groups . comparisons of wbb scores and ipod touch level belt results between the fracture patients and controls can be seen in figures 3 and 4 , respectively . the purpose of this study was to identify inexpensive , noninvasive , portable tools that can be used to assess potential deficits that may put elderly patients at risk for falls . current techniques used to measure gait and balance often require highly specialized , expensive , and cumbersome equipment . these factors make it difficult to identify balance deficits in elderly patients conveniently and efficiently.28 the torso twist evaluation on the wbb is able to assess a patient s real - time balance during a reaching exercise , very similar to situations that occur when actual falls take place . in addition , the ipod touch level belt is able to measure real - time sway during normal walking . handgrip dynamometry uses a simulation of gripping handrails and has been shown to be highly predictive of functional limitations and disability later in life.28,31 these clinical assessment tools are able to assess patients in situations very closely mimicking normal daily activities , thus allowing the results from this study to be highly clinically relevant . in addition , commonly used balance evaluations , such as the berg balance scale and the timed up and go test , do exist ; however , these evaluation tools are limited in their ability to detect subtle changes and often can not distinguish between fallers and nonfallers.22,29,32 in contrast , the clinical assessment tools used in this study were able to detect differences in postural sway between fragility fracture patients and controls . all of the clinical assessment tools were easily transported into nearly any small - sized exam room and could be set up within 10 minutes . a previous pilot study conducted with nine advanced practice providers demonstrated that all nine participants could be trained in under an hour to effectively and confidently use the clinical assessment tools to evaluate patients for fall risk in a clinical setting.33 this study demonstrates that these clinical assessment tools identified are portable , affordable , and efficient to use in clinical settings . for example , these tools can be used to identify a patient s specific weaknesses in stability and strength , from which an individualized rehabilitation plan can be developed to address each patient s specific functional needs . clinical assessment tools can continue to be involved in the rehabilitation process , as well in the assessment of a patient s recovery process . in our analysis of participant demographics , the daily activity level of the fracture patients was significantly decreased when compared to controls . given that fragility fractures have been shown to limit patients ability to complete activities of daily living , this finding seemed logical.4,5 coinciding with our initial hypothesis , the clinical assessment tools were able to detect both increased ml sway and lower scores on the wbb torso twist task in fracture patients when compared to controls . while dominant and nondominant handgrip strength for fracture patients were approximately 3 kg lower than the controls , the differences were not statistically significant ( p=0.334 , 0.268 ) . unexpectedly , the fracture patients had significantly higher scores on the left leg and total components of the wbb single leg stand task than the controls , indicating decreased postural sway . the right leg component for the fracture patients , although not significant , was still higher than the scores for the controls ( p=0.085 ) . paralleling these findings , the fracture patients recorded significantly less faults during the left leg task and less total faults when compared to the controls ( p=0.003 , 0.085 ) . one possible explanation for this difference is that the fracture patients concentrate harder on staying balanced because they are more concerned about falling than the control patients . a similar phenomenon was also reported in a study measuring postural coordination in athletes who have sustained an anterior cruciate ligament injury.36 interestingly , when looking at a more functional dynamic task of gait compared to single leg balance , these same postural sway deficits actually reversed and fragility fracture patients had decreased postural sway control ( increased ml sway ) . this is compelling evidence that variability of movement during different tasks may reveal functional deficits in fragility fracture patients that may put them at risk for future fracture and falls . the fragility fracture population has the potential for baseline balance strength and stability deficits that put them at risk for future falls . this study revealed that this population differs from healthy control subjects in functions measured by the clinical assessment tools , and these tools identified some deficits associated with a high risk for falling . while the risk factors identified in this pathologic population were not their baseline measures associated with falling risk , these tests are promising to be used in future longitudinal study to predict the risk for future falls . additional information about the subjects such as socioeconomic status , prefracture activity levels , and motivation levels could improve risk stratification in our fragility fracture group . however , one of the main goals of this study was to identify tools that could assess fragility fracture patients in a clinical setting both quickly and efficiently . both the wii and handheld dynamometer tests are well validated in the literature.10,14,23,28,37 the ipod touch level belt has also been validated ; however , it is relatively a newer technology and more validation studies are emerging each year related to the technology.24 the analysis for the ipod touch level belt was performed by the techniques detailed by the inventors , and this emerging technology is being used in high level sport and rehabilitation settings.24 similar accelerometer - based testing using the ipod touch has demonstrated high validity and reliability during gait and posture testing in adults of all ages.25 the differences in handgrip strength may not have reached significance due to the smaller sample size , and future work will investigate this further . the fragility fracture population has the potential for baseline balance strength and stability deficits that put them at risk for future falls . this study revealed that this population differs from healthy control subjects in functions measured by the clinical assessment tools , and these tools identified some deficits associated with a high risk for falling . while the risk factors identified in this pathologic population were not their baseline measures associated with falling risk , these tests are promising to be used in future longitudinal study to predict the risk for future falls . additional information about the subjects such as socioeconomic status , prefracture activity levels , and motivation levels could improve risk stratification in our fragility fracture group . however , one of the main goals of this study was to identify tools that could assess fragility fracture patients in a clinical setting both quickly and efficiently . in addition , portable technology inherently has some limitations , such as the possible need for calibration with each use and the simplification of internal equipment . both the wii and handheld dynamometer tests are well validated in the literature.10,14,23,28,37 the ipod touch level belt has also been validated ; however , it is relatively a newer technology and more validation studies are emerging each year related to the technology.24 the analysis for the ipod touch level belt was performed by the techniques detailed by the inventors , and this emerging technology is being used in high level sport and rehabilitation settings.24 similar accelerometer - based testing using the ipod touch has demonstrated high validity and reliability during gait and posture testing in adults of all ages.25 the differences in handgrip strength may not have reached significance due to the smaller sample size , and future work will investigate this further . the clinical assessment tools utilized in this study are relatively cheap , portable , and were able to detect differences in postural sway between fragility fracture patients and controls . in the orthopedic trauma setting , fractures in the elderly population lead to profound morbidity , mortality , and social and financial consequences for the patients and the orthopedic community . utilization of clinical assessment tools to identify patients at risk for falls and future fractures may help clinicians develop targeted intervention strategies to help prevent future falls and fragility fractures .

hypertension ( htn ) prevalence in the normal pediatric and adolescent population ranges from 3.2% to 4.7% ( 1 - 4 ) . childhood htn has been linked to target - organ damage in young adults ( 5 ) . htn is often asymptomatic in both children and adolescents ; when persistent and long - standing , it could be a significant risk factor for kidney damage and increased glomerular permeability ( 6 , 7 ) . proteinuria and/or urinary red blood cells ( rbcs ) are important urinalysis manifestations of kidney damage and increased glomerular permeability . such evidence of bilateral kidney damage is an indication of chronic kidney disease if protracted for three months and may progress to end - stage renal disease ( esrd ) if not recognized and treated early ( 6 ) . renal replacement therapy , the standard treatment for esrd , is rarely available and affordable for many patients in developing countries ( 8 - 11 ) . it was postulated that hypertension - related kidney damage is caused by glomerular ischemia and hypoperfusion secondary to progressive narrowing of preglomerular arteries and arterioles ( 12 ) . animal models of htn showed that glomerular capillary htn and hyperperfusion are the principal factors causing glomerular damage and progressive loss of kidney function ( 13 ) . as a compensatory mechanism , direct transmission of high systemic blood pressure ( bp ) to the glomerular capillary network is physiologically blocked by an increase in afferent arteriolar resistance ( 14 ) . the failure of this mechanism leads to increased glomerular capillary pressure with resultant high glomerular filtration rate , increased transglomerular passage of proteins , and mesangial influx of proteins and other macromolecules . the ultimate renal histopathology is glomerular sclerosis secondary to the activation of mesangial cells and upregulation of proinflammatory cytokines and growth factors ( 14 ) . this study prospectively determined the burden of hypertension and its impact on glomerular permeability in randomly recruited primary school children . this study was conducted between june 3 and november 15 , 2013 , among primary - school pupils , in ile - ife , state of osun , nigeria . the study location is semiurban and has two local government areas ( lgas ) , namely ife central ( population , n = 167,254 ) and ife east ( population , n = 188,087 ) . these lgas cover land - mass areas of 111 km and 172 km , respectively . the study was approved by the ethics and research committee of our institution and the local inspectorates of education in the two lgas where it was carried out . the study conformed to the helsinki declaration of 1975 on research involving human subjects , as revised in 2000 . a multistage random sampling technique was employed to select 12 schools from a list of schools in both lgas . six primary schools , consisting of three private and three government - owned schools , were selected from each lga . the school register was consulted for enrollment figures by age and gender in each of the selected schools . a composite register was drawn for subjects aged 6 - 14 years whose parent(s)/guardian(s ) had consented . the subjects were categorized according to their classes ; a table of random numbers was used to select the pupils . the research protocol was administered on each pupil for biodata and anthropometric and clinical information . a general physical examination was carried out on each pupil before the midday break ( 1100 hours ) in a classroom . following the recommendation of the task force on high bp in children and adolescents ( 7 ) , resting bp was determined by the auscultation method in the right arm after a 10-minute resting period using the mercury gravity sphygmomanometer with bladder cuff sizes 17.0 - 19.0 cm ( length ) by 7.5 - 9.0 cm ( width ) . onset of the first tapping sound ( korotkoff sound 1 ) indicated the systolic bp ( sbp ) , whereas the point of complete disappearance of the sound ( korotkoff sound 5 ) indicated the diastolic bp ( dbp ) . for each pupil , the average of two measurements was taken after an initial bp trial run to allay fear and anxiety . pupils with high bp had their bps checked monthly for three months in their schools ; those with persistently elevated bp were thereafter referred to our pediatric nephrology clinic for further assessment and treatment . htn was defined as sbp and/or dbp 95th percentile for age , gender , and height on three different occasions ( 7 ) . sbp and/or dbp 95th but not > 99th percentile by > 5 mmhg was regarded as stage i htn , whereas stage ii htn was defined as sbp and/or dbp > 99th percentile by more than 5 mmhg for age , gender , and height ( 7 ) . bp percentile charts based on age , gender , and height developed by the task force on the diagnosis , evaluation , and treatment of high bp in children and adolescents ( 7 ) was used to determine whether a pupil was hypertensive ; the same chart was used for staging of hypertension . weight was measured using the seca weighing machine ( seca gmbh & co.kg , germany ) . height was measured with a leicester height stadiometer ( marsden weighing machine group , uk ) mounted on a vertical wall with the pupil barefoot and with head held in the frankfort plane . body mass index ( bmi ) was calculated as weight divided by the height squared ( kg / m ) . the international obesity task force cutoff points of > 25 kg / m and > 30 kg / m for overweight and obesity , respectively , were used ( 15 ) , whereas underweight and normal weight were defined using the bmi - for - age profiles developed by the national center for health statistics for boys and girls aged 2 - 20 years ( 16 ) . the presence of protein and/or red blood cells ( rbcs ) in the urine was assessed using the uriscreen combi 10 dipstick ( yercon diagnostic co. , ltd . freshly voided urine samples were collected into plane universal bottles prior to the pupils midday break . the test strip was dipped into the fresh urine for approximately 1 sec and then drawn across the edge of the container to remove excess urine . after 30 seconds , the test strip was compared with the color scale , and the result was immediately recorded . urine protein level was assessed as negative , trace , 1 + ( 30 mg / dl ) , 2 + ( 100 mg / dl ) , and 3 + ( 500 mg / dl ) . in this study , significant proteinuria by dipstick testing was defined as urinary protein 30 mg / dl ( 1 + ) . the pupils urine was examined monthly for significant proteinuria and/or rbcs for three months . according to the national kidney foundation - kidney / disease outcome quality initiative ( 6 ) , persistent significant proteinuria and/or urinary rbcs determined by the dipstick test for three months or more statistical analysis was performed using spss 16 for windows , evaluation version ( 2006 spss inc . ) . the comparative statistics used were student s t - test , and pearson s correlation ( r ) . this study was conducted between june 3 and november 15 , 2013 , among primary - school pupils , in ile - ife , state of osun , nigeria . the study location is semiurban and has two local government areas ( lgas ) , namely ife central ( population , n = 167,254 ) and ife east ( population , n = 188,087 ) . these lgas cover land - mass areas of 111 km and 172 km , respectively . the study was approved by the ethics and research committee of our institution and the local inspectorates of education in the two lgas where it was carried out . the study conformed to the helsinki declaration of 1975 on research involving human subjects , as revised in 2000 . a multistage random sampling technique was employed to select 12 schools from a list of schools in both lgas . six primary schools , consisting of three private and three government - owned schools , were selected from each lga . the school register was consulted for enrollment figures by age and gender in each of the selected schools . a composite register was drawn for subjects aged 6 - 14 years whose parent(s)/guardian(s ) had consented . the subjects were categorized according to their classes ; a table of random numbers was used to select the pupils . the research protocol was administered on each pupil for biodata and anthropometric and clinical information . a general physical examination was carried out on each pupil before the midday break ( 1100 hours ) in a classroom . following the recommendation of the task force on high bp in children and adolescents ( 7 ) , resting bp was determined by the auscultation method in the right arm after a 10-minute resting period using the mercury gravity sphygmomanometer with bladder cuff sizes 17.0 - 19.0 cm ( length ) by 7.5 - 9.0 cm ( width ) . onset of the first tapping sound ( korotkoff sound 1 ) indicated the systolic bp ( sbp ) , whereas the point of complete disappearance of the sound ( korotkoff sound 5 ) indicated the diastolic bp ( dbp ) . for each pupil , the average of two measurements was taken after an initial bp trial run to allay fear and anxiety . pupils with high bp had their bps checked monthly for three months in their schools ; those with persistently elevated bp were thereafter referred to our pediatric nephrology clinic for further assessment and treatment . htn was defined as sbp and/or dbp 95th percentile for age , gender , and height on three different occasions ( 7 ) . sbp and/or dbp 95th but not > 99th percentile by > 5 mmhg was regarded as stage i htn , whereas stage ii htn was defined as sbp and/or dbp > 99th percentile by more than 5 mmhg for age , gender , and height ( 7 ) . bp percentile charts based on age , gender , and height developed by the task force on the diagnosis , evaluation , and treatment of high bp in children and adolescents ( 7 ) was used to determine whether a pupil was hypertensive ; the same chart was used for staging of hypertension . weight was measured using the seca weighing machine ( seca gmbh & co.kg , germany ) . height was measured with a leicester height stadiometer ( marsden weighing machine group , uk ) mounted on a vertical wall with the pupil barefoot and with head held in the frankfort plane . body mass index ( bmi ) was calculated as weight divided by the height squared ( kg / m ) . the international obesity task force cutoff points of > 25 kg / m and > 30 kg / m for overweight and obesity , respectively , were used ( 15 ) , whereas underweight and normal weight were defined using the bmi - for - age profiles developed by the national center for health statistics for boys and girls aged 2 - 20 years ( 16 ) . the presence of protein and/or red blood cells ( rbcs ) in the urine was assessed using the uriscreen combi 10 dipstick ( yercon diagnostic co. , ltd . freshly voided urine samples were collected into plane universal bottles prior to the pupils midday break . the test strip was dipped into the fresh urine for approximately 1 sec and then drawn across the edge of the container to remove excess urine . after 30 seconds , the test strip was compared with the color scale , and the result was immediately recorded . urine protein level was assessed as negative , trace , 1 + ( 30 mg / dl ) , 2 + ( 100 mg / dl ) , and 3 + ( 500 mg / dl ) . in this study , significant proteinuria by dipstick testing was defined as urinary protein 30 mg / dl ( 1 + ) . a dipstick value ca . 5 - 10 erythrocytes/l was taken as significant hematuria . like their bp , the pupils urine was examined monthly for significant proteinuria and/or rbcs for three months . according to the national kidney foundation - kidney / disease outcome quality initiative ( 6 ) , persistent significant proteinuria and/or urinary rbcs determined by the dipstick test for three months or more is regarded as evidence of abnormal glomerular permeability . statistical analysis was performed using spss 16 for windows , evaluation version ( 2006 spss inc . ) . the comparative statistics used were student s t - test , and pearson s correlation ( r ) . other data were presented as numbers and percentages . a p value < 0.05 was regarded as statistically significant . the study population included more females ( n = 687 ; 51.5% ) than males ( n = 648 ; 48.5% ) . of 1,335 ( 1.9% ) pupils , 25 had prehypertension , and 33 ( 2.5% ) pupils aged 6 - 14 ( 10.0 2.4 ) years were hypertensive ; the majority of them were females ( 29/33 ; 88.0% ) , resulting in a male - to - female ratio of 1:7 . the mean sbp / dbp in the hypertensive pupils was 125.6 6.5/81.7 3.3 mmhg ( range : 114.0 - 140.0/80.0 - 90.0 ; > 95th to > 99th/ > 95th to 99th percentile ) . there was a weak but significant correlation between sbp ( r = + 0.334 , p = 0.01 ) , dbp ( r = + 0.278 , p = 0.01 ) , and age . gender comparisons of mean sbp and dbp by age group in normotensive and hypertensive school pupils are respectively , summarized in figures 1 and 2 . table 1 shows the number of pupils per htn stage by age group and gender , and table 2 summarizes the pattern and stages of htn in the pupils . the number in each column represents the sample size for both systolic and diastolic blood pressure . * p = 0.157 , * * p = 0.218 , p = 0.545 , p = 0.292 , # p = 0.000 , and # # p = 0.000 . the letters m and f denote male and female gender , respectively . the number in each column represents the sample size for both systolic and diastolic blood pressure . * p = 0.596 , * * p = 0.104 , p = 0.122 , p = 0.242 , # the overall prevalence of overweight and obesity among the 1,335 pupils was 0.4% and 1.4% , respectively , but none of the hypertensive pupils was obese . htn was , however , detected in 25 ( 75.8% ) normal weight and seven ( 21.2% ) underweight pupils and in one ( 3.0% ) overweight pupil . the mean heights for the hypertensive male and female subjects were 132.4 6.91 cm and 138.80 10.44 cm , respectively ( p = 0.04 ) , and the mean bmis were 16.12 2.11 kg / m and 15.91 1.76 kg / m , respectively ( p = 0.72 ) . overall , sbp ( r = + 0.213 ; p = 0.01 ) and dbp ( r = + 0.148 ; p = 0.01 ) correlated weakly with bmi . sbp ( r = + 0.434 ; p = 0.01 ) and dbp ( r = + 0.389 ; p = 0.01 ) , however , correlated fairly well with height . analyses for gender difference showed poor correlations between sbp , dbp , mean arterial pressure ( map ) , and bmi as well as height ( table 3 ) . urinalysis by dipstick revealed no evidence of abnormal glomerular permeability , because none of the hypertensive pupils had significant proteinuria and/or urinary rbcs . trace proteinuria , however , was found in three of the 33 ( 9.1% ) hypertensive pupils . the 2.5% prevalence of htn among school children in this study , determined based on the fourth task - force criterion ( 7 ) , is similar to the 2.2% prevalence reported among swiss school children by chiolero et al . ( 17 ) but is 1.3 times lower than the 3.2% prevalence reported four years earlier by adegoke et al . the disparity is probably due to the higher upper - age limit of 18 ( range : 6 - 18 ) years in the earlier study compared to 14 ( range : 6 - 14 ) years in the current study . in both studies , htn was diagnosed mostly for the first time in adolescent pupils , thus underlining the need for early screening of school children for hypertension , because target - organ damage in adulthood had been traced to childhood htn ( 5 , 18 ) . isolated htn was more frequent ( 72.7% ) than combined systolic and diastolic htn ( 27.3% ) in this study . furthermore , isolated systolic htn ( shtn ) prevalence ( 42.4% ) was higher than isolated diastolic htn ( dhtn ; 30.3% ) , confirming earlier reports that isolated htn is not rare ( 19 , 20 ) . ( 19 ) ( shtn , 4.4% vs. dhtn , 3.2% ) and sorof et al . ( 20 ) ( shtn , 47% vs. dhtn , 17% ) revealed that isolated shtn is more common than isolated dhtn . the pathophysiology of isolated htn in children is not yet clear ; however , studies have shown that isolated shtn is more frequently linked with end - organ damage than is isolated dhtn ( 21 , 22 ) . as shown in figure 1 , the mean bp of the normotensive school children tended to increase with increasing age , especially as the children approached pubertal age . this is similar to findings in earlier studies ( 1 , 4 , 23 - 26 ) . this pattern , which can be imputed to normal physiological hormonal changes that attend puberty and the increase of peripheral arterial resistance and cardiac output with age ( 27 ) , was not replicated in the hypertensive children . in the latter , bp rise was haphazard and failed to correlate with rising age ( figure 2 ) . the mean hypertension age was 10 years , and the majority of the hypertensive pupils had stage i htn ( 60.0% ) . whether they had stage i or ii htn , the female children tended to be more hypertensive . this may also , in part , be due to earlier puberty onset in females than in males . however , the very high prevalence of stage ii htn ( 40.0% ) in this study indicates the possibility of a secondary etiology for hypertension in a good number of the pupils . in one study , 35% - 50% of hypertensive adolescents were obese , with a positive correlation established between obesity and htn as early as 5 years of age . the study argued that obesity causes and sustains childhood essential hypertension ( 28 ) . in another study , htn prevalence was found to increase progressively with increasing bmi , and approximately 30% of overweight school children had htn ( bmi > 95th percentile ) ( 29 ) . although these findings agree with those from port harcourt ( 18 ) , a highly westernized cosmopolitan city in nigeria where the prevalence of overweight and that of obesity among school children were 5.7% and 5.9% , respectively , they are at variance with the findings from this study . the prevalence of overweight and that of obesity were 0.4% and 1.4% , respectively , but none of the hypertensive pupils was obese ; bmi correlated poorly and inversely with bp in the hypertensive pupils in this study . in fact , the majority of the hypertensive pupils had either normal or low bmi . this is consistent with a us study that revealed high bp at low bmi among black children , indicating a weaker effect of bmi on bp levels in blacks ( 19 ) . it is not clear why the majority of the hypertensive pupils in this study had either normal or low bmi . perhaps an investigation of the etiology of htn would have made the reasons for this obvious . environmental factors , sleep disorders , low birth weight / reduced nephron number , positive family history of htn / cardiovascular disease , and genetic disorders are a few of the factors that have been associated with htn ( 30 ) . some congenital anomalies of the kidneys and urinary tract and even renal artery stenosis and endocrine disorders could have been missed in the children , because the study was not designed to establish the etiology of htn . although all of these may constitute limitations of this study , they point to future avenues of research on htn in school children . future studies should , therefore , focus not only on htn prevalence but also on htn etiology so that management plans can be more robust and definitive . evidence for abnormal glomerular permeability could not be established in this study , because none of the hypertensive pupils , including those with stage ii htn , had significant proteinuria and/or urinary rbcs . this may indicate that htn was not long - standing at the time of the study or that the method used was not sensitive enough to detect abnormal glomerular permeability in htn . ( 31 ) showed that young adults with stage i htn and hyperfiltration developed microalbuminuria three times more often than normally filtrating patients after eight years of follow - up , supporting the argument that the impact of increased bp on renal structures depends not only on high bp levels , but also on how long the bp has been elevated ( 14 ) . trace proteinuria , a risk factor for esrd ( 32 ) , was found in 9.1% of the hypertensive pupils . the pupils with trace proteinuria who were regarded as normal in this study may have had microalbuminuria , but the convention of regarding trace proteinuria as negative has the tendency to exclude a large number of people with microalbuminuria ( 33 ) . trace proteinuria may serve as an important marker of microalbuminuria in both the general population and those at high risk of cardiovascular disease ( 33 ) . the dipstick test of + 1 that is considered positive evidence of proteinuria has been reported as an unsuitable test in the general population because of its low sensitivity for urinary abnormalities ( 34 , 35 ) . trace proteinuria should therefore be an indication for exclusion of microalbuminuria in the hypertensive person . the dipstick test showing trace proteinuria may therefore be a useful tool for that purpose ( 36 - 38 ) . the burden of htn was 2.5% , with high bp commonly found at normal bmi . young females were more frequently hypertensive than male school children , indicating the need for routine bp checks in school - aged female children . abnormal glomerular permeability was rare in the young hypertensive school children , suggesting either recent hypertension or an insufficiently sensitive evaluation method . testing for microalbuminuria and a urine microscopy examination for rbcs and other urine sediments might be more informative and predictive with respect to abnormal glomerular permeability in hypertension . the burden of htn was 2.5% , with high bp commonly found at normal bmi . young females were more frequently hypertensive than male school children , indicating the need for routine bp checks in school - aged female children . abnormal glomerular permeability was rare in the young hypertensive school children , suggesting either recent hypertension or an insufficiently sensitive evaluation method . testing for microalbuminuria and a urine microscopy examination for rbcs and other urine sediments might be more informative and predictive with respect to abnormal glomerular permeability in hypertension .

backgroundchildhood hypertension has been associated with target - organ damage in young adults . it is often asymptomatic in both children and adolescents ; when persistent , and long - standing , it could be a significant risk factor for kidney damage and increased glomerular permeability.objectivesburden of hypertension and its impact on glomerular permeability were prospectively determined in randomly recruited primary school children.patients and methodsblood pressure ( bp ) measurement was performed by the auscultation method , and abnormal glomerular permeability was assessed by dipstick testing of urine for persistent proteinuria and/or hematuria for three months in hypertensive children.resultsof 1,335 pupils aged 10.0 2.4 ( 6.0 - 14.0 ) years , 33 ( 2.5% ) were hypertensive . overall mean systolic / diastolic bp was 125.6 6.5/81.7 3.3 ( range : 114.0 - 140.0/80.0 - 90.0 ) mmhg . nine ( 27.3% ) had combined systolic and diastolic hypertension , 126.7 5.7/80.0 - 80.0 0.0 ( 120.0 - 130.0/80.0 - 80.0 ) mmhg . isolated systolic hypertension , 125.4 6.7 ( 114.0 - 140.0 ) mmhg , was present in 14 ( 42.4% ) , whereas 10 ( 30.3% ) had isolated diastolic hypertension , 82.0 3.5 ( 80.0 - 90.0 ) mmhg . mean systolic and diastolic bp were 131.0 3.3 ( 130.0 - 140.0 ) mmhg and 86.5 4.43 ( 80.0 - 90.0 ) mmhg , respectively . according to the dipstick test , none of the hypertensive pupils showed urinalysis evidence of proteinuria and/or hematuria after three months of testing.conclusionsalthough the burden of hypertension was 2.5% , the dipstick method did not detect any hypertension - related abnormal glomerular permeability in the school children .

1. Background 2. Objectives 3. Patients and Methods 3.1. Study Area and Population 3.2. Ethics 3.3. Sampling Technique and Research Protocol 3.4. Blood Pressure Measurement 3.5. Anthropometric Measurement 3.6. Urinalysis 3.7. Data Analysis 4. Results 5. Discussion 5.1. Conclusion

hypertension ( htn ) prevalence in the normal pediatric and adolescent population ranges from 3.2% to 4.7% ( 1 - 4 ) . childhood htn has been linked to target - organ damage in young adults ( 5 ) . htn is often asymptomatic in both children and adolescents ; when persistent and long - standing , it could be a significant risk factor for kidney damage and increased glomerular permeability ( 6 , 7 ) . proteinuria and/or urinary red blood cells ( rbcs ) are important urinalysis manifestations of kidney damage and increased glomerular permeability . such evidence of bilateral kidney damage is an indication of chronic kidney disease if protracted for three months and may progress to end - stage renal disease ( esrd ) if not recognized and treated early ( 6 ) . renal replacement therapy , the standard treatment for esrd , is rarely available and affordable for many patients in developing countries ( 8 - 11 ) . it was postulated that hypertension - related kidney damage is caused by glomerular ischemia and hypoperfusion secondary to progressive narrowing of preglomerular arteries and arterioles ( 12 ) . animal models of htn showed that glomerular capillary htn and hyperperfusion are the principal factors causing glomerular damage and progressive loss of kidney function ( 13 ) . as a compensatory mechanism , direct transmission of high systemic blood pressure ( bp ) to the glomerular capillary network is physiologically blocked by an increase in afferent arteriolar resistance ( 14 ) . the failure of this mechanism leads to increased glomerular capillary pressure with resultant high glomerular filtration rate , increased transglomerular passage of proteins , and mesangial influx of proteins and other macromolecules . the ultimate renal histopathology is glomerular sclerosis secondary to the activation of mesangial cells and upregulation of proinflammatory cytokines and growth factors ( 14 ) . this study prospectively determined the burden of hypertension and its impact on glomerular permeability in randomly recruited primary school children . these lgas cover land - mass areas of 111 km and 172 km , respectively . the study was approved by the ethics and research committee of our institution and the local inspectorates of education in the two lgas where it was carried out . the study conformed to the helsinki declaration of 1975 on research involving human subjects , as revised in 2000 . a multistage random sampling technique was employed to select 12 schools from a list of schools in both lgas . the school register was consulted for enrollment figures by age and gender in each of the selected schools . a composite register was drawn for subjects aged 6 - 14 years whose parent(s)/guardian(s ) had consented . following the recommendation of the task force on high bp in children and adolescents ( 7 ) , resting bp was determined by the auscultation method in the right arm after a 10-minute resting period using the mercury gravity sphygmomanometer with bladder cuff sizes 17.0 - 19.0 cm ( length ) by 7.5 - 9.0 cm ( width ) . onset of the first tapping sound ( korotkoff sound 1 ) indicated the systolic bp ( sbp ) , whereas the point of complete disappearance of the sound ( korotkoff sound 5 ) indicated the diastolic bp ( dbp ) . for each pupil , the average of two measurements was taken after an initial bp trial run to allay fear and anxiety . pupils with high bp had their bps checked monthly for three months in their schools ; those with persistently elevated bp were thereafter referred to our pediatric nephrology clinic for further assessment and treatment . sbp and/or dbp 95th but not > 99th percentile by > 5 mmhg was regarded as stage i htn , whereas stage ii htn was defined as sbp and/or dbp > 99th percentile by more than 5 mmhg for age , gender , and height ( 7 ) . bp percentile charts based on age , gender , and height developed by the task force on the diagnosis , evaluation , and treatment of high bp in children and adolescents ( 7 ) was used to determine whether a pupil was hypertensive ; the same chart was used for staging of hypertension . height was measured with a leicester height stadiometer ( marsden weighing machine group , uk ) mounted on a vertical wall with the pupil barefoot and with head held in the frankfort plane . body mass index ( bmi ) was calculated as weight divided by the height squared ( kg / m ) . the international obesity task force cutoff points of > 25 kg / m and > 30 kg / m for overweight and obesity , respectively , were used ( 15 ) , whereas underweight and normal weight were defined using the bmi - for - age profiles developed by the national center for health statistics for boys and girls aged 2 - 20 years ( 16 ) . the presence of protein and/or red blood cells ( rbcs ) in the urine was assessed using the uriscreen combi 10 dipstick ( yercon diagnostic co. , ltd . freshly voided urine samples were collected into plane universal bottles prior to the pupils midday break . the test strip was dipped into the fresh urine for approximately 1 sec and then drawn across the edge of the container to remove excess urine . after 30 seconds , the test strip was compared with the color scale , and the result was immediately recorded . urine protein level was assessed as negative , trace , 1 + ( 30 mg / dl ) , 2 + ( 100 mg / dl ) , and 3 + ( 500 mg / dl ) . in this study , significant proteinuria by dipstick testing was defined as urinary protein 30 mg / dl ( 1 + ) . the pupils urine was examined monthly for significant proteinuria and/or rbcs for three months . according to the national kidney foundation - kidney / disease outcome quality initiative ( 6 ) , persistent significant proteinuria and/or urinary rbcs determined by the dipstick test for three months or more statistical analysis was performed using spss 16 for windows , evaluation version ( 2006 spss inc . ) the comparative statistics used were student s t - test , and pearson s correlation ( r ) . the study location is semiurban and has two local government areas ( lgas ) , namely ife central ( population , n = 167,254 ) and ife east ( population , n = 188,087 ) . the study was approved by the ethics and research committee of our institution and the local inspectorates of education in the two lgas where it was carried out . the study conformed to the helsinki declaration of 1975 on research involving human subjects , as revised in 2000 . a multistage random sampling technique was employed to select 12 schools from a list of schools in both lgas . the school register was consulted for enrollment figures by age and gender in each of the selected schools . a composite register was drawn for subjects aged 6 - 14 years whose parent(s)/guardian(s ) had consented . the subjects were categorized according to their classes ; a table of random numbers was used to select the pupils . following the recommendation of the task force on high bp in children and adolescents ( 7 ) , resting bp was determined by the auscultation method in the right arm after a 10-minute resting period using the mercury gravity sphygmomanometer with bladder cuff sizes 17.0 - 19.0 cm ( length ) by 7.5 - 9.0 cm ( width ) . onset of the first tapping sound ( korotkoff sound 1 ) indicated the systolic bp ( sbp ) , whereas the point of complete disappearance of the sound ( korotkoff sound 5 ) indicated the diastolic bp ( dbp ) . pupils with high bp had their bps checked monthly for three months in their schools ; those with persistently elevated bp were thereafter referred to our pediatric nephrology clinic for further assessment and treatment . htn was defined as sbp and/or dbp 95th percentile for age , gender , and height on three different occasions ( 7 ) . sbp and/or dbp 95th but not > 99th percentile by > 5 mmhg was regarded as stage i htn , whereas stage ii htn was defined as sbp and/or dbp > 99th percentile by more than 5 mmhg for age , gender , and height ( 7 ) . bp percentile charts based on age , gender , and height developed by the task force on the diagnosis , evaluation , and treatment of high bp in children and adolescents ( 7 ) was used to determine whether a pupil was hypertensive ; the same chart was used for staging of hypertension . height was measured with a leicester height stadiometer ( marsden weighing machine group , uk ) mounted on a vertical wall with the pupil barefoot and with head held in the frankfort plane . body mass index ( bmi ) was calculated as weight divided by the height squared ( kg / m ) . the international obesity task force cutoff points of > 25 kg / m and > 30 kg / m for overweight and obesity , respectively , were used ( 15 ) , whereas underweight and normal weight were defined using the bmi - for - age profiles developed by the national center for health statistics for boys and girls aged 2 - 20 years ( 16 ) . the presence of protein and/or red blood cells ( rbcs ) in the urine was assessed using the uriscreen combi 10 dipstick ( yercon diagnostic co. , ltd . the test strip was dipped into the fresh urine for approximately 1 sec and then drawn across the edge of the container to remove excess urine . after 30 seconds , the test strip was compared with the color scale , and the result was immediately recorded . urine protein level was assessed as negative , trace , 1 + ( 30 mg / dl ) , 2 + ( 100 mg / dl ) , and 3 + ( 500 mg / dl ) . in this study , significant proteinuria by dipstick testing was defined as urinary protein 30 mg / dl ( 1 + ) . like their bp , the pupils urine was examined monthly for significant proteinuria and/or rbcs for three months . according to the national kidney foundation - kidney / disease outcome quality initiative ( 6 ) , persistent significant proteinuria and/or urinary rbcs determined by the dipstick test for three months or more is regarded as evidence of abnormal glomerular permeability . statistical analysis was performed using spss 16 for windows , evaluation version ( 2006 spss inc . ) the comparative statistics used were student s t - test , and pearson s correlation ( r ) . the study population included more females ( n = 687 ; 51.5% ) than males ( n = 648 ; 48.5% ) . of 1,335 ( 1.9% ) pupils , 25 had prehypertension , and 33 ( 2.5% ) pupils aged 6 - 14 ( 10.0 2.4 ) years were hypertensive ; the majority of them were females ( 29/33 ; 88.0% ) , resulting in a male - to - female ratio of 1:7 . the mean sbp / dbp in the hypertensive pupils was 125.6 6.5/81.7 3.3 mmhg ( range : 114.0 - 140.0/80.0 - 90.0 ; > 95th to > 99th/ > 95th to 99th percentile ) . there was a weak but significant correlation between sbp ( r = + 0.334 , p = 0.01 ) , dbp ( r = + 0.278 , p = 0.01 ) , and age . table 1 shows the number of pupils per htn stage by age group and gender , and table 2 summarizes the pattern and stages of htn in the pupils . the number in each column represents the sample size for both systolic and diastolic blood pressure . * p = 0.157 , * * p = 0.218 , p = 0.545 , p = 0.292 , # p = 0.000 , and # # p = 0.000 . the number in each column represents the sample size for both systolic and diastolic blood pressure . * p = 0.596 , * * p = 0.104 , p = 0.122 , p = 0.242 , # the overall prevalence of overweight and obesity among the 1,335 pupils was 0.4% and 1.4% , respectively , but none of the hypertensive pupils was obese . the mean heights for the hypertensive male and female subjects were 132.4 6.91 cm and 138.80 10.44 cm , respectively ( p = 0.04 ) , and the mean bmis were 16.12 2.11 kg / m and 15.91 1.76 kg / m , respectively ( p = 0.72 ) . sbp ( r = + 0.434 ; p = 0.01 ) and dbp ( r = + 0.389 ; p = 0.01 ) , however , correlated fairly well with height . analyses for gender difference showed poor correlations between sbp , dbp , mean arterial pressure ( map ) , and bmi as well as height ( table 3 ) . urinalysis by dipstick revealed no evidence of abnormal glomerular permeability , because none of the hypertensive pupils had significant proteinuria and/or urinary rbcs . trace proteinuria , however , was found in three of the 33 ( 9.1% ) hypertensive pupils . the 2.5% prevalence of htn among school children in this study , determined based on the fourth task - force criterion ( 7 ) , is similar to the 2.2% prevalence reported among swiss school children by chiolero et al . the disparity is probably due to the higher upper - age limit of 18 ( range : 6 - 18 ) years in the earlier study compared to 14 ( range : 6 - 14 ) years in the current study . in both studies , htn was diagnosed mostly for the first time in adolescent pupils , thus underlining the need for early screening of school children for hypertension , because target - organ damage in adulthood had been traced to childhood htn ( 5 , 18 ) . isolated htn was more frequent ( 72.7% ) than combined systolic and diastolic htn ( 27.3% ) in this study . furthermore , isolated systolic htn ( shtn ) prevalence ( 42.4% ) was higher than isolated diastolic htn ( dhtn ; 30.3% ) , confirming earlier reports that isolated htn is not rare ( 19 , 20 ) . the pathophysiology of isolated htn in children is not yet clear ; however , studies have shown that isolated shtn is more frequently linked with end - organ damage than is isolated dhtn ( 21 , 22 ) . as shown in figure 1 , the mean bp of the normotensive school children tended to increase with increasing age , especially as the children approached pubertal age . this pattern , which can be imputed to normal physiological hormonal changes that attend puberty and the increase of peripheral arterial resistance and cardiac output with age ( 27 ) , was not replicated in the hypertensive children . the mean hypertension age was 10 years , and the majority of the hypertensive pupils had stage i htn ( 60.0% ) . whether they had stage i or ii htn , the female children tended to be more hypertensive . however , the very high prevalence of stage ii htn ( 40.0% ) in this study indicates the possibility of a secondary etiology for hypertension in a good number of the pupils . in another study , htn prevalence was found to increase progressively with increasing bmi , and approximately 30% of overweight school children had htn ( bmi > 95th percentile ) ( 29 ) . although these findings agree with those from port harcourt ( 18 ) , a highly westernized cosmopolitan city in nigeria where the prevalence of overweight and that of obesity among school children were 5.7% and 5.9% , respectively , they are at variance with the findings from this study . the prevalence of overweight and that of obesity were 0.4% and 1.4% , respectively , but none of the hypertensive pupils was obese ; bmi correlated poorly and inversely with bp in the hypertensive pupils in this study . in fact , the majority of the hypertensive pupils had either normal or low bmi . it is not clear why the majority of the hypertensive pupils in this study had either normal or low bmi . environmental factors , sleep disorders , low birth weight / reduced nephron number , positive family history of htn / cardiovascular disease , and genetic disorders are a few of the factors that have been associated with htn ( 30 ) . some congenital anomalies of the kidneys and urinary tract and even renal artery stenosis and endocrine disorders could have been missed in the children , because the study was not designed to establish the etiology of htn . although all of these may constitute limitations of this study , they point to future avenues of research on htn in school children . evidence for abnormal glomerular permeability could not be established in this study , because none of the hypertensive pupils , including those with stage ii htn , had significant proteinuria and/or urinary rbcs . this may indicate that htn was not long - standing at the time of the study or that the method used was not sensitive enough to detect abnormal glomerular permeability in htn . ( 31 ) showed that young adults with stage i htn and hyperfiltration developed microalbuminuria three times more often than normally filtrating patients after eight years of follow - up , supporting the argument that the impact of increased bp on renal structures depends not only on high bp levels , but also on how long the bp has been elevated ( 14 ) . trace proteinuria , a risk factor for esrd ( 32 ) , was found in 9.1% of the hypertensive pupils . trace proteinuria may serve as an important marker of microalbuminuria in both the general population and those at high risk of cardiovascular disease ( 33 ) . the dipstick test of + 1 that is considered positive evidence of proteinuria has been reported as an unsuitable test in the general population because of its low sensitivity for urinary abnormalities ( 34 , 35 ) . trace proteinuria should therefore be an indication for exclusion of microalbuminuria in the hypertensive person . the dipstick test showing trace proteinuria may therefore be a useful tool for that purpose ( 36 - 38 ) . the burden of htn was 2.5% , with high bp commonly found at normal bmi . abnormal glomerular permeability was rare in the young hypertensive school children , suggesting either recent hypertension or an insufficiently sensitive evaluation method . testing for microalbuminuria and a urine microscopy examination for rbcs and other urine sediments might be more informative and predictive with respect to abnormal glomerular permeability in hypertension . the burden of htn was 2.5% , with high bp commonly found at normal bmi . abnormal glomerular permeability was rare in the young hypertensive school children , suggesting either recent hypertension or an insufficiently sensitive evaluation method . testing for microalbuminuria and a urine microscopy examination for rbcs and other urine sediments might be more informative and predictive with respect to abnormal glomerular permeability in hypertension .

contrast thresholds for detecting the presence of a sinewave grating with horizontal bars were determined using a two temporal - interval forced - choice paradigm combined with a two - down one - up staircase procedure that tracked performance at 71% accuracy . grating stimuli were generated using a vsg2/5 video card ( cambridge research systems , rochester , uk ) and presented on a sony 24 graphics display ( model # gdm - fw900 ; park ridge , nj , usa ) at a mean luminance of 75 cd / m . on each trial , a full - field grating with at least five cycles visible , was presented in one of the two temporal intervals that lasted 1 second each with abrupt onset and offset , with a 0.5 second separation between the two intervals . the two temporal intervals were denoted with a high- and low - pitch tone , respectively . observers indicated the temporal interval in which the grating was presented , guessing if necessary . the contrast of the grating decreased by one step following two consecutive trials with correct responses , and increased by one step following a single incorrect response . the step sizes were 0.2 log units for trials before the first two reversals of the staircase and 0.1 log units for subsequent trials . each staircase terminated after 10 reversals and only the thresholds at the last eight reversals were used for calculating the mean contrast threshold . each spatial frequency was tested twice , in two different sessions . between six and eight spatial frequencies the exact range of spatial frequencies was chosen for each eye such that there were at least two spatial frequencies left of the peak of the csf and two on the right . for these eyes , the contrast threshold reported for each spatial frequency represents the geometric mean of a total of 16 reversals from two staircases . to construct the normal template , we tested five normally - sighted observers using the same experimental paradigm with the following changes . first , contrast thresholds were measured for 10 spatial frequencies ranging from 0.4 to 25 c / deg , in steps of 0.2 log units . second , four staircases were tested for each spatial frequency and each observer , instead of two for low - vision eyes . thus , at each spatial frequency , the contrast threshold of the normal data represents the geometric mean of 160 staircase reversals ( 8 reversals per staircase 4 staircases per observer 5 observers ) . twenty observers with low vision ( 12 males and 8 females , mean age = 70.6 11.12 years ) and five observers with normal vision ( 2 males and 3 females , mean age = 25.8 9.9 years ) participated in this study . the five observers with normal vision all had best - corrected visual acuity ( bcva ) of at least 20/20 in each eye , normal binocular vision and ocular motility function , and no history of any eye diseases . all observers in the low vision group had bilateral vision loss , with a bcva of 0.14 logmar ( 20/25 snellen equivalent ) or worse in the better eye , with a confirmed diagnosis of an eye disease that led to reduced acuity . the diagnoses included amd , stargardt disease , glaucoma and optic neuropathy ; all of which have been reported to lead to acuity and contrast deficits , and are common etiologies of patients attending low - vision clinics . these observers had a complete low vision evaluation within the 3 months prior to their participation in the study . all observers , normal or low vision , gave informed oral and written consent before the commencement of data collection . the research followed the tenets of the declaration of helsinki and was approved by the institutional review board at the first author 's institution . all observers were tested monocularly , with the nontested eye covered using a standard black - cloth eye - patch . visual characteristics of the 20 eyes with low vision we chose to use young adults , instead of age - matched older adults , for constructing the normal - vision template mainly for two reasons . most importantly , our hypothesis of a shift of the normal template to account for acuity and contrast deficits is not age - specific . second , because of our need to construct a high - quality normal template , it was more practical to test young , normally - sighted adults on the more rigorous and time - consuming csf data collection . although many functions have been used to describe normal csfs ( see watson and ahumada for a comparison of the different functions ) , we adopted an asymmetric parabolic function , which is simple to construct , and provides an excellent fit to our normal - vision data . the function , which requires four parameters , is given by the following equation : where f(sf ) is the contrast sensitivity at a spatial frequency sf , csp is the peak contrast sensitivity , sfp is the spatial frequency at which csp occurs , and widthl and widthr are the curvatures of the left and right branches of the asymmetric parabolic function , respectively . the values for contrast sensitivity and spatial frequency were log - transformed before curve - fitting . curve - fitting was performed using igor pro ( wavemetrics , inc . , lake oswego , or , usa ) , which uses a levenberg - marquardt iterative algorithm ( a form of nonlinear least - squares fitting ) to minimize the error between the experimental and the model fit . to evaluate whether the shapes of the low - vision csfs were the same as that of the normal - vision csf , we first constructed a normal template by determining the four parameter values yielding the best - fit function to the normal - vision data using equation 1 . then we compared two fitting methods , free - fit versus template - fit , for each low - vision csf . free - fit was accomplished by fitting a low - vision csf using equation 1 and allowing all four parameters to vary . template - fit also made use of equation 1 but with the two parameters , widthl and widthr , constrained to the values returned for the best - fit function for the normal - vision data . the template fit allowed only variation in the peak contrast sensitivity csp and the spatial - frequency at the peak sfp . the effect of this restriction is to retain the shape of the csf while permitting only translation of the function horizontally and vertically along the log spatial frequency and log contrast sensitivity axes . model comparisons of the four- and two - parameter fits for the low - vision csfs were performed using the akaike information criterion ( aic ) , which takes into account the goodness - of - fit of each fitting method , given the number of free parameters used . according to akaike , the model with the smaller overall aic value is the model of choice . besides comparing the goodness - of - fit , we also compared several key parameters of the fitted functions derived from the two fitting methods , to determine how well these parameters correlate or agree with each other . the degree of agreement was examined using the bland - altman analysis . if the shape of low - vision csfs is the same as that for the normal - vision csf , then the normal template should fit the low - vision data just as well as the free - fit . the goodness - of - fit of the two fitting methods would be similar , and that the key parameters derived from the two methods would show a strong correlation and agreement with each other . twenty observers with low vision ( 12 males and 8 females , mean age = 70.6 11.12 years ) and five observers with normal vision ( 2 males and 3 females , mean age = 25.8 9.9 years ) participated in this study . the five observers with normal vision all had best - corrected visual acuity ( bcva ) of at least 20/20 in each eye , normal binocular vision and ocular motility function , and no history of any eye diseases . all observers in the low vision group had bilateral vision loss , with a bcva of 0.14 logmar ( 20/25 snellen equivalent ) or worse in the better eye , with a confirmed diagnosis of an eye disease that led to reduced acuity . the diagnoses included amd , stargardt disease , glaucoma and optic neuropathy ; all of which have been reported to lead to acuity and contrast deficits , and are common etiologies of patients attending low - vision clinics . these observers had a complete low vision evaluation within the 3 months prior to their participation in the study . all observers , normal or low vision , gave informed oral and written consent before the commencement of data collection . the research followed the tenets of the declaration of helsinki and was approved by the institutional review board at the first author 's institution . all observers were tested monocularly , with the nontested eye covered using a standard black - cloth eye - patch . visual characteristics of the 20 eyes with low vision we chose to use young adults , instead of age - matched older adults , for constructing the normal - vision template mainly for two reasons . most importantly , our hypothesis of a shift of the normal template to account for acuity and contrast deficits is not age - specific . second , because of our need to construct a high - quality normal template , it was more practical to test young , normally - sighted adults on the more rigorous and time - consuming csf data collection . although many functions have been used to describe normal csfs ( see watson and ahumada for a comparison of the different functions ) , we adopted an asymmetric parabolic function , which is simple to construct , and provides an excellent fit to our normal - vision data . the function , which requires four parameters , is given by the following equation : where f(sf ) is the contrast sensitivity at a spatial frequency sf , csp is the peak contrast sensitivity , sfp is the spatial frequency at which csp occurs , and widthl and widthr are the curvatures of the left and right branches of the asymmetric parabolic function , respectively . the values for contrast sensitivity and spatial frequency were log - transformed before curve - fitting . curve - fitting was performed using igor pro ( wavemetrics , inc . , lake oswego , or , usa ) , which uses a levenberg - marquardt iterative algorithm ( a form of nonlinear least - squares fitting ) to minimize the error between the experimental and the model fit . to evaluate whether the shapes of the low - vision csfs were the same as that of the normal - vision csf , we first constructed a normal template by determining the four parameter values yielding the best - fit function to the normal - vision data using equation 1 . then we compared two fitting methods , free - fit versus template - fit , for each low - vision csf . free - fit was accomplished by fitting a low - vision csf using equation 1 and allowing all four parameters to vary . template - fit also made use of equation 1 but with the two parameters , widthl and widthr , constrained to the values returned for the best - fit function for the normal - vision data . the template fit allowed only variation in the peak contrast sensitivity csp and the spatial - frequency at the peak sfp . the effect of this restriction is to retain the shape of the csf while permitting only translation of the function horizontally and vertically along the log spatial frequency and log contrast sensitivity axes . model comparisons of the four- and two - parameter fits for the low - vision csfs were performed using the akaike information criterion ( aic ) , which takes into account the goodness - of - fit of each fitting method , given the number of free parameters used . according to akaike , the model with the smaller overall aic value is the model of choice . besides comparing the goodness - of - fit , we also compared several key parameters of the fitted functions derived from the two fitting methods , to determine how well these parameters correlate or agree with each other . the degree of agreement was examined using the bland - altman analysis . if the shape of low - vision csfs is the same as that for the normal - vision csf , then the normal template should fit the low - vision data just as well as the free - fit . the goodness - of - fit of the two fitting methods would be similar , and that the key parameters derived from the two methods would show a strong correlation and agreement with each other . we will first present the csf data of the normal vision observers , to be used in constructing the normal template . then we will compare the fitting of the normal template and the free - fit to the csfs of the low vision observers . key parameters derived from the template fit and the free - fit will also be compared . figure 2 shows the contrast sensitivity versus spatial frequency data of our group of normal vision observers . each data point represents the contrast sensitivity , the reciprocal of the geometric mean of contrast thresholds across the five normal vision observers , with 32 staircase reversals for each observer ( eight reversals per staircase and four staircases per observer ) . error bars represent the sd of the mean sensitivity based on 1000 bootstrap resamplings . to derive the normal - vision csf , we fit the data set using the asymmetric parabolic function ( equation 1 ) . the best - fit function returns the following parameters : peak contrast sensitivity , csp = 2.22 log units ( corresponds to 166 ) ; spatial frequency at which peak contrast sensitivity occurs , sfp = 0.4 log c / deg ( corresponds to 2.5 c / deg ) ; widthl = 0.68 and widthr = 1.28 . 2 ) , with the width parameters constrained to the values of the best - fit curve , forms our normal template . contrast sensitivity is plotted as a function of spatial frequency ( c / deg ) for the five normal - vision control observers . each data point represents the sensitivity ( reciprocal of contrast threshold ) based on eight reversals per staircase , four staircases per observer , and five observers . these data are color - coded : red for those with central field loss and blue for those with intact central field . we categorized the low - vision observers in this way because the presence or absence of central field loss is often associated with substantial differences in reading and other real - world activities . each low - vision observer 's csf data were fit separately using the two fitting methods , the best - fit curve using the free - fit method is given in each panel in the same color as the symbols and the best - fit curve using the template - fit is given as the gray solid line . contrast sensitivity is plotted as a function of spatial frequency ( c / deg ) for the 20 eyes with low vision . data are color - coded : red for observers with central field loss and blue for observers with intact central field . each data point represents the sensitivity ( reciprocal of contrast threshold ) based on 16 reversals ( 8 reversals per staircase for 2 staircases ) . the best - fit curves according to the free - fit and template - fit are given as the color ( red or blue ) and gray solid curves , respectively . the normal - vision csf is also given in each panel as the gray dashed curve . we first evaluated which of the two fitting methods provided a better description of individual low - vision csfs . to do so , we compared the sum of the aic of the two fitting methods across all low - vision eyes , with aic given by where k is the number of free parameters ( 4 for free - fit and 2 for template - fit ) . using this method , the sum of aic is 231.2 for the free - fit method and 231.1 for the template - fit , implying that the two fitting methods provide equally good fits to the low - vision data . we examined the correlation and agreement of three key parameters derived from the two fitting methods , the peak contrast sensitivity ( csp ) , the spatial frequency at peak contrast sensitivity ( sfp ) , and the high cutoff spatial frequency . csp and sfp are parameters returned by the two fitting methods , while the high cutoff spatial frequency was derived from equation 1 , by setting f(sf ) to a value of 1 and solving for the corresponding spatial frequency . the top panels in figure 4 show how well each of these parameters derived from the two fitting methods correlate with each other . a t - test on the correlation coefficients ( r ranging from 0.880.99 ) shows that all of these correlations are statistically significant ( p < 0.0001 ) , indicating that the parameters from the two fitting methods correlate strongly with each other . because high correlation does not necessarily mean good numerical agreement , we also examined the data in bland - altman plots ( bottom panels in fig . 4 ) to determine how well each of these parameters derived from the two fitting methods agree with each other . we reason that if the 95% limits of agreement ( plotted as dashed lines in each of the bottom panels ) are comparable with the test retest reliability of similar measurements , then the two fitting methods are in good agreement with each other . the 95% limits of agreement for csp are 0.06 log units , which are smaller than the published data on test - retest reliability of contrast sensitivity measurement using the pelli - robson chart or the mars chart , with values ranging from 0.15 to 0.33 log units . for sfp , the 95% limits of agreement are 0.17 log units , but because we know of no published data on the test - retest reliability of sfp , or of similar measurement , we can not compare our measurements with previous data . the 95% limits of agreement for the high cutoff spatial frequency are 0.05 log units , smaller than the test - retest reliability of visual acuity measurement ( equivalent to the high cutoff spatial frequency ) , which ranges between 0.1 and 0.2 log units . thus , in general , the parameters derived from the two fitting methods are in good agreement . values derived from the free - fit and template - fit methods are compared for the peak contrast sensitivity ( csp ) , the spatial frequency that yields the peak contrast sensitivity ( sfp ) and the high cutoff spatial frequency . in the top panels , we examine how well these values from the two fitting methods correlate with each other . data are color - coded , and the black line in each panel represents the best - fit regression line . the gray dashed line in each panel represents the 1:1 line . in the bottom panels , we examine , in the form of bland - altman plots , the black solid and dashed lines in each panel represent the mean of the difference of log values between the two fitting methods and the 95% limits of agreement , respectively . note that the main results do not change if we consider only data for eyes with central field loss . the correlation coefficients for the values estimated for the two fitting methods remain unchanged ( 0.99 for csp , 0.88 for sfp , and 0.99 for the high cutoff spatial frequency ) , and remain statistically significant ( p < 0.0001 ) . in addition , bland - altman plots show that the values derived from the two fitting methods still agree well with each other . these additional analyses ensure that our conclusion that the free - fit method provides a good fit to the low - vision csf data is still valid if we consider only patients with central field loss . given our hypothesis of low - vision csfs having the same shape as the normal csf , it is important to compare the parameters that govern the shape of the fitted functions . these are the widthl and widthr parameters . in figure 5 , we represent the distributions of the widthl and widthr parameters for the low - vision csfs , derived from the free - fit method in box - and - whisker plots . a test for normality based on the shapiro - wilk test showed that the distribution for widthl does not follow a normal distribution ( w = 0.89 , p = 0.03 ) but the distribution for widthr is not different from a normal distribution ( w = 0.94 , p = 0.28 ) . therefore , we used the mann - whitney u test to evaluate whether these distributions , derived from the free - fit method , are different from the parameter values for the normal csf . while values for the parameter widthr do not differ between the free - fit method and the constrained value ( u = 200 , p = 1.0 ) , values for widthl are different between the free - fit method and the constrained value ( u = 100 , p = 0.004 ) . indeed , figure 3 shows that for several data - sets , the free - fit method yields a fitted curve with a shallower low - frequency decline than that returned by the template fit , especially for eyes c , d , f , g , and i. however , the means of the two distributions , represented by the red circles , are very close to the constrained values . box - and - whisker plots comparing the values of widthl and widthr derived from the free - fit method with the constrained values based on the normal csf . the upper and lower bound of each box represent the 75th and 25th percentiles of the distribution , and the median is represented by the thick line within the box . the top and bottom ends of the whisker represent the 95th and 5th percentiles of the distribution , respectively . note that the mean values are very similar to the parameter values for the normal template , shown as gray dashed lines . results from these analyses show that the free - fit method provides a description of the low - vision csf data that is very similar to the template - fit , which is derived from the normal - vision data . we acknowledge that some of the low - vision csfs are more low - pass in shape , resulting in the sample distribution of the parameter widthl being different from that of the constrained value , however , the mean of the sample distribution ( 0.64 ) is very similar to that of the constrained value ( 0.68 ) . considering that the template fit only has two free parameters , our results suggest that low - vision csfs can be related to a normal csf with two additional measurements . a practical question is whether letter - chart acuity , a clinical measurement performed on every clinical patient , can be one of the two additional measurements . here , we seek to determine if letter - chart acuity can predict some of the key parameters of the fitted csf functions . to do so , we examined if there exists a significant correlation between letter - chart acuity , measured using a bailey - lovie chart with acuity expressed in logmar , and each of the three key parameters : csp , sfp , and the high cutoff spatial frequency , as derived from the free - fit method . as shown in figure 6 , the correlation between csp and acuity is weak ( r = 0.07 ; p = 0.78 ) , but there is a high correlation between sfp and acuity ( r = 0.76 ; p < 0.0001 ) , and between the high cutoff spatial frequency and acuity ( r = 0.79 ; p < 0.0001 ) . in other words , letter - chart acuity can explain 58% and 62% of the variances in sfp and the high cutoff spatial frequency . the correlation between csp ( left ) , sfp ( middle ) , and the high cutoff sf ( right ) with letter - chart acuity . once again , when we consider only data for eyes with central field loss , the results remain practically the same . the correlation between csp and acuity remains weak and insignificant ( r = 0.05 ; p = 0.84 ) , while the correlations remain high between sfp and acuity ( r = 0.76 ; p = 0.0006 ) , and between the high cutoff spatial frequency and acuity ( r = 0.75 ; p = 0.0009 ) . the high cutoff spatial frequency of the csf represents the smallest detail that can be resolved , thus theoretically it should agree well with letter - chart acuity . the bland - altman plot in figure 7 shows how well the high cutoff spatial frequency derived from the free - fit method agrees with the letter - chart acuity . the high cutoff spatial frequencies were converted to their equivalent logmar acuities by assuming that individual bars of the sinewave grating are the spatial details to be resolved . as an example , the critical feature of a 20/20 letter ( logmar 0.0 ) subtends 1 arc min , and each light and dark bar of a 30 c / deg grating subtends 1 arc min . therefore , a cutoff spatial frequency of 30 c / deg is often taken to correspond to a letter acuity of 20/20 . this method essentially assumes that the letter frequency for identification is 2.5 c / letter , although the critical frequency for letter identification may vary depending on conditions such as letter size , retinal eccentricity , and so on . the 95% limits of agreement are 0.28 log units , larger than the test - retest reliability of visual acuity measurement of 0.1 to 0.2 log units . in addition , the mean acuity difference between the high cutoff spatial frequency and chart acuity is 0.15 logmar , which is statistically different from a null difference ( p = 0.0002 ) . these results show that in general , using the conversion that 20/20 letters correspond to 30 c / deg , the letter - chart yields a poorer acuity than the high cutoff spatial frequency . therefore , attempts to derive the full csf using the letter - chart acuity as one of the two measurements need to take into account this constant difference . letter - chart acuity and the high cutoff spatial frequency derived from the free - fit method ( converted to logmar acuity ) are compared in a bland - altman plot . the mean difference between the two methods is 0.15 logmar ( black solid line ) and is statistically different from 0 ( gray dotted line ) . contrast sensitivities and visual acuities are affected in many ocular diseases that lead to impaired vision , thus leading to many reports that the csfs of individuals with diseases such as macular disorders , glaucoma , and optic neuropathy are different from that of a pelli et al . was the first investigation to test the hypothesis that low - vision csfs have the same shape as that of a normal - vision csf . they measured the csfs of 32 low - vision observers and fit each set of log cs versus log sf data using a single parabola that was derived from four normally sighted observers . by constraining the shape of the parabolic template but allowing it to shift vertically and horizontally along the log cs and log sf axes , pelli et al . found that the parabolic template provided a good fit to all the low - vision csfs . subsequently , rohaly and owsley applied the method of pelli et al . and fit a parabolic function to the data of 100 older adults aged between 53 and 85 years , with an average acuity of 20/25 ; and found that the parabolic template did not provide a good fit for 20% of their observers . these authors also attempted to fit their data using an exponential function but again found that the function did not provide a good fit to their data . this led the authors to conclude that the csfs of older adults could not be described by a single parabolic or exponential function with two or three free parameters . differences in the observer characteristics ( low vision versus older adults with relatively good vision ) aside , there are several key methodologic differences between our study and that of rohaly and owsley . rohaly and owsley had their observers adjust the contrast of a grating stimulus until they just detected the grating ( method of adjustment ) . we used a two temporal interval forced - choice paradigm combined with a staircase procedure . it is well known that a criterion - free forced - choice method usually provides more reliable measurements of contrast threshold than the method of adjustment . this might account for why the shapes of the csfs for some of their observers were unusual , as shown in their paper . most noticeably , in several figures in their paper , contrast sensitivities were practically invariant for a large range of low- to high - spatial frequencies , and the contrast sensitivity at the highest - spatial frequency ( 22.5 c / deg ) was still much higher than expected ( based on published data ) . this unusual trend of contrast sensitivity data , and the lack of a clear fall - off of sensitivity at high - spatial frequencies make it difficult for any function with a high - frequency roll - off to provide a good fit . it was indeed because of this reason that we tested a range of spatial frequencies that included at least two frequencies lower than , and at least two frequencies higher than the peak of the csf . more recently , watson and ahumada compared 11 functions to describe a large set of csf data obtained from normally sighted observers using a variety of stimuli ( the modelfest data ) , and found that many of the functions fit the data similarly well . these authors cautioned the use of a template that is based on a set of average data in fitting an individual observer 's data . nevertheless , in our study , we found that a template based on the average data of a group of normally - sighted observers provides a reasonably good fit to individual data of 20 low - vision eyes . the close coupling between the low- and normal - vision csfs does not even require the normal - vision template to be derived from age - matched control observers . presumably , shifting the normal template to fit the low - vision data accounts for the visual deficits of the individual , regardless of whether the deficits are due to ocular diseases or normal aging . had we found that the shifted normal template did not provide a good fit to the low - vision data , the age difference between our young normal and low - vision groups would have been a possible factor accounting for the difference , and that the use of a normal template derived from normally sighted observers age - matched with those of the low - vision group would have been necessary . it is well known that the rates of fall - off of contrast sensitivity with spatial frequency differ for the left and right branches of the csf with respect to csp , thus in this study , we chose an asymmetric parabolic function to fit our data because it allows for different rates of fall - off ( or the width parameters in equation 1 ) for the left and right branches of the csf . an asymmetric parabolic function has four free parameters . by constraining two of these parameters that determine the shape ( width ) of the function , recently , pelli and bex suggested that a template with four free parameters , as in the many models evaluated by watson and ahumada , might provide a better fit to individual data . here , we showed that a model with only two free parameters might be sufficient , and in fact , the template fit with only two free parameters yielded an overall aic value that is virtually identical to that of the free - fit with four free parameters . a practical advantage of modeling csfs with a template that requires only two free parameters is that we only need two measurements in addition to the template to derive a full csf , thus offering an efficient method for the determination of the csf . if our concern is to obtain an estimate of the csf of a patient with low vision , then one of these two measurements could be the conventional acuity measurement , since acuity is measured for every patient in the clinic , and that letter chart acuity correlates strongly with both the spatial frequency at the peak of the csf and the high cutoff spatial frequency ( fig . there are many clinical tests of contrast sensitivity , such as the bailey - lovie low contrast acuity chart or regan charts , which measure acuity for letters at a fixed low contrast ; the pelli - robson chart or the mars chart , which provide a contrast sensitivity measurement close to the peak of the csf ; and other contrast sensitivity tests such as the melbourne edge test , and the berkeley discs contrast sensitivity test ( bailey il , et al . the differences in acuity and a clinical contrast sensitivity measurement between a low - vision individual and the norms could then be used to shift a normal csf horizontally along the log sf axis and vertically along the log cs axis . as an example , assume a low - vision individual had an acuity of 0.4 logmar and a contrast sensitivity as measured using the pelli - robson chart of 1.35 log units . now , let us further assume that the normal values for acuity and contrast sensitivity measurements using the same charts are 0.1 logmar and 2.1 log units ; then the differences in acuity and contrast sensitivity between the low - vision individual and normals become 0.5 and 0.75 log units , respectively . in this case , the csf of the low - vision individual can be approximated by shifting a normal csf template leftward along the log sf axis by 0.5 log units to account for the difference in acuity , and downward along the log cs axis by 0.75 log units to account for the difference in contrast sensitivity . whether or not our conjecture of using just two clinical measurements , visual acuity and contrast sensitivity , can yield a full csf closely resembles that measured using the gold standard ( using grating stimuli ) would need to be empirically validated in future studies . first , the specific shape of a csf is stimulus - dependent and may change with stimulus parameters such as luminance , retinal eccentricity , field size , duration , and the temporal characteristics of the gratings . for example , the shape of a csf changes from band - pass to low - pass with a reduction in the mean luminance of the gratings from photopic to scotopic level , an increase in retinal eccentricity , a briefer presentation of the grating stimulus or when there is an increased temporal modulation . the close coupling between the low- and normal - vision csfs observed in this study applies to the set of stimulus conditions used in this study . given that our set of stimulus parameters is fairly generic , it is likely that the close coupling is quite general and would apply to measurements made with other stimulus parameters nevertheless , future investigations may wish to establish their own normal template to ensure the best fitting of their low - vision data , especially if these investigations are going to use stimulus parameters drastically different from those used in this study . second , although our group of low - vision observers includes etiologies of ocular disease commonly seen in low - vision clinics , it did not include patients with cataracts or neurologic disorders such as multiple sclerosis . patients with cataracts whose disability is more due to glare may show a selective reduction in contrast sensitivity at low spatial frequencies . multiple sclerosis , although infrequently seen in low - vision clinics , has been shown to produce notches in the csf in the midfrequencies , which could make it difficult for any function to provide a good description of the csf . it remains to be verified whether or not a normal template , ours or others , can be used to fit the csf of these conditions , or other conditions that lead to the shape of the csf being different from that of the normal template . an efficient method of determining a full csf is useful for individuals with normal vision , but it is especially useful for low vision as the peak contrast sensitivity is a good predictor of daily activities such as mobility , face recognition , and reading . in the laboratory , full low - vision csfs can be used as filters to recreate visual stimuli that simulate what individuals with low vision can detect . this may be a useful tool for informal evaluation of the impact of vision impairment , especially acuity loss and reduced contrast sensitivity , on visibility of real - world objects and scenes . in the clinic , filtering visual scenes using the csf of a low - vision patient may help family members understand what the patient can see or why there are difficulties with certain tasks . it should be recognized , however , that using the csf as a linear filter has significant limitations in simulating low - vision visibility . more sophisticated models of feature visibility , such as that of peli , may include multiple spatial - frequency channels and the ability to represent local luminance - dependent variations in contrast sensitivity across the visual field . in this study , we have learned that the contrast sensitivity functions ( csfs ) of many low - vision subjects have the same shape as the csfs of normally - sighted subjects , but differ from the normal csfs in two values , the spatial frequency and contrast sensitivity at the peak of the curve . this means that by coupling our knowledge of the characteristic csf curves of normally - sighted subjects with two readily available clinical measures from a patient with low vision , we can infer a rich and informative description of the patient 's pattern vision .

purposethe contrast sensitivity function ( csf ) provides a detailed description of an individual 's spatial - pattern detection capability . we tested the hypothesis that the csfs of people with low vision differ from a normal csf only in their horizontal and vertical positions along the spatial frequency ( sf ) and contrast sensitivity ( cs ) axes.methodscontrast sensitivity for detecting horizontal sinewave gratings was measured with a two temporal - interval forced - choice staircase procedure , for a range of sfs spanning 5 to 6 octaves , for 20 low - vision observers and five adults with normal vision . an asymmetric parabolic function was used to fit the aggregate data of the normal - vision observers , yielding the normal template . each of the 20 low - vision csfs was fit in two ways , by using a shape - invariant version of the normal template ( with the width parameters fixed ) that was shifted along the log - sf and log - cs axes , and by an unconstrained asymmetric parabolic function ( free - fit).resultsthe two fitting methods yielded values of the peak cs , the sf corresponding to peak cs , and the high cut - off sf that were highly correlated and in good agreement with each other . in addition , the width parameters of the low - vision csfs were comparable with those of the normal template , implying that low - vision csfs are similar in shape to the normal csf.conclusionsthe excellent agreement of parameters estimated by the two fitting methods suggests that low - vision csfs can be approximated by a normal csf shifted along the log - sf and log - cs axes to account for the impaired acuity and contrast sensitivity .

Methods Observers Curve-Fitting and Statistical Analyses Results Discussion

contrast thresholds for detecting the presence of a sinewave grating with horizontal bars were determined using a two temporal - interval forced - choice paradigm combined with a two - down one - up staircase procedure that tracked performance at 71% accuracy . to construct the normal template , we tested five normally - sighted observers using the same experimental paradigm with the following changes . twenty observers with low vision ( 12 males and 8 females , mean age = 70.6 11.12 years ) and five observers with normal vision ( 2 males and 3 females , mean age = 25.8 9.9 years ) participated in this study . the diagnoses included amd , stargardt disease , glaucoma and optic neuropathy ; all of which have been reported to lead to acuity and contrast deficits , and are common etiologies of patients attending low - vision clinics . visual characteristics of the 20 eyes with low vision we chose to use young adults , instead of age - matched older adults , for constructing the normal - vision template mainly for two reasons . most importantly , our hypothesis of a shift of the normal template to account for acuity and contrast deficits is not age - specific . although many functions have been used to describe normal csfs ( see watson and ahumada for a comparison of the different functions ) , we adopted an asymmetric parabolic function , which is simple to construct , and provides an excellent fit to our normal - vision data . the function , which requires four parameters , is given by the following equation : where f(sf ) is the contrast sensitivity at a spatial frequency sf , csp is the peak contrast sensitivity , sfp is the spatial frequency at which csp occurs , and widthl and widthr are the curvatures of the left and right branches of the asymmetric parabolic function , respectively . to evaluate whether the shapes of the low - vision csfs were the same as that of the normal - vision csf , we first constructed a normal template by determining the four parameter values yielding the best - fit function to the normal - vision data using equation 1 . then we compared two fitting methods , free - fit versus template - fit , for each low - vision csf . template - fit also made use of equation 1 but with the two parameters , widthl and widthr , constrained to the values returned for the best - fit function for the normal - vision data . the template fit allowed only variation in the peak contrast sensitivity csp and the spatial - frequency at the peak sfp . the effect of this restriction is to retain the shape of the csf while permitting only translation of the function horizontally and vertically along the log spatial frequency and log contrast sensitivity axes . model comparisons of the four- and two - parameter fits for the low - vision csfs were performed using the akaike information criterion ( aic ) , which takes into account the goodness - of - fit of each fitting method , given the number of free parameters used . besides comparing the goodness - of - fit , we also compared several key parameters of the fitted functions derived from the two fitting methods , to determine how well these parameters correlate or agree with each other . if the shape of low - vision csfs is the same as that for the normal - vision csf , then the normal template should fit the low - vision data just as well as the free - fit . the goodness - of - fit of the two fitting methods would be similar , and that the key parameters derived from the two methods would show a strong correlation and agreement with each other . twenty observers with low vision ( 12 males and 8 females , mean age = 70.6 11.12 years ) and five observers with normal vision ( 2 males and 3 females , mean age = 25.8 9.9 years ) participated in this study . all observers in the low vision group had bilateral vision loss , with a bcva of 0.14 logmar ( 20/25 snellen equivalent ) or worse in the better eye , with a confirmed diagnosis of an eye disease that led to reduced acuity . the diagnoses included amd , stargardt disease , glaucoma and optic neuropathy ; all of which have been reported to lead to acuity and contrast deficits , and are common etiologies of patients attending low - vision clinics . visual characteristics of the 20 eyes with low vision we chose to use young adults , instead of age - matched older adults , for constructing the normal - vision template mainly for two reasons . most importantly , our hypothesis of a shift of the normal template to account for acuity and contrast deficits is not age - specific . although many functions have been used to describe normal csfs ( see watson and ahumada for a comparison of the different functions ) , we adopted an asymmetric parabolic function , which is simple to construct , and provides an excellent fit to our normal - vision data . the function , which requires four parameters , is given by the following equation : where f(sf ) is the contrast sensitivity at a spatial frequency sf , csp is the peak contrast sensitivity , sfp is the spatial frequency at which csp occurs , and widthl and widthr are the curvatures of the left and right branches of the asymmetric parabolic function , respectively . to evaluate whether the shapes of the low - vision csfs were the same as that of the normal - vision csf , we first constructed a normal template by determining the four parameter values yielding the best - fit function to the normal - vision data using equation 1 . then we compared two fitting methods , free - fit versus template - fit , for each low - vision csf . template - fit also made use of equation 1 but with the two parameters , widthl and widthr , constrained to the values returned for the best - fit function for the normal - vision data . the template fit allowed only variation in the peak contrast sensitivity csp and the spatial - frequency at the peak sfp . the effect of this restriction is to retain the shape of the csf while permitting only translation of the function horizontally and vertically along the log spatial frequency and log contrast sensitivity axes . model comparisons of the four- and two - parameter fits for the low - vision csfs were performed using the akaike information criterion ( aic ) , which takes into account the goodness - of - fit of each fitting method , given the number of free parameters used . besides comparing the goodness - of - fit , we also compared several key parameters of the fitted functions derived from the two fitting methods , to determine how well these parameters correlate or agree with each other . if the shape of low - vision csfs is the same as that for the normal - vision csf , then the normal template should fit the low - vision data just as well as the free - fit . the goodness - of - fit of the two fitting methods would be similar , and that the key parameters derived from the two methods would show a strong correlation and agreement with each other . we will first present the csf data of the normal vision observers , to be used in constructing the normal template . then we will compare the fitting of the normal template and the free - fit to the csfs of the low vision observers . figure 2 shows the contrast sensitivity versus spatial frequency data of our group of normal vision observers . each data point represents the contrast sensitivity , the reciprocal of the geometric mean of contrast thresholds across the five normal vision observers , with 32 staircase reversals for each observer ( eight reversals per staircase and four staircases per observer ) . to derive the normal - vision csf , we fit the data set using the asymmetric parabolic function ( equation 1 ) . 2 ) , with the width parameters constrained to the values of the best - fit curve , forms our normal template . contrast sensitivity is plotted as a function of spatial frequency ( c / deg ) for the five normal - vision control observers . each low - vision observer 's csf data were fit separately using the two fitting methods , the best - fit curve using the free - fit method is given in each panel in the same color as the symbols and the best - fit curve using the template - fit is given as the gray solid line . contrast sensitivity is plotted as a function of spatial frequency ( c / deg ) for the 20 eyes with low vision . we first evaluated which of the two fitting methods provided a better description of individual low - vision csfs . to do so , we compared the sum of the aic of the two fitting methods across all low - vision eyes , with aic given by where k is the number of free parameters ( 4 for free - fit and 2 for template - fit ) . using this method , the sum of aic is 231.2 for the free - fit method and 231.1 for the template - fit , implying that the two fitting methods provide equally good fits to the low - vision data . we examined the correlation and agreement of three key parameters derived from the two fitting methods , the peak contrast sensitivity ( csp ) , the spatial frequency at peak contrast sensitivity ( sfp ) , and the high cutoff spatial frequency . csp and sfp are parameters returned by the two fitting methods , while the high cutoff spatial frequency was derived from equation 1 , by setting f(sf ) to a value of 1 and solving for the corresponding spatial frequency . the top panels in figure 4 show how well each of these parameters derived from the two fitting methods correlate with each other . a t - test on the correlation coefficients ( r ranging from 0.880.99 ) shows that all of these correlations are statistically significant ( p < 0.0001 ) , indicating that the parameters from the two fitting methods correlate strongly with each other . 4 ) to determine how well each of these parameters derived from the two fitting methods agree with each other . we reason that if the 95% limits of agreement ( plotted as dashed lines in each of the bottom panels ) are comparable with the test retest reliability of similar measurements , then the two fitting methods are in good agreement with each other . the 95% limits of agreement for the high cutoff spatial frequency are 0.05 log units , smaller than the test - retest reliability of visual acuity measurement ( equivalent to the high cutoff spatial frequency ) , which ranges between 0.1 and 0.2 log units . thus , in general , the parameters derived from the two fitting methods are in good agreement . values derived from the free - fit and template - fit methods are compared for the peak contrast sensitivity ( csp ) , the spatial frequency that yields the peak contrast sensitivity ( sfp ) and the high cutoff spatial frequency . in the top panels , we examine how well these values from the two fitting methods correlate with each other . in the bottom panels , we examine , in the form of bland - altman plots , the black solid and dashed lines in each panel represent the mean of the difference of log values between the two fitting methods and the 95% limits of agreement , respectively . the correlation coefficients for the values estimated for the two fitting methods remain unchanged ( 0.99 for csp , 0.88 for sfp , and 0.99 for the high cutoff spatial frequency ) , and remain statistically significant ( p < 0.0001 ) . in addition , bland - altman plots show that the values derived from the two fitting methods still agree well with each other . these additional analyses ensure that our conclusion that the free - fit method provides a good fit to the low - vision csf data is still valid if we consider only patients with central field loss . given our hypothesis of low - vision csfs having the same shape as the normal csf , it is important to compare the parameters that govern the shape of the fitted functions . in figure 5 , we represent the distributions of the widthl and widthr parameters for the low - vision csfs , derived from the free - fit method in box - and - whisker plots . indeed , figure 3 shows that for several data - sets , the free - fit method yields a fitted curve with a shallower low - frequency decline than that returned by the template fit , especially for eyes c , d , f , g , and i. however , the means of the two distributions , represented by the red circles , are very close to the constrained values . box - and - whisker plots comparing the values of widthl and widthr derived from the free - fit method with the constrained values based on the normal csf . note that the mean values are very similar to the parameter values for the normal template , shown as gray dashed lines . results from these analyses show that the free - fit method provides a description of the low - vision csf data that is very similar to the template - fit , which is derived from the normal - vision data . we acknowledge that some of the low - vision csfs are more low - pass in shape , resulting in the sample distribution of the parameter widthl being different from that of the constrained value , however , the mean of the sample distribution ( 0.64 ) is very similar to that of the constrained value ( 0.68 ) . considering that the template fit only has two free parameters , our results suggest that low - vision csfs can be related to a normal csf with two additional measurements . to do so , we examined if there exists a significant correlation between letter - chart acuity , measured using a bailey - lovie chart with acuity expressed in logmar , and each of the three key parameters : csp , sfp , and the high cutoff spatial frequency , as derived from the free - fit method . in other words , letter - chart acuity can explain 58% and 62% of the variances in sfp and the high cutoff spatial frequency . in addition , the mean acuity difference between the high cutoff spatial frequency and chart acuity is 0.15 logmar , which is statistically different from a null difference ( p = 0.0002 ) . letter - chart acuity and the high cutoff spatial frequency derived from the free - fit method ( converted to logmar acuity ) are compared in a bland - altman plot . was the first investigation to test the hypothesis that low - vision csfs have the same shape as that of a normal - vision csf . they measured the csfs of 32 low - vision observers and fit each set of log cs versus log sf data using a single parabola that was derived from four normally sighted observers . by constraining the shape of the parabolic template but allowing it to shift vertically and horizontally along the log cs and log sf axes , pelli et al . found that the parabolic template provided a good fit to all the low - vision csfs . this led the authors to conclude that the csfs of older adults could not be described by a single parabolic or exponential function with two or three free parameters . we used a two temporal interval forced - choice paradigm combined with a staircase procedure . most noticeably , in several figures in their paper , contrast sensitivities were practically invariant for a large range of low- to high - spatial frequencies , and the contrast sensitivity at the highest - spatial frequency ( 22.5 c / deg ) was still much higher than expected ( based on published data ) . this unusual trend of contrast sensitivity data , and the lack of a clear fall - off of sensitivity at high - spatial frequencies make it difficult for any function with a high - frequency roll - off to provide a good fit . it was indeed because of this reason that we tested a range of spatial frequencies that included at least two frequencies lower than , and at least two frequencies higher than the peak of the csf . nevertheless , in our study , we found that a template based on the average data of a group of normally - sighted observers provides a reasonably good fit to individual data of 20 low - vision eyes . presumably , shifting the normal template to fit the low - vision data accounts for the visual deficits of the individual , regardless of whether the deficits are due to ocular diseases or normal aging . had we found that the shifted normal template did not provide a good fit to the low - vision data , the age difference between our young normal and low - vision groups would have been a possible factor accounting for the difference , and that the use of a normal template derived from normally sighted observers age - matched with those of the low - vision group would have been necessary . it is well known that the rates of fall - off of contrast sensitivity with spatial frequency differ for the left and right branches of the csf with respect to csp , thus in this study , we chose an asymmetric parabolic function to fit our data because it allows for different rates of fall - off ( or the width parameters in equation 1 ) for the left and right branches of the csf . here , we showed that a model with only two free parameters might be sufficient , and in fact , the template fit with only two free parameters yielded an overall aic value that is virtually identical to that of the free - fit with four free parameters . a practical advantage of modeling csfs with a template that requires only two free parameters is that we only need two measurements in addition to the template to derive a full csf , thus offering an efficient method for the determination of the csf . if our concern is to obtain an estimate of the csf of a patient with low vision , then one of these two measurements could be the conventional acuity measurement , since acuity is measured for every patient in the clinic , and that letter chart acuity correlates strongly with both the spatial frequency at the peak of the csf and the high cutoff spatial frequency ( fig . there are many clinical tests of contrast sensitivity , such as the bailey - lovie low contrast acuity chart or regan charts , which measure acuity for letters at a fixed low contrast ; the pelli - robson chart or the mars chart , which provide a contrast sensitivity measurement close to the peak of the csf ; and other contrast sensitivity tests such as the melbourne edge test , and the berkeley discs contrast sensitivity test ( bailey il , et al . the differences in acuity and a clinical contrast sensitivity measurement between a low - vision individual and the norms could then be used to shift a normal csf horizontally along the log sf axis and vertically along the log cs axis . now , let us further assume that the normal values for acuity and contrast sensitivity measurements using the same charts are 0.1 logmar and 2.1 log units ; then the differences in acuity and contrast sensitivity between the low - vision individual and normals become 0.5 and 0.75 log units , respectively . in this case , the csf of the low - vision individual can be approximated by shifting a normal csf template leftward along the log sf axis by 0.5 log units to account for the difference in acuity , and downward along the log cs axis by 0.75 log units to account for the difference in contrast sensitivity . it remains to be verified whether or not a normal template , ours or others , can be used to fit the csf of these conditions , or other conditions that lead to the shape of the csf being different from that of the normal template . an efficient method of determining a full csf is useful for individuals with normal vision , but it is especially useful for low vision as the peak contrast sensitivity is a good predictor of daily activities such as mobility , face recognition , and reading . in the laboratory , full low - vision csfs can be used as filters to recreate visual stimuli that simulate what individuals with low vision can detect . in this study , we have learned that the contrast sensitivity functions ( csfs ) of many low - vision subjects have the same shape as the csfs of normally - sighted subjects , but differ from the normal csfs in two values , the spatial frequency and contrast sensitivity at the peak of the curve . this means that by coupling our knowledge of the characteristic csf curves of normally - sighted subjects with two readily available clinical measures from a patient with low vision , we can infer a rich and informative description of the patient 's pattern vision .

fungi play a central role in most ecosystems and seem to dominate the microbial biomass in soil habitats ( joergensen and wichern 2008 ) , where they are important decomposers and occupy a notable position in the natural carbon , nitrogen and phosphorus cycles ( christensen 1989 ) . mycorrhizal and parasitic communities in different habitats are well characterised at the molecular level ( ryberg et al . 2009 ) , and they directly affect plant community composition and productivity ( klironomos 2002 ; van der heijden et al . in contrast , fungal species inventories from agricultural soils are so far mainly known from cultivation studies ( domsch and gams 1970 ; domsch et al . 1993 ; hagn et al . 2003 ) , while there are only few studies employing cultivation - independent techniques ( de castro et al . 2008 ; lynch and thorn 2006 ) . a solid knowledge of the fungal community in agricultural soils provides the basis for functional studies about specific processes carried out by members of this group . the main contributions of the fungal community to functioning of the agroecosystem are soil stabilization and nutrient cycling ( stromberger 2005 ) . the presented study is part of a larger effort to elucidate the microbial processes in fertilizer nitrogen transformations . to gain a better insight into the role of fungi in the nutrient cycling processes in agricultural soils , we took an inventory of this important group , which we showed previously by quantitative real - time pcr to constitute a dominant microbial community in two agriculatural soils ( inselsbacher et al . the soils studied here derived from different locations in lower austria in the vicinity of vienna . four of the soils are used as agricultural fields , while one is a grassland . several microbial parameters and nitrogen dynamics were investigated in previous studies ( inselsbacher et al . all five soils support higher nitrification rates than gross nitrogen mineralization rates leading to a rapid conversion of ammonium to nitrate . accordingly , nitrate dominates over ammonium in the soil inorganic nitrogen pools ( inselsbacher et al . 2010 ; inselsbacher et al . following fertilization more inorganic nitrogen was translocated to the microbial biomass compared to plants at the short term , but after 2 days plants accumulated higher amounts of applied fertilizer nitrogen ( inselsbacher et al . . rapid uptake of inorganic nitrogen by microbes prevents losses due to leaching and denitrification ( jackson et al . 2008 ) . the aims of the presented work were ( i ) to identify the most prominent members of the fungal communities in agricultural soils , and ( ii ) to address the issue of fungal biodiversity in agroecosystems . knowledge of community structure and composition will allow assessing the beneficial role of fungi in agriculture besides their well established role as major phytopathogens . to this end the most prominent members of the fungal communities in four arable soils and one grassland in lower austria were identified by sequencing of cloned pcr products comprising the its- and partial lsu - region . the obtained dataset of fungal species present in the different agricultural soils provides the basis for future work on specific functions of fungi in agroecosystems . soils were collected from four different arable fields and one grassland in lower austria ( austria ) . the soils were selected to represent different bedrocks , soil textures , ph values , water , and humus contents . for a detailed description of the soils sampling site riederberg ( r ) is a grassland for hay production , while sampling sites maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) are arable fields . grassland soil r as well as arable field soil p were covered with vegetation ( grasses and winter barley , resp . ) at the time of sampling , while arable field soils m , n and t were bare . at each site five randomized samples of 5 kg each were taken from an area of 400 m from the a horizon ( 010 cm depth ) and mixed . soils were sampled on april , 11th 2006 and immediately stored at 4c until further analysis . soils were homogenised , sieved ( < 2 mm ) and kept at 4c before processing . dna was extracted in triplicate from each soil ( 1 g fresh weight per extraction ) using the ultra clean soil dna isolation kit ( mobio ) according to the manufacturer s instructions and further purified with the qiaquick pcr purification kit ( qiagen ) . fungal its - region and partial lsu were amplified with its1f ( gardes and bruns 1993 ) , which is specific for fungi , and the universal eukaryotic primer tw13 ( taylor and bruns 1999 ) . the lsu region serves for higher order identification of fungi without homologous its reference sequences in public databases . pcrs contained gotaq green master mix ( promega ) , 1 m of each primer , 0.5 mg / ml bsa and 0.5 l soil dna in a total volume of 20 l . the following thermocycling program was used : 95c for 230 ( 1 cycle ) ; 94c for 3054c for 3072c for 130 ( 30 cycles ) ; and 72c for 5 ( 1 cycle ) . the nine replicate pcr products for each soil ( three dnas for each soil times three replicas for each dna ) were pooled before ligation to minimize effects from spatial heterogeneity and variability during pcr amplification ( schwarzenbach et al . a clone library ( 96 independent clones each ) of its / lsu - pcr - products was constructed in plasmid ptz57r / t ( fermentas ) according to manufacturer s instructions . insert pcr products ( its1f / tw13 ) from individual clones were directly subjected to rflp analyses . the reaction was performed with the restriction endonuclease bsuri ( fermentas , isoschizomere of haeiii ) for 2 h at 37c and the fragments were separated on a 3% high resolution agarose gel . initially up to 4 randomly selected clones that produced an identical pattern were sequenced ( big dye terminator v3.1 , cycle sequencing kit , abi ) using the primers its1f , its3 ( white et al . sequencing reactions were purified over sephadex - g50 in microtiterplates and separated on a dna sequencer ( abi 3100 genetic analyzer , pop69 , bdv3.1 ) at the department of applied genetics und cell biology , university of natural resources and applied life sciences , vienna ( austria ) . where sequencing of more than one representative of one rflp - pattern resulted in sequences with less than 97% identity in the its region or less than 99% identity in the lsu region ( see cut - off values for species delineation below ) , all clones from the particular pattern were sequenced . general molecular genetic manipulations were carried out according to sambrook and russell ( 2001 ) . forward and reverse sequence reads were assembled using the commercial software vector nti advance 10 for windows , version 10.3.0 . mended contig sequences were checked for chimeras by bellerophon ( huber et al . 2004 ) and submitted to a nucleotide blast search ( altschul et al . blast searches were performed separately with parts of the sequence corresponding to the its and partial lsu region , respectively . its- and lsu - taxonomies were compared for consistency to detect chimeras left undetected by bellerophon . reference hits from blast searches were scrutinised concerning their reliability ( e.g. sequences from strains from collections like cbs were preferably taken as reliable references ) . in cases in which sequences could not be identified to a certain taxonomic level , the lowest common affiliation of reliable reference sequences was taken . cut - off for distinct species was set to 97% for the its region ( hughes et al . 2009 ) and 99% for the lsu region , unless blast results for two closely related sequences gave distinct hits to well characterised strains . sequences are deposited at genbank under accession numbers gu055518gu055547 ( soil m ) , gu055548gu055606 ( soil n ) , gu055607gu055649 ( soil p ) , gu055650gu055710 ( soil r ) and gu055711gu055747 ( soil t ) . the data from each clone library were used for the calculation of estimates of species richness and diversity with estimates ( version 8.2.0 , r. k. colwell , http://purl.oclc.org/estimates ) . in addition to chimeric sequences , one sequence of eukaryotic but non - fungal origin ( ng_r_f10 , acc . gu055695 ) from soil r was also removed prior to data analysis to obtain estimates of fungal richness and diversity . richness estimators available in estimates 8.2.0 were compared to each other and gave comparable results for each of the five different soils . only results for the chao2 richness estimator ( chao 1987 ) are shown in table 1 . for comparison , richness and diversity indices were calculated from published sequence datasets from a natural grassland at the sourhope research station , scotland ( anderson et al . 2003 ) and from a soybean plantation in cristalina , brazil ( de castro et al . sourhope research station : libraries a and b comprising overlapping 18s rrna fragments were cured from non - fungal and chimeric sequences and richness and diversity was estimated from the combined a and b dataset as described above . similarly , species richness and diversity was calculated from sourhope research station its library d. the cut - off was also set to 99% , since there was no difference in predicted species richness and diversity between cut - off values of 9599% . soybean plantation cristalina : the published dataset did not contain chimeric or non - fungal sequences . table 1fungal richness and diversity indices for agricultural and grassland soilssoilmanagementlibraryclonessobschao2 sd% cov.shann.simp.maissauarable fieldits / lsu961920.4 3.192.82.337.37niederschleinzarable fieldits / lsu923451.3 12.066.33.2728.09purkersdorfarable fieldits / lsu943244.9 9.571.33.1823.76riederberggrasslandits / lsu923141.4 7.177.32.8410.76tullnarable fieldits / lsu892432.9 8.072.92.8415.48sourhope ( uk)grasslandssu531847.8 22.437.71.933.62sourhope ( uk)grasslandits452251.3 20.542.92.537.50cristalina ( bra)arable field ( soy)ssu1042230.9 7.671.21.872.87data for the soils sourhope from the sourhope research station in scotland , uk ( anderson et al . 2003 ) and cristalina from the district cristalina in gois , brazil ( de castro et al . 2008 ) were taken from the respective publicationslibrary indicates on which region from rrna - encoding cluster profiling of the fungal community was doneclones : number of analysed clones for each soil;sobs : number of observed species in the clone libraries;chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2;% cov . : estimated coverage of the libraries based on observed and estimated species richness;shann . : shannon diversity indexsimp . : simpson diversity index fungal richness and diversity indices for agricultural and grassland soils data for the soils sourhope from the sourhope research station in scotland , uk ( anderson et al . 2003 ) and cristalina from the district cristalina in gois , brazil ( de castro et al . 2008 ) were taken from the respective publications library indicates on which region from rrna - encoding cluster profiling of the fungal community was done clones : number of analysed clones for each soil ; sobs : number of observed species in the clone libraries ; chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2 ; % cov . : estimated coverage of the libraries based on observed and estimated species richness ; shann . : simpson diversity index unifrac was used to compare the phylogenetic structures of the fungal communities from soils m , n , p , r and t ( lozupone et al . 2007 ) , and a neighbor - joining tree was calculated from the aligned partial lsu sequences . the its - region was excluded , since it can not be unambiguously aligned over such a broad phylogenetic distance . data were weighted for abundance and normalized for branch length for calculating the unifrac metric of the distance between each pair of soil samples ( lozupone et al . soils were collected from four different arable fields and one grassland in lower austria ( austria ) . the soils were selected to represent different bedrocks , soil textures , ph values , water , and humus contents . for a detailed description of the soils sampling site riederberg ( r ) is a grassland for hay production , while sampling sites maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) are arable fields . grassland soil r as well as arable field soil p were covered with vegetation ( grasses and winter barley , resp . ) at the time of sampling , while arable field soils m , n and t were bare . at each site five randomized samples of 5 kg each were taken from an area of 400 m from the a horizon ( 010 cm depth ) and mixed . soils were sampled on april , 11th 2006 and immediately stored at 4c until further analysis . soils were homogenised , sieved ( < 2 mm ) and kept at 4c before processing . dna was extracted in triplicate from each soil ( 1 g fresh weight per extraction ) using the ultra clean soil dna isolation kit ( mobio ) according to the manufacturer s instructions and further purified with the qiaquick pcr purification kit ( qiagen ) . fungal its - region and partial lsu were amplified with its1f ( gardes and bruns 1993 ) , which is specific for fungi , and the universal eukaryotic primer tw13 ( taylor and bruns 1999 ) . the lsu region serves for higher order identification of fungi without homologous its reference sequences in public databases . pcrs contained gotaq green master mix ( promega ) , 1 m of each primer , 0.5 mg / ml bsa and 0.5 l soil dna in a total volume of 20 l . the following thermocycling program was used : 95c for 230 ( 1 cycle ) ; 94c for 3054c for 3072c for 130 ( 30 cycles ) ; and 72c for 5 ( 1 cycle ) . the nine replicate pcr products for each soil ( three dnas for each soil times three replicas for each dna ) were pooled before ligation to minimize effects from spatial heterogeneity and variability during pcr amplification ( schwarzenbach et al . a clone library ( 96 independent clones each ) of its / lsu - pcr - products was constructed in plasmid ptz57r / t ( fermentas ) according to manufacturer s instructions . insert pcr products ( its1f / tw13 ) from individual clones were directly subjected to rflp analyses . the reaction was performed with the restriction endonuclease bsuri ( fermentas , isoschizomere of haeiii ) for 2 h at 37c and the fragments were separated on a 3% high resolution agarose gel . initially up to 4 randomly selected clones that produced an identical pattern were sequenced ( big dye terminator v3.1 , cycle sequencing kit , abi ) using the primers its1f , its3 ( white et al . 1990 ) and tw13 . sequencing reactions were purified over sephadex - g50 in microtiterplates and separated on a dna sequencer ( abi 3100 genetic analyzer , pop69 , bdv3.1 ) at the department of applied genetics und cell biology , university of natural resources and applied life sciences , vienna ( austria ) . where sequencing of more than one representative of one rflp - pattern resulted in sequences with less than 97% identity in the its region or less than 99% identity in the lsu region ( see cut - off values for species delineation below ) , all clones from the particular pattern were sequenced . general molecular genetic manipulations were carried out according to sambrook and russell ( 2001 ) . forward and reverse sequence reads were assembled using the commercial software vector nti advance 10 for windows , version 10.3.0 . mended contig sequences were checked for chimeras by bellerophon ( huber et al . 2004 ) and submitted to a nucleotide blast search ( altschul et al . blast searches were performed separately with parts of the sequence corresponding to the its and partial lsu region , respectively . its- and lsu - taxonomies were compared for consistency to detect chimeras left undetected by bellerophon . reference hits from blast searches were scrutinised concerning their reliability ( e.g. sequences from strains from collections like cbs were preferably taken as reliable references ) . in cases in which sequences could not be identified to a certain taxonomic level , the lowest common affiliation of reliable reference sequences was taken . cut - off for distinct species was set to 97% for the its region ( hughes et al . 2009 ) and 99% for the lsu region , unless blast results for two closely related sequences gave distinct hits to well characterised strains . sequences are deposited at genbank under accession numbers gu055518gu055547 ( soil m ) , gu055548gu055606 ( soil n ) , gu055607gu055649 ( soil p ) , gu055650gu055710 ( soil r ) and gu055711gu055747 ( soil t ) . the data from each clone library were used for the calculation of estimates of species richness and diversity with estimates ( version 8.2.0 , r. k. colwell , http://purl.oclc.org/estimates ) . in addition to chimeric sequences , one sequence of eukaryotic but non - fungal origin ( ng_r_f10 , acc . gu055695 ) from soil r was also removed prior to data analysis to obtain estimates of fungal richness and diversity . richness estimators available in estimates 8.2.0 were compared to each other and gave comparable results for each of the five different soils . only results for the chao2 richness estimator ( chao 1987 ) for comparison , richness and diversity indices were calculated from published sequence datasets from a natural grassland at the sourhope research station , scotland ( anderson et al . 2003 ) and from a soybean plantation in cristalina , brazil ( de castro et al . 2008 ) . sourhope research station : libraries a and b comprising overlapping 18s rrna fragments were cured from non - fungal and chimeric sequences and richness and diversity was estimated from the combined a and b dataset as described above . similarly , species richness and diversity was calculated from sourhope research station its library d. the cut - off was also set to 99% , since there was no difference in predicted species richness and diversity between cut - off values of 9599% . soybean plantation cristalina : the published dataset did not contain chimeric or non - fungal sequences . table 1fungal richness and diversity indices for agricultural and grassland soilssoilmanagementlibraryclonessobschao2 sd% cov.shann.simp.maissauarable fieldits / lsu961920.4 3.192.82.337.37niederschleinzarable fieldits / lsu923451.3 12.066.33.2728.09purkersdorfarable fieldits / lsu943244.9 9.571.33.1823.76riederberggrasslandits / lsu923141.4 7.177.32.8410.76tullnarable fieldits / lsu892432.9 8.072.92.8415.48sourhope ( uk)grasslandssu531847.8 22.437.71.933.62sourhope ( uk)grasslandits452251.3 20.542.92.537.50cristalina ( bra)arable field ( soy)ssu1042230.9 7.671.21.872.87data for the soils sourhope from the sourhope research station in scotland , uk ( anderson et al . 2003 ) and cristalina from the district cristalina in gois , brazil ( de castro et al . 2008 ) were taken from the respective publicationslibrary indicates on which region from rrna - encoding cluster profiling of the fungal community was doneclones : number of analysed clones for each soil;sobs : number of observed species in the clone libraries;chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2;% cov . : estimated coverage of the libraries based on observed and estimated species richness;shann . : simpson diversity index fungal richness and diversity indices for agricultural and grassland soils data for the soils sourhope from the sourhope research station in scotland , uk ( anderson et al . 2003 ) and cristalina from the district cristalina in gois , brazil ( de castro et al . 2008 ) were taken from the respective publications library indicates on which region from rrna - encoding cluster profiling of the fungal community was done clones : number of analysed clones for each soil ; sobs : number of observed species in the clone libraries ; chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2 ; % cov . : estimated coverage of the libraries based on observed and estimated species richness ; shann . : shannon diversity index simp . : simpson diversity index unifrac was used to compare the phylogenetic structures of the fungal communities from soils m , n , p , r and t ( lozupone et al . 2006 ) . to this end sequences were aligned with the clustalw algorithm in mega4 ( tamura et al . 2007 ) , and a neighbor - joining tree was calculated from the aligned partial lsu sequences . the its - region was excluded , since it can not be unambiguously aligned over such a broad phylogenetic distance . data were weighted for abundance and normalized for branch length for calculating the unifrac metric of the distance between each pair of soil samples ( lozupone et al . soil characteristics of the five soils used in the present study are given in inselsbacher et al . all soil parameters are within the range for typical arable land as used for cultivation of barley in this area . fungal communities were analysed by direct amplification of fungal its / partial lsu regions with primer pair its1f and tw13 . cloned pcr products from each soil were grouped by rflp and up to four representatives from each rflp type were sequenced . by this approach even closely related sequences ( e.g. four different tetracladium species from soil p with a maximum sequence difference of 3.7% ) could be dissected . while the its region provides excellent resolution down to the species level , the partial lsu region provides good resolution at higher taxonomic levels when sufficiently identified its reference data in public databases are missing ( urban et al . 2008 ) . by this combined approach of rflp typing and sequencing a total of 116 ribotypes were detected in the five soils . from the 115 fungal ribotypes , 42 could be classified to the species level , an additional 24 at least to the genus level , while the remaining 49 fungal sequences could only be classified to the family or higher taxonomic level . richness ranged from 19 to 34 for detected and from 20.5 to 51.3 for estimated species numbers ( chao2 ; chao 1987 ) per sampling site . coverage of the libraries ranged from 66.3 to 92.8% of estimated species numbers ( see table 1 ) . as in a few cases sequencing of more than one representative clone from the same rflp pattern resulted in closely related but dissimilar sequences , the species numbers given here most likely slightly underestimate the true fungal diversity in the investigated soils . unifrac analysis could not detect significant differences between the phylogenetic structures of the fungal communities from the herein studied soils . bonferroni corrected p - values for pairwise comparisons were all above or equal to 0.1 . no clustering of spatially close locations could be found ( the distance between sampling sites m and n , p and r respectively r and t is less then 10 km ) . all five soils are dominated by ascomycota , which are represented by 77.7 to 88.2% of the clones in the respective libraries , followed by basidiomycota , which are represented by 7.5 to 21.3% of the clones in the respective libraries ( fig . 1 ) . other phyla ( chytridiomycota , blastocladiomycota as well as mucoromycotina ) were only detected occasionally and at low frequencies . no sequences belonging to the glomeromycota were found . at all taxonomic levels from phylum to species soil m showed the lowest observed richness ( see fig . 1 and table 2 ) . similarly , predicted species richness , several diversity indices ( magurran 2004 ) and evenness were lowest for soil m ( see table 1 ) . the dominant species in soil m a species related to trichocladium asperum was represented by nearly 30% of all analysed clones ( see table 2 ) . relative abundances at the phylum ( or where appropriate alternative taxonomic ranks ; left part ) and ordinal ( right part ) level of clones from libraries from arable soils maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) and grassland soil riederberg ( r)table 2species list of fungi from arable and grassland soils in lower austriasoilcloneacc.no.identificationorderphy.racomng_m_a03gu055520trichocladium asperum relatedsordarialesa29,2mng_m_a01gu055518myrothecium sp . m_a01hypocrealesa14,6mng_m_a06gu055523cercophora costaricensissordarialesa13,5mng_m_b07gu055525scleroderma bovistaboletalesb8,3mng_m_a04gu055521hapsidospora irregularishypocrealesa5,2mng_m_d07gu055530podospora dimorphasordarialesa4,2mng_m_c04gu055528cercophora coprophila / terricolasordarialesa3,1mng_m_h03gu055544fusarium merismoides var . merism.hypocrealesa3,1n , rmng_m_d12gu055532hebeloma pallidoluctuosumagaricalesb3,1mng_m_c08gu055529lasiosphaeriaceae m_g03sordarialesa3,1mng_m_g01gu055537cyphellophora laciniatachaetothyrialesa2,1nmng_m_h01gu055543minimedusa polysporacantharellalesb2,1n , pmng_m_g11gu055542paecilomyces carneushypocrealesa2,1mng_m_g04gu055539cryptococcus terricolatremellalesb1,0pmng_m_e04gu055534hypocreales m_e04hypocrealesa1,0mng_m_d10gu055531lasiosphaeriaceae m_d10sordarialesa1,0rmng_m_h07gu055546periconia macrospinosamicroascalesa1,0rmng_m_a02gu055519thielavia hyalocarpa relatedsordarialesa1,0mng_m_e08gu055535trichosporon dulcitumtremellalesb1,0nng_n_a02gu055548fusarium merismoides var . merism.hypocrealesa8,7m , rnng_n_a06gu055552pyrenophora tritici - repentispleosporalesa7,6nng_n_a09gu055554stachybotrys chartarumhypocrealesa7,6nng_n_a03gu055549chaetomiaceae n_a03chaetosphaerialesa6,5nng_n_a04gu055550hypocreales n_a04hypocrealesa5,4nng_n_e02gu055577verticillium nigrescensphyllachoralesa5,4nng_n_b06gu055559botryotinia fuckelianahelotialesa4,3nng_n_e10gu055583cyphellophora laciniatachaetothyrialesa4,3mnng_n_b09gu055561fusarium incarnatumhypocrealesa4,3nng_n_e07gu055581tetracladium maxilliformehelotialesa4,3p , rnng_n_c08gu055568thanatephorus cucumeriscantharellalesb4,3nng_n_a08gu055553acremonium strictumhypocrealesa3,3nng_n_b01gu055557pleosporales n_b01pleosporalesa3,3nng_n_b08gu055560sordariales n_b08sordarialesa3,3nng_n_e04gu055579fusarium solanihypocrealesa2,2rnng_n_e01gu055576lasiosphaeriaceae n_e01sordarialesa2,2nng_n_a12gu055556minimedusa polysporacantharellalesb2,2 m , pnng_n_d07gu055573nectria mauritiicolahypocrealesa2,2pnng_n_e06gu055580pleosporales n_e06pleosporalesa2,2nng_n_e09gu055582chaetomium globosum relatedsordarialesa1,1nng_n_b12gu055562acremonium strictum relatedhypocrealesa1,1nng_n_g10gu055599alternaria sp . n_g10pleosporalesa1,1nng_n_c01gu055563chytridiomycota n_c01chytridiomycota i.s.c1,1nng_n_g11gu055600cladosporium herbarum complexcapnodialesa1,1r , tnng_n_c04gu055565fungus n_c04fungi i.s.f1,1nng_n_h08gu055604guehomyces pullulanscystofilobasidialesb1,1nng_n_d09gu055575hypocrea lixii relatedhypocrealesa1,1nng_n_h02gu055603hypocreales n_h02hypocrealesa1,1nng_n_g12gu055601lasiosphaeriaceae n_g12sordarialesa1,1pnng_n_f01gu055586monographella nivalisxylarialesa1,1nng_n_c12gu055570mortierella alpinamortierellalesm1,1nng_n_f11gu055593spizellomycetales n_f11spizellomycetalesc1,1nng_n_g09gu055598tetracladium sp . n_g09helotialesa1,1nng_n_e12gu055585tetracladium sp . p_e08helotialesa1,1ppng_p_b05gu055621corticium related p_b05corticialesb10,6png_p_a12gu055616exophiala sp . rsem07_18chaetothyrialesa9,6png_p_d08gu055634tetracladium sp . p_d08helotialesa8,5png_p_a04gu055610cryptococcus terricolatremellalesb5,3mpng_p_c08gu055628helotiales p_c08helotialesa5,3tpng_p_a07gu055613schizothecium vesticolasordarialesa5,3tpng_p_e09gu055641tetracladium sp . p_e09helotialesa5,3tpng_p_b01gu055617byssonectria sp . p_b01pezizalesa4,3png_p_a11gu055615coniochaetaceae p_a11coniochaetalesa4,3png_p_f03gu055642kotlabaea sp . p_f03pezizalesa4,3rpng_p_c02gu055626nectria mauritiicolahypocrealesa3,2npng_p_a02gu055608pucciniomycotina p_a02pucciniomycotina i.s.b3,2png_p_c09gu055629tetracladium furcatumhelotialesa3,2rpng_p_b03gu055619tetracladium maxilliformehelotialesa3,2n , rpng_p_c01gu055625chaetomiaceae p_c01sordarialesa2,1png_p_d07gu055633helotiales p_d07helotialesa2,1png_p_e05gu055637leptodontidium orchidicolahelotialesa2,1png_p_b06gu055622minimedusa polysporacantharellalesb2,1 m , npng_p_b04gu055620neonectria radicicolahypocrealesa2,1rpng_p_h08gu055649arthrinium phaeospermumsordariomycetidae i.s.a1,1png_p_h06gu055647bionectriaceae p_h06hypocrealesa1,1png_p_e02gu055635chaetomium sp . p_e02sordarialesa1,1png_p_b10gu055623chalara sp . p_b10helotialesa1,1png_p_e03gu055636fusarium sp . p_e03hypocrealesa1,1png_p_b11gu055624helotiales p_b11helotialesa1,1png_p_d03gu055632helotiales p_d03helotialesa1,1png_p_c03gu055627lasiosphaeriaceae n_g12sordarialesa1,1npng_p_b02gu055618mortierellaceae p_b02mortierellalesm1,1png_p_g05gu055644ramularia sp . p_g05capnodialesa1,1png_p_e06gu055638sordariomycetes p_e06sordariomycetes i.s.a1,1png_p_e08gu055640tetracladium sp . p_e08helotialesa1,1npng_p_h07gu055648trichoderma spiralehypocrealesa1,1rng_r_b12gu055661tetracladium maxilliformehelotialesa22,6n , prng_r_h09gu055707scgi r_h09scgi i.s.a18,3rng_r_e08gu055685cladosporium herbarum complexcapnodialesa5,4n , trng_r_c06gu055666cryptococcus aeriustremellalesb4,3trng_r_e09gu055686fusarium oxysporumhypocrealesa4,3trng_r_b03gu055656hypocreales r_b03hypocrealesa4,3rng_r_d03gu055673lasiosphaeriaceae m_d10sordarialesa4,3mrng_r_d10gu055679agaricomycotina r_e03agaricomycotina i.s.b2,2rng_r_f02gu055690fungus r_f02fungi i.s.f2,2rng_r_g12gu055703fusarium r_g12hypocrealesa2,2rng_r_b09gu055660kotlabaea sp . p_f03pezizalesa2,2prng_r_d04gu055674lasiosphaeriaceae r_d04sordarialesa2,2rng_r_f04gu055692neonectria radicicolahypocrealesa2,2prng_r_b08gu055659pyronemataceae r_b08pezizalesa2,2rng_r_c09gu055668tetracladium furcatumhelotialesa2,2prng_r_d12gu055681tetracladium sp . r_d12helotialesa2,2rng_r_b04gu055657agaricomycotina r_b04agaricomycotina i.s.b1,1rng_r_d01gu055671agaricomycotina r_d01agaricomycotina i.s.b1,1rng_r_c01gu055662auxarthron umbrinumonygenalesa1,1rng_r_d09gu055678blastocladiomycota r_d09blastocladiomycota i.s.bc1,1rng_r_d02gu055672cryptococcus tephrensistremellalesb1,1rng_r_f10gu055695eukaryote r_f10eukaryota i.s.e1,1rng_r_d07gu055677exophiala sp . r_h11phyllachoralesa1,1rng_r_g01gu055697scgi r_g01scgi i.s.a1,1rng_r_g03gu055699sordariomycetes r_g03sordariomycetes i.s.a1,1tng_t_b06gu055716chaetomiaceae t_b06sordarialesa16,9tng_t_a04gu055713schizothecium vesticolasordarialesa10,1ptng_t_a01gu055711lasiosphaeriaceae t_a01sordarialesa9,0tng_t_a06gu055714exophiala sp . rsem07_18chaetothyrialesa6,7rtng_t_h11gu055747fusarium oxysporumhypocrealesa6,7rtng_t_c10gu055724helotiales t_c10helotialesa5,6tng_t_b11gu055717pleosporales t_b11pleosporalesa5,6tng_t_h09gu055745trichocladium asperumsordarialesa5,6tng_t_d07gu055729cladosporium herbarum complexcapnodialesa4,5n , rtng_t_c05gu055721coprinellus sp . t_c05agaricalesb4,5tng_t_e09gu055733mortierellales t_e09mortierellalesm4,5tng_t_e04gu055732pyronemataceae t_e04pezizalesa3,4tng_t_f08gu055736cryptococcus aeriustremellalesb2,2rtng_t_c01gu055718nectria ramulariaehypocrealesa2,2tng_t_d03gu055727psathyrella sp . t_d03agaricalesb2,2tng_t_a03gu055712apodus deciduussordarialesa1,1tng_t_f11gu055737chytridiomycota t_f11chytridiomycota i.s.c1,1tng_t_h01gu055742helotiales p_c08helotialesa1,1ptng_t_d02gu055726helotiales t_d02helotialesa1,1tng_t_d06gu055728helotiales t_d06helotialesa1,1tng_t_d01gu055725hypocreales t_d01hypocrealesa1,1tng_t_h06gu055743sordariomycetes t_h06sordariomycetes i.s.a1,1tng_t_c03gu055720stephanosporaceae t_c03agaricalesb1,1tng_t_h10gu055746tetracladium sp . p_e09helotialesa1,1pm , maissau ; n , niederschleinz ; p , purkersdorf ; r , riederberg ; t , tullnrepresentative sequenced clone from libraryacc.no . , accession number at genbanksequence identification based on separate blast searches of the its - region and the partial lsu - sequence ; clone epithets are used to distinguish different species were identification to the species - level was not possible ( e.g. hypocreales m_e04 is different from hypocreales n_a02)phylogenetic affiliation to a phylum ( or other higher taxonomic ranks where appropriate ) ; a , ascomycota ; b , basidiomycota ; bc , blastocladiomycota ; c , chytridiomycota ; e , eukaryota ; f , fungi ; m , mucoromycotinara : relative abundance in percent of analysed clones per soil type based on rflp and sequencing dataco : co - occurence of the same species in a second ( and third ) soili.s . , incertae sedis relative abundance of fungal groups in arable and grassland soils . relative abundances at the phylum ( or where appropriate alternative taxonomic ranks ; left part ) and ordinal ( right part ) level of clones from libraries from arable soils maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) and grassland soil riederberg ( r ) species list of fungi from arable and grassland soils in lower austria m , maissau ; n , niederschleinz ; p , purkersdorf ; r , riederberg ; t , tulln representative sequenced clone from library acc.no . , accession number at genbank sequence identification based on separate blast searches of the its - region and the partial lsu - sequence ; clone epithets are used to distinguish different species were identification to the species - level was not possible ( e.g. hypocreales m_e04 is different from hypocreales n_a02 ) phylogenetic affiliation to a phylum ( or other higher taxonomic ranks where appropriate ) ; a , ascomycota ; b , basidiomycota ; bc , blastocladiomycota ; c , chytridiomycota ; e , eukaryota ; f , fungi ; m , mucoromycotina ra : relative abundance in percent of analysed clones per soil type based on rflp and sequencing data co : co - occurence of the same species in a second ( and third ) soil the most abundant orders for all soils were the sordariales , hypocreales and helotiales , although helotiales could not be detected in soil m. additionally , the ascomycetous soil clone group i ( scgi ; porter et al . 2008 ) was found at a relatively high abundance in the grassland soil r , represented by 18.3% of all clones from the library , but was absent from the four libraries from arable soils . scgi could be detected at a similar level in a published dataset from a study analysing fungal communities in a natural grassland : 17.5% of clones from the ssu library ( a and b combined , and after removal of non - fungal and chimeric sequences ) belonged to scgi ( anderson et al . 2003 ) . the most abundant genus was tetracladium , which could be found at all sites , except in soil m. t. maxilliforme was the most abundant species in the grassland soil r , represented by 22.6% of clones from the library . another important group found in all soil samples are potentially phytopathogenic fungi , e.g. from the genera fusarium and nectria . from the 116 species detected in the five soil samples , 17 species could be detected in two soils , and four species could even be detected in three soils ( co - occurring species are indicated in table 2 ) . while there is a plenitude of data available on fungal communities in different natural soil habitats ( anderson et al . 2003 ; buee et al . 2009 ; curlevski et al . 2010 ; fierer et al . 2007 ; urich et al . 2002 ) , much less is so far known about fungal communities in agricultural soil ( de castro et al . 2008 ; domsch and gams 1970 ; lynch and thorn 2006 ; stromberger 2005 ) . molecular fingerprinting approaches like dgge or t - rflp allow rapid profiling of distinct communities and are especially useful for comparative analyses of numerous samples , but provide no information on species identities ( kennedy and clipson 2003 ) . cloning and sequencing , on the other hand , is more labour - intensive but allows identification of the community members . care must , however , be taken when using genbank for species identification , since many sequences are incorrectly named ( for a case study see e.g. cai et al . 2009 ) . in this study we obtained by sequencing of its / partial lsu clones from four arable and one grassland soil a dataset of 115 fungal species , of which this species inventory contains both , actively growing mycelium and dormant structures like spores ( anderson and cairney 2004 ) . the majority of fungal sequences belonged to the ascomycota , which is not unusual for soil habitats lacking ectomycorrhizal host plants ( schadt et al . 2003 ) and is in good agreement with findings from a soy bean plantation site ( de castro et al . 2008 ) and from numerous studies using cultivation techniques to describe agricultural soil fungal communities ( domsch and gams 1970 ) . dominance of ascomycota is probably enhanced by relatively high nitrogen contents of all soils analysed herein ( nemergut et al . 2008 ) . the grassland soil analysed by anderson et al . ( 2003 ) , however , was dominated by basidiomycota ( 60% of the clones in the combined ssu library and 47% in the its library ) , while basidiomycota were only the second most abundant group in all five soil samples from our study ( 7.521.3% of the analysed clones ) . a similar distribution of sequences between fungal phyla was observed in a sandy lawn by a metatranscriptomic approach , which assessed abundance of soil rnas by pyrosequencing ( urich et al . surprisingly , the inventory of agricultural soil fungal taxa found by cultivation techniques ( domsch and gams 1970 ) correlates well with the molecular data obtained from our cultivation - independent survey as there is e.g. the dominance of ascomycota or frequent occurrence of fungi from the orders sordariales , hypocreales and helotiales . even at the genus and species level many fungi found in our study were already previously described to occur in agricultural soils , as is the case e.g. for the genus tetracladium and for the potentially phytopathogenic genera fusarium and nectria . it should , however , be considered that 49 of the 115 fungal species in our study could not be classified below family level . this group of 49 species is probably composed of formally described fungal species for which no its or lsu reference sequences are deposited in genbank and for another part harbours species not yet formally described . no attempts for a cultivation - dependent description of the soil fungal communities were undertaken in our study . the relatively good correlation between cultivation - dependent and -independent techniques for fungal communities in agricultural soils is not unprecedented for environments dominated by ascomycetes ( gtz et al . traditional soil bacterial genera known from cultivation techniques make up only 2.7 to 3.7% of the total community investigated by cultivation independent techniques ( janssen 2006 ) . tetracladium , which was the most prominent genus found in the soils from our study , is mainly known to occur in aquatic ecosystems , where it is involved in leaf litter decay ( brlocher 1992 ) , or as plant endophyte ( selosse et al . 2008 ) . nevertheless , this genus has been found also in agricultural soils ( domsch and gams 1970 ; domsch et al . 1993 ) , where it is most likely involved in plant debris degradation . a survey of insufficiently identified sequences from environmental samples in emerencia ( ryberg et al . 2009 ) revealed that tetracladium actually commonly occurs in soil samples or associated with plant roots . in our study , tetracladium was only absent from soil m , the soil with the lowest clay content ( see inselsbacher et al . similarly , relatively dry soil conditions and consequently good aeration resulted in highest nitrification activities and highest no3-n / nh4 + -n ratios in soil m ( inselsbacher et al . 2009 ) . predicted species richness ( chao2 ; chao 1987 ) for the soils studied here ranged from 20.4 to 51.3 , which is in a similar range as found in comparable studies ( see table 1 ) , but substantially lower than fungal richness estimations from studies employing high throughput sequencing ( buee et al . in addition , richness estimation is strongly dependent on the prediction model ( fierer et al . . for these reasons predicted species richness allows direct comparison of datasets similar in size analysed by identical models , but gives little information about the actual number of species present in a sample . predicted species richness , diversity and the phylogenetic composition of fungal communities from arable soils did not differ from the grassland soil r ( see table 1 ) , although soil r showed higher levels of microbial biomass and activity compared to the four arable soils ( inselsbacher et al . likewise , vegetation cover at sampling time did , within the limits of our experimental resolution , not substantially influence richness , diversity and phylogenetic composition of soil fungi . ( 2006 ) who showed that aboveground plant richness does not directly influence belowground fungal richness . while there does not seem to be a difference in general parameters of fungal communities between arable and grassland soils , the most striking difference is the obvious absence of scgi from arable soil , a group of fungi that could be found at high frequencies in grassland soils ( soil r and natural grassland field site at the sourhope research station ( anderson et al . scgi is an only recently detected subphylum at the base of the ascomycota with thus far no cultivated members ( porter et al . presence in grassland and absence in arable soil could be an indication that scgi fungi directly depend on a continuous plant cover , which is in good agreement with the list published by porter et al . ( although site characteristics ranged from tundra to forest and from tropical to boreal , not a single arable site was included in this listing . 2002 ) or ectomycorrhizal root tips ( izzo et al . 2005 ; menkis et al . 2005 ; rosling et al . 2003 ; urban et al . 2008 ) , further pointing to an obligate - biotrophic lifestyle , which was already proposed by porter et al . such a direct dependence of the fungus on living plants could be the reason for the hitherto inability to cultivate scgi fungi . ( 2007 ) suggested that diversity is independent of soil parameters but an intrinsic feature of microbe types , the fungal specific simpson s diversity index being 134 39 . this value is however far above the values found in our study ( 7.3728.09 ) , in brazilian soy bean plantation soil ( 2.87 ; de castro et al . 2008 ) , scottish grassland soil ( 3.627.50 ; anderson et al . 2003 ) or soil with mixed grass - legume - shrub vegetation in tennessee ( 2.5641.67 ; castro et al . underestimation of diversity indices due to smaller sizes of libraries is unlikely to be the cause for this discrepancy , since predictions for the diversity indices of soils m , n , p , r and t stabilised after analysis of a maximum of 50 sequences . this is in good agreement with a comparative evaluation of diversity indices by giavelli et al . ( 1986 ) , who found that simpson s diversity index is least sensitive to small sample size . while the diversity in our study is potentially underestimated due to the use of rflp for clone selection , even lower diversity indices were found in published studies for grassland ( anderson et al . 2008 ) soil by directly sequencing ssu libraries without preselection by rflp ( see table 1 ) , an approach adopted at larger scale by fierer et al . underestimation of diversity at the species level by analysing ssu libraries is expected since the phylogenetic resolution of the fungal ssu is commonly thought to be restricted to the genus or family level but not to be sufficient for species identification ( anderson and cairney 2004 ; seena et al . more comparative studies are needed to give a solid answer whether arable and grassland soils indeed sustain a lower fungal diversity compared to desert , prairie or rainforest soils , which are the ecosystems studied by fierer et al . our study provides a fungal community inventory of agricultural soils and reveals the most prominent species . considering , however , the known seasonal dynamics of soil fungal communities and the diversity of agricultural practices , further studies are needed to extend and corroborate the presented initial findings . at least at the regional scale some general conclusions can be drawn from this study , i.e. ( i ) different agricultural soils harbour common fungal taxa from the species to the phylum level ; ( ii ) the fungal biodiversity of our four investigated arable soils was in a similar range as one investigated and one reference grassland soils , and ( iii ) scgi fungi seem to be absent from agricultural soils . these findings will certainly facilitate future studies on the relationship between fungal community structure and function and how these fungal - specific functions influence microbial nutrient cycling and the soil food web . the culturability of the majority of agricultural soil fungi opens the possibility for laboratory culture experiments to study genetics and molecular physiology of a number of potentially important species and thus to better determine their role in agroecosystems .

a culture - independent survey of fungal diversity in four arable soils and one grassland in lower austria was conducted by rflp and sequence analysis of clone libraries of the partial its / lsu - region . all soils were dominated by the ascomycetous orders sordariales , hypocreales and helotiales , taxa that are known from traditional cultivation approaches to occur in agricultural soils . the most abundant genus in the investigated soils was tetracladium , a hyphomycete which has been described as occurring predominantly in aquatic habitats , but was also found in agricultural soils . additionally , soil clone group i ( scgi ) , a subphylum at the base of the ascomycota with so far no cultivated members , was identified at high frequency in the grassland soil but was below detection limit in the four arable fields . in addition to this striking difference , general fungal community parameters like richness , diversity and evenness were similar between cropland and grassland soils . the presented data provide a fungal community inventory of agricultural soils and reveal the most prominent species .

Introduction Materials and methods Field sites and soil sampling DNA extraction and PCR Sequence analysis Statistical analysis Results Discussion

fungi play a central role in most ecosystems and seem to dominate the microbial biomass in soil habitats ( joergensen and wichern 2008 ) , where they are important decomposers and occupy a notable position in the natural carbon , nitrogen and phosphorus cycles ( christensen 1989 ) . mycorrhizal and parasitic communities in different habitats are well characterised at the molecular level ( ryberg et al . in contrast , fungal species inventories from agricultural soils are so far mainly known from cultivation studies ( domsch and gams 1970 ; domsch et al . 2003 ) , while there are only few studies employing cultivation - independent techniques ( de castro et al . a solid knowledge of the fungal community in agricultural soils provides the basis for functional studies about specific processes carried out by members of this group . the main contributions of the fungal community to functioning of the agroecosystem are soil stabilization and nutrient cycling ( stromberger 2005 ) . to gain a better insight into the role of fungi in the nutrient cycling processes in agricultural soils , we took an inventory of this important group , which we showed previously by quantitative real - time pcr to constitute a dominant microbial community in two agriculatural soils ( inselsbacher et al . the soils studied here derived from different locations in lower austria in the vicinity of vienna . accordingly , nitrate dominates over ammonium in the soil inorganic nitrogen pools ( inselsbacher et al . following fertilization more inorganic nitrogen was translocated to the microbial biomass compared to plants at the short term , but after 2 days plants accumulated higher amounts of applied fertilizer nitrogen ( inselsbacher et al . the aims of the presented work were ( i ) to identify the most prominent members of the fungal communities in agricultural soils , and ( ii ) to address the issue of fungal biodiversity in agroecosystems . to this end the most prominent members of the fungal communities in four arable soils and one grassland in lower austria were identified by sequencing of cloned pcr products comprising the its- and partial lsu - region . the obtained dataset of fungal species present in the different agricultural soils provides the basis for future work on specific functions of fungi in agroecosystems . soils were collected from four different arable fields and one grassland in lower austria ( austria ) . the soils were selected to represent different bedrocks , soil textures , ph values , water , and humus contents . for a detailed description of the soils sampling site riederberg ( r ) is a grassland for hay production , while sampling sites maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) are arable fields . fungal its - region and partial lsu were amplified with its1f ( gardes and bruns 1993 ) , which is specific for fungi , and the universal eukaryotic primer tw13 ( taylor and bruns 1999 ) . a clone library ( 96 independent clones each ) of its / lsu - pcr - products was constructed in plasmid ptz57r / t ( fermentas ) according to manufacturer s instructions . sequencing reactions were purified over sephadex - g50 in microtiterplates and separated on a dna sequencer ( abi 3100 genetic analyzer , pop69 , bdv3.1 ) at the department of applied genetics und cell biology , university of natural resources and applied life sciences , vienna ( austria ) . where sequencing of more than one representative of one rflp - pattern resulted in sequences with less than 97% identity in the its region or less than 99% identity in the lsu region ( see cut - off values for species delineation below ) , all clones from the particular pattern were sequenced . in addition to chimeric sequences , one sequence of eukaryotic but non - fungal origin ( ng_r_f10 , acc . gu055695 ) from soil r was also removed prior to data analysis to obtain estimates of fungal richness and diversity . for comparison , richness and diversity indices were calculated from published sequence datasets from a natural grassland at the sourhope research station , scotland ( anderson et al . 2008 ) were taken from the respective publicationslibrary indicates on which region from rrna - encoding cluster profiling of the fungal community was doneclones : number of analysed clones for each soil;sobs : number of observed species in the clone libraries;chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2;% cov . : simpson diversity index fungal richness and diversity indices for agricultural and grassland soils data for the soils sourhope from the sourhope research station in scotland , uk ( anderson et al . 2008 ) were taken from the respective publications library indicates on which region from rrna - encoding cluster profiling of the fungal community was done clones : number of analysed clones for each soil ; sobs : number of observed species in the clone libraries ; chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2 ; % cov . soils were collected from four different arable fields and one grassland in lower austria ( austria ) . the soils were selected to represent different bedrocks , soil textures , ph values , water , and humus contents . for a detailed description of the soils sampling site riederberg ( r ) is a grassland for hay production , while sampling sites maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) are arable fields . fungal its - region and partial lsu were amplified with its1f ( gardes and bruns 1993 ) , which is specific for fungi , and the universal eukaryotic primer tw13 ( taylor and bruns 1999 ) . a clone library ( 96 independent clones each ) of its / lsu - pcr - products was constructed in plasmid ptz57r / t ( fermentas ) according to manufacturer s instructions . sequencing reactions were purified over sephadex - g50 in microtiterplates and separated on a dna sequencer ( abi 3100 genetic analyzer , pop69 , bdv3.1 ) at the department of applied genetics und cell biology , university of natural resources and applied life sciences , vienna ( austria ) . where sequencing of more than one representative of one rflp - pattern resulted in sequences with less than 97% identity in the its region or less than 99% identity in the lsu region ( see cut - off values for species delineation below ) , all clones from the particular pattern were sequenced . in addition to chimeric sequences , one sequence of eukaryotic but non - fungal origin ( ng_r_f10 , acc . gu055695 ) from soil r was also removed prior to data analysis to obtain estimates of fungal richness and diversity . only results for the chao2 richness estimator ( chao 1987 ) for comparison , richness and diversity indices were calculated from published sequence datasets from a natural grassland at the sourhope research station , scotland ( anderson et al . similarly , species richness and diversity was calculated from sourhope research station its library d. the cut - off was also set to 99% , since there was no difference in predicted species richness and diversity between cut - off values of 9599% . 2008 ) were taken from the respective publicationslibrary indicates on which region from rrna - encoding cluster profiling of the fungal community was doneclones : number of analysed clones for each soil;sobs : number of observed species in the clone libraries;chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2;% cov . : simpson diversity index fungal richness and diversity indices for agricultural and grassland soils data for the soils sourhope from the sourhope research station in scotland , uk ( anderson et al . 2008 ) were taken from the respective publications library indicates on which region from rrna - encoding cluster profiling of the fungal community was done clones : number of analysed clones for each soil ; sobs : number of observed species in the clone libraries ; chao2 sd : estimated species richness standard deviation for the sampling site based on the chao2 richness estimator ( chao 1987 ) implemented in estimates 8.2 ; % cov . soil characteristics of the five soils used in the present study are given in inselsbacher et al . fungal communities were analysed by direct amplification of fungal its / partial lsu regions with primer pair its1f and tw13 . cloned pcr products from each soil were grouped by rflp and up to four representatives from each rflp type were sequenced . as in a few cases sequencing of more than one representative clone from the same rflp pattern resulted in closely related but dissimilar sequences , the species numbers given here most likely slightly underestimate the true fungal diversity in the investigated soils . all five soils are dominated by ascomycota , which are represented by 77.7 to 88.2% of the clones in the respective libraries , followed by basidiomycota , which are represented by 7.5 to 21.3% of the clones in the respective libraries ( fig . similarly , predicted species richness , several diversity indices ( magurran 2004 ) and evenness were lowest for soil m ( see table 1 ) . relative abundances at the phylum ( or where appropriate alternative taxonomic ranks ; left part ) and ordinal ( right part ) level of clones from libraries from arable soils maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) and grassland soil riederberg ( r)table 2species list of fungi from arable and grassland soils in lower austriasoilcloneacc.no.identificationorderphy.racomng_m_a03gu055520trichocladium asperum relatedsordarialesa29,2mng_m_a01gu055518myrothecium sp . , accession number at genbanksequence identification based on separate blast searches of the its - region and the partial lsu - sequence ; clone epithets are used to distinguish different species were identification to the species - level was not possible ( e.g. hypocreales m_e04 is different from hypocreales n_a02)phylogenetic affiliation to a phylum ( or other higher taxonomic ranks where appropriate ) ; a , ascomycota ; b , basidiomycota ; bc , blastocladiomycota ; c , chytridiomycota ; e , eukaryota ; f , fungi ; m , mucoromycotinara : relative abundance in percent of analysed clones per soil type based on rflp and sequencing dataco : co - occurence of the same species in a second ( and third ) soili.s . , incertae sedis relative abundance of fungal groups in arable and grassland soils . relative abundances at the phylum ( or where appropriate alternative taxonomic ranks ; left part ) and ordinal ( right part ) level of clones from libraries from arable soils maissau ( m ) , niederschleinz ( n ) , purkersdorf ( p ) and tulln ( t ) and grassland soil riederberg ( r ) species list of fungi from arable and grassland soils in lower austria m , maissau ; n , niederschleinz ; p , purkersdorf ; r , riederberg ; t , tulln representative sequenced clone from library acc.no . , accession number at genbank sequence identification based on separate blast searches of the its - region and the partial lsu - sequence ; clone epithets are used to distinguish different species were identification to the species - level was not possible ( e.g. hypocreales m_e04 is different from hypocreales n_a02 ) phylogenetic affiliation to a phylum ( or other higher taxonomic ranks where appropriate ) ; a , ascomycota ; b , basidiomycota ; bc , blastocladiomycota ; c , chytridiomycota ; e , eukaryota ; f , fungi ; m , mucoromycotina ra : relative abundance in percent of analysed clones per soil type based on rflp and sequencing data co : co - occurence of the same species in a second ( and third ) soil the most abundant orders for all soils were the sordariales , hypocreales and helotiales , although helotiales could not be detected in soil m. additionally , the ascomycetous soil clone group i ( scgi ; porter et al . 2008 ) was found at a relatively high abundance in the grassland soil r , represented by 18.3% of all clones from the library , but was absent from the four libraries from arable soils . the most abundant genus was tetracladium , which could be found at all sites , except in soil m. t. maxilliforme was the most abundant species in the grassland soil r , represented by 22.6% of clones from the library . another important group found in all soil samples are potentially phytopathogenic fungi , e.g. 2002 ) , much less is so far known about fungal communities in agricultural soil ( de castro et al . in this study we obtained by sequencing of its / partial lsu clones from four arable and one grassland soil a dataset of 115 fungal species , of which this species inventory contains both , actively growing mycelium and dormant structures like spores ( anderson and cairney 2004 ) . the majority of fungal sequences belonged to the ascomycota , which is not unusual for soil habitats lacking ectomycorrhizal host plants ( schadt et al . the grassland soil analysed by anderson et al . ( 2003 ) , however , was dominated by basidiomycota ( 60% of the clones in the combined ssu library and 47% in the its library ) , while basidiomycota were only the second most abundant group in all five soil samples from our study ( 7.521.3% of the analysed clones ) . surprisingly , the inventory of agricultural soil fungal taxa found by cultivation techniques ( domsch and gams 1970 ) correlates well with the molecular data obtained from our cultivation - independent survey as there is e.g. the dominance of ascomycota or frequent occurrence of fungi from the orders sordariales , hypocreales and helotiales . even at the genus and species level many fungi found in our study were already previously described to occur in agricultural soils , as is the case e.g. the relatively good correlation between cultivation - dependent and -independent techniques for fungal communities in agricultural soils is not unprecedented for environments dominated by ascomycetes ( gtz et al . traditional soil bacterial genera known from cultivation techniques make up only 2.7 to 3.7% of the total community investigated by cultivation independent techniques ( janssen 2006 ) . tetracladium , which was the most prominent genus found in the soils from our study , is mainly known to occur in aquatic ecosystems , where it is involved in leaf litter decay ( brlocher 1992 ) , or as plant endophyte ( selosse et al . nevertheless , this genus has been found also in agricultural soils ( domsch and gams 1970 ; domsch et al . predicted species richness ( chao2 ; chao 1987 ) for the soils studied here ranged from 20.4 to 51.3 , which is in a similar range as found in comparable studies ( see table 1 ) , but substantially lower than fungal richness estimations from studies employing high throughput sequencing ( buee et al . for these reasons predicted species richness allows direct comparison of datasets similar in size analysed by identical models , but gives little information about the actual number of species present in a sample . predicted species richness , diversity and the phylogenetic composition of fungal communities from arable soils did not differ from the grassland soil r ( see table 1 ) , although soil r showed higher levels of microbial biomass and activity compared to the four arable soils ( inselsbacher et al . likewise , vegetation cover at sampling time did , within the limits of our experimental resolution , not substantially influence richness , diversity and phylogenetic composition of soil fungi . while there does not seem to be a difference in general parameters of fungal communities between arable and grassland soils , the most striking difference is the obvious absence of scgi from arable soil , a group of fungi that could be found at high frequencies in grassland soils ( soil r and natural grassland field site at the sourhope research station ( anderson et al . scgi is an only recently detected subphylum at the base of the ascomycota with thus far no cultivated members ( porter et al . this value is however far above the values found in our study ( 7.3728.09 ) , in brazilian soy bean plantation soil ( 2.87 ; de castro et al . 2008 ) , scottish grassland soil ( 3.627.50 ; anderson et al . while the diversity in our study is potentially underestimated due to the use of rflp for clone selection , even lower diversity indices were found in published studies for grassland ( anderson et al . 2008 ) soil by directly sequencing ssu libraries without preselection by rflp ( see table 1 ) , an approach adopted at larger scale by fierer et al . underestimation of diversity at the species level by analysing ssu libraries is expected since the phylogenetic resolution of the fungal ssu is commonly thought to be restricted to the genus or family level but not to be sufficient for species identification ( anderson and cairney 2004 ; seena et al . more comparative studies are needed to give a solid answer whether arable and grassland soils indeed sustain a lower fungal diversity compared to desert , prairie or rainforest soils , which are the ecosystems studied by fierer et al . our study provides a fungal community inventory of agricultural soils and reveals the most prominent species . considering , however , the known seasonal dynamics of soil fungal communities and the diversity of agricultural practices , further studies are needed to extend and corroborate the presented initial findings . ( i ) different agricultural soils harbour common fungal taxa from the species to the phylum level ; ( ii ) the fungal biodiversity of our four investigated arable soils was in a similar range as one investigated and one reference grassland soils , and ( iii ) scgi fungi seem to be absent from agricultural soils . these findings will certainly facilitate future studies on the relationship between fungal community structure and function and how these fungal - specific functions influence microbial nutrient cycling and the soil food web . the culturability of the majority of agricultural soil fungi opens the possibility for laboratory culture experiments to study genetics and molecular physiology of a number of potentially important species and thus to better determine their role in agroecosystems .

with the increasing burden of patients with multiple disease states , the drug therapy has grown more complex . the complex therapeutic regimens increase the risk of drug - drug interaction ( ddi ) to a great extent . ddi is said to occur when the effect of one drug is altered by the concurrent administration of other . pharmacokinetic interaction occurs when either of the concurrently administered drugs have potential to alter other 's pattern of absorption , distribution , metabolism and excretion . ddi is said to account for a number of severe adverse drug reactions ( adr ) resulting in hospitalizations and emergency department visits . it is estimated that ddi contribute to about 6 - 30% of all adrs . furthermore , adr due to ddi accounts for about 2.8% of hospital admission every year . they are regarded as a leading cause of deaths world - wide . in nepal , cardiovascular disease furthermore , patients with cardiovascular disorders are even at higher risk of ddi due to the number and types of drug they receive and the influence of heart disease on drug metabolism . the potential of cardiovascular drug in the involvement of ddi is relatively higher as shown in the studies conducted world - wide . a prospective study conducted in one of the teaching hospitals in india indicated that the incidence of potential drug interaction amongst cardiac drugs in hospitalized patients is 30.67% . a study in palestine among patients receiving antihypertensive medications came up with 433 different unique pairs of potential drug interactions among 867 patients . another study from nepal regarding ddi indicated that 53% of the patients admitted to the department of internal medicine experience one or more ddis . yet another study in nepal to evaluate the pattern of ddi amongst diabetic outpatients also found that 47.5% of medications potentially interacting with antidiabetics were cardiovascular drugs . while similar studies are reported to be common world - wide there have been few reports of drug interaction and the factors associated with it among south asian cardiac patients . hence , this study was conducted to evaluate the pattern of potential drug - drug interaction ( pddi ) and to identify the associated risk factors among hospitalized cardiac patients in a tertiary care hospital in nepal . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . patient profile form was used for collecting the socio - demographic variables and medication profile of patients . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . micromedex is an electronic database that contains a separate section on ddi known as the drug - reax system . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects ) moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . poor : documentation is scant ; however , the possibility of a clinical conflict exists . their demographic and medical details were properly documented in the self - designed patient profile form . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . all the statistical analysis was carried out with statistical package for social sciences ( ibm corporation ) , version 16.0 considering p < 0.01 as statistically significant . socio - demographic data was obtained from the patients after obtaining their verbal informed consent . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . patient profile form was used for collecting the socio - demographic variables and medication profile of patients . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . micromedex is an electronic database that contains a separate section on ddi known as the drug - reax system . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects ) moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . poor : documentation is scant ; however , the possibility of a clinical conflict exists . their demographic and medical details were properly documented in the self - designed patient profile form . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . all the statistical analysis was carried out with statistical package for social sciences ( ibm corporation ) , version 16.0 considering p < 0.01 as statistically significant . socio - demographic data was obtained from the patients after obtaining their verbal informed consent . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . patient profile form was used for collecting the socio - demographic variables and medication profile of patients . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . micromedex is an electronic database that contains a separate section on ddi known as the drug - reax system . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects ) moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . poor : documentation is scant ; however , the possibility of a clinical conflict exists . their demographic and medical details were properly documented in the self - designed patient profile form . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . all the statistical analysis was carried out with statistical package for social sciences ( ibm corporation ) , version 16.0 considering p < 0.01 as statistically significant . socio - demographic data was obtained from the patients after obtaining their verbal informed consent . a total of 150 cardiac patients admitted to the cardiac unit of general medicine ward were enrolled . among them , prescriptions of 32 patients were identified which had least one potentially interacting drug combination . the overall incidence rate of potential drug interaction was 21.3% . among 150 study patients , majority were in the age group of 61 - 70 years . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . hence cardiovascular medicines constituted 44.8% of total medicines . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) a total of 48 pddis were identified . among the total of 150 patients , majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . interactions encountered were analyzed on the basis of mechanism of interaction . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . interacting pairs of major severity with the potentially hazardous effect and documentation status some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . statistical analysis by pearson correlation co - efficient revealed that there was a significant linear relationship between these factors and the occurrence of ddis [ table 4 ] . among 150 study patients , majority were in the age group of 61 - 70 years . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) a total of 48 pddis were identified . among the total of 150 patients , majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . interactions encountered were analyzed on the basis of mechanism of interaction . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . interacting pairs of major severity with the potentially hazardous effect and documentation status some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . statistical analysis by pearson correlation co - efficient revealed that there was a significant linear relationship between these factors and the occurrence of ddis [ table 4 ] . among 150 study patients , majority were in the age group of 61 - 70 years . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . interactions encountered were analyzed on the basis of mechanism of interaction . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . statistical analysis by pearson correlation co - efficient revealed that there was a significant linear relationship between these factors and the occurrence of ddis [ table 4 ] . it may pose a significant health hazard to patients when the risk - benefit ratio of combining interacting drugs is not accurately estimated . it has already been approximated that the effect of drug interactions can range from any minor morbidity to fatal consequences . the present study identified the pattern of pddis among patients admitted to cardiac unit of general medicine ward . the value obtained in the present study is relatively less compared with the study by patel et al . in india who reported an incidence rate of 30.23% . these differences might be because our study took into consideration only the potential drug interactions of major and moderate severity in contrast to the other studies that considered drug interactions of all severity . data regarding the incidence of pddi in cardiac patients is not sufficient but previous studies in general medicine in mth had identified the maximum involvement of cardiovascular medicines in drug interactions . on analyzing the mechanism of drug interaction identified here , pharmacokinetic type of reactions ( 58.3% ) was found in higher number compared to pharmacodynamic type ( 33.3% ) . aparasu et al . who reported 76% of pharmacokinetic and 22% of pharmacodynamic interactions respectively . of the total pddis identified , the interacting combination of moderate severity ( 62.5% ) constituted majority of pddi . the most common interacting pairs identified were atorvastatin / azithromycin , enalapril / metformin , enalapril / potassium chloride , atorvastatin / clarithromycin and furosemide / gentamicin . the pddi involving atorvastatin ( 16 [ 33.3% ] ) was the highest among all followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . the values obtained here are quite different from the study in india where patel et al . reported aspirin ( 44.85% ) , heparin ( 42.78% ) , clopidogrel ( 22.16% ) , warfarin ( 11.59% ) , atorvastatin ( 7.22% ) and ramipril ( 6.95% ) as the highest risk drug categories for ddi . there was maximum involvement of atorvastatin ( 16 [ 33.3% ] ) in pddi encountered in this study . this might be because atorvastatin ( 68 [ 14.7% ] ) was one of the most commonly prescribed medicines in the present study . a significant positive linear relationship was found between the length of hospital stay and pddis ( r = 0.63 , p < 0.01 ) . our finding well resembles to the finding by several studies which have also shown that increased incidence of pddi corresponds with an increase in duration of hospital stay . the reason might be that the likelihood of getting the multiple drugs increases with the increased length of hospital stay which in turn will increase the likelihood of pddi . similar positive linear relationship was also found between the number of medicines prescribed and pddi ( r = 0.5 , p < 0.01 ) and with concurrent illness and pddi ( r = 0.62 , p < 0.01 ) . the findings well correlate with the fact that polypharmacy increases the likelihood of ddis to a great extent as shown by several studies . this study shows that cardiac in - patients are at a high risk of hazardous ddi . this emphasizes the need to consider pddi during therapeutic planning , protect patients from consequence of drug interactions . in addition , providing ddi related information to the prescribers and drug interaction alert software to the dispensing pharmacist can play a vital role in minimizing the incidence rate of ddi . in the present study , monitor the occurrences of ddi clinically along with pharmacokinetic estimation of the drug is suggested , especially for moderate to major pddi .

aim : drug - drug interaction ( ddi ) is of major concern in patients with complex therapeutic regimens . the involvement of cardiovascular medicines in drug interaction is even higher . however , reports of ddi between these groups of drugs are few . the study aims to identify the potential ddi among hospitalized cardiac patients . furthermore , we assessed the possible risk factors associated with these interactions.subjects and methods : the type of study prospective observational study was conducted from may 2012 to august 2012 among hospitalized cardiac patients . cardiac patients who were taking at least two drugs and who had a hospital stay of at least 24 h were enrolled . the medications of the patients were analyzed for possible interactions using the standard drug interaction database - micromedex -2 ( thomson reuters ) 2.0.results:from a total of 150 enrolled patients , at least one interacting drug combination was identified among 32 patients . the incidence of potential ddi was 21.3% . a total of 48 potentially hazardous drug interactions were identified . atorvastatin / azithromycin ( 10.4% ) , enalapril / metformin ( 10.4% ) , enalapril / potassium chloride ( 10.4% ) , atorvastatin / clarithromycin ( 8.3% ) and furosemide / gentamicin ( 6.3% ) were the most common interacting pairs . drugs most commonly involved were atorvastatin , enalapril , digoxin , furosemide , clopidogrel and warfarin . majority of interactions were of moderate severity ( 62.5% ) and pharmacokinetic ( 58.3% ) in nature . increased number of medicines , prolonged hospital stays and comorbid conditions were the risk factors found associated with the potential ddi.conclusions:this study highlighted the need of intense monitoring of patients who have identified risk factors to help detect and prevent them from serious health hazards associated with drug interactions .

Introduction Subjects and Methods None Setting and Study Design Study Population Tools Used Operational Modality Statistical Analysis Ethics Results None Characteristics of Study Population Potential Drug Interactions Risk Factors Discussion Conclusion

with the increasing burden of patients with multiple disease states , the drug therapy has grown more complex . the complex therapeutic regimens increase the risk of drug - drug interaction ( ddi ) to a great extent . in nepal , cardiovascular disease furthermore , patients with cardiovascular disorders are even at higher risk of ddi due to the number and types of drug they receive and the influence of heart disease on drug metabolism . the potential of cardiovascular drug in the involvement of ddi is relatively higher as shown in the studies conducted world - wide . a prospective study conducted in one of the teaching hospitals in india indicated that the incidence of potential drug interaction amongst cardiac drugs in hospitalized patients is 30.67% . another study from nepal regarding ddi indicated that 53% of the patients admitted to the department of internal medicine experience one or more ddis . while similar studies are reported to be common world - wide there have been few reports of drug interaction and the factors associated with it among south asian cardiac patients . hence , this study was conducted to evaluate the pattern of potential drug - drug interaction ( pddi ) and to identify the associated risk factors among hospitalized cardiac patients in a tertiary care hospital in nepal . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . on the basis of severity micromedex classifies ddi as major , moderate and minor as follows : major : potentially life - threatening ; requires medical intervention to minimize or prevent the serious adverse effects)moderate : results in potential deterioration of patients clinical condition and may require an alteration in therapy.minor : the effects are usually mild and may not require change in therapy . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . the prospective observational study was undertaken for the period of 4 months from may 1 , 2012 to august 28 at the department of internal medicine of manipal teaching hospital ( mth ) , a tertiary care teaching hospital in western nepal . cardiac patients aged 18 years or older admitted to the cardiac unit of general medicine wards with a hospital stay of at least 24 h and those prescribed two or more drugs were enrolled for the study . ddi database system ( micromede 2.0 ) was used to identify and analyze the pattern of potential ddis . on entering the list of medications , it enlists all the potentially hazardous drug interactions on the basis of severity , onset and documentation status . it also classifies potential ddi as excellent , good , fair , poor or unlikely on the basis of documentation status as mentioned follows : excellent : the existence of the drug interaction has been clearly established by the controlled studies.good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking.fair : available documentation is poor.poor : documentation is scant ; however , the possibility of a clinical conflict exists.unlikely : documentation as well as a sound pharmacological basis is lacking . excellent : the existence of the drug interaction has been clearly established by the controlled studies . good : the existence of drug interaction is suggested by documentation , but well - controlled studies are lacking . the medications taken by the patients during their hospital stay were analyzed for possible drug interaction via the electronic database - micromedex 2.0 . there included a number of medicines , length of hospital stay and concurrent illnesses as the risk factors studied . pearson correlation was used to find the association between length of hospital stay and pddi , number of medicines prescribed and pddis and concurrent illness and pddis . a total of 150 cardiac patients admitted to the cardiac unit of general medicine ward were enrolled . among them , prescriptions of 32 patients were identified which had least one potentially interacting drug combination . the overall incidence rate of potential drug interaction was 21.3% . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) a total of 48 pddis were identified . among the total of 150 patients , majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . interacting pairs of major severity with the potentially hazardous effect and documentation status some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) a total of 48 pddis were identified . among the total of 150 patients , majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . interacting pairs of major severity with the potentially hazardous effect and documentation status some factors were assessed to determine their association with the likelihood of occurrence of ddis . length of hospital stay , number of medicines and concurrent illness were the factors studied . male patients ( 91 [ 60.7% ] ) were in higher number compared with females ( 59 [ 39.3% ) ] . majority of patients ( 115 [ 76.7% ] ) had cardiovascular disease as their primary admission disease and rest ( 35 [ 23.3% ] ) had it as concurrent illness . hypertension ( 80 [ 53.3% ] ) was the most common diagnosis in the study population followed by coronary heart disease ( 29 [ 19.33% ] ) [ table 1 ] . hence the average number of medicines prescribed per patient was 6.9 . among the total of 1037 medicines , 464 were cardiovascular drugs . the length of hospital stay of the patient was found to be 6.16 2.3 days . diagnosis of patients with cardiovascular diseases in a tertiary care hospital in nepal ( n=150 ) cardiovascular medicines prescribed to the patients admitted to a tertiary care hospital in nepal ( n=1037 ) majority of patients ( 20 [ 62.5% ] ) were encountered with single interacting combination . this was followed by the patients who encountered two ( 8 [ 25% ] ) and three interactions ( 4 [ 12.5% ] ) . significant proportion of potential drug interactions identified was of moderate severity ( 30 [ 62.5% ] ) while just 18 interacting combinations identified were of major severity ( 37.5% ) . the interacting pairs of major severity along with the potentially hazardous effects and documentation status are enlisted in the table 3 . among 48 pddi identified , 6 ( 12.5% ) had excellent status of documentation , 36 ( 75.5% ) had good documentation status and 6 ( 12.0% ) were of fair documentation status . in total , 28 ( 58.3% ) pddi was of pharmacokinetic type , 16 ( 33.3% ) were of pharmacodynamic type and remaining 4 ( 8.4% ) were of unknown mechanism . the most common interacting pairs identified in this study were atorvastatin / azithromycin ( 5 [ 10.4% ] ) , enalapril / metformin ( 5 [ 10.4% ] ) , enalapril / potassium chloride ( 5 [ 10.4% ] ) , atorvastatin / clarithromycin ( 4 [ 8.3% ] ) and furosemide / gentamicin ( 3 [ 6.3% ] ) . atorvastatin ( 16 [ 33.3% ] ) was the topmost drug found to be involved in potential ddi followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . length of hospital stay , number of medicines and concurrent illness were the factors studied . the value obtained in the present study is relatively less compared with the study by patel et al . these differences might be because our study took into consideration only the potential drug interactions of major and moderate severity in contrast to the other studies that considered drug interactions of all severity . data regarding the incidence of pddi in cardiac patients is not sufficient but previous studies in general medicine in mth had identified the maximum involvement of cardiovascular medicines in drug interactions . on analyzing the mechanism of drug interaction identified here , pharmacokinetic type of reactions ( 58.3% ) was found in higher number compared to pharmacodynamic type ( 33.3% ) . of the total pddis identified , the interacting combination of moderate severity ( 62.5% ) constituted majority of pddi . the most common interacting pairs identified were atorvastatin / azithromycin , enalapril / metformin , enalapril / potassium chloride , atorvastatin / clarithromycin and furosemide / gentamicin . the pddi involving atorvastatin ( 16 [ 33.3% ] ) was the highest among all followed by enalapril ( 15 [ 31.2% ] ) , digoxin ( 4 [ 8.3% ] ) , furosemide ( 4 [ 8.3% ] ) , clopidogrel ( 3 [ 6.3% ] ) and warfarin ( 3 [ 6.3% ] ) . reported aspirin ( 44.85% ) , heparin ( 42.78% ) , clopidogrel ( 22.16% ) , warfarin ( 11.59% ) , atorvastatin ( 7.22% ) and ramipril ( 6.95% ) as the highest risk drug categories for ddi . there was maximum involvement of atorvastatin ( 16 [ 33.3% ] ) in pddi encountered in this study . this might be because atorvastatin ( 68 [ 14.7% ] ) was one of the most commonly prescribed medicines in the present study . the reason might be that the likelihood of getting the multiple drugs increases with the increased length of hospital stay which in turn will increase the likelihood of pddi . similar positive linear relationship was also found between the number of medicines prescribed and pddi ( r = 0.5 , p < 0.01 ) and with concurrent illness and pddi ( r = 0.62 , p < 0.01 ) . this emphasizes the need to consider pddi during therapeutic planning , protect patients from consequence of drug interactions . in addition , providing ddi related information to the prescribers and drug interaction alert software to the dispensing pharmacist can play a vital role in minimizing the incidence rate of ddi . in the present study , monitor the occurrences of ddi clinically along with pharmacokinetic estimation of the drug is suggested , especially for moderate to major pddi .

sodium chloride ( nacl ) is the most abundant salt on earth and its crystallization is a key factor in many processes . apart from its essential role in the food industry , it is one of the key components for paper and soda ash production . sodium chloride is also commonly used in oil well drilling and is the primary deicing compound on roads during winter with an average use of 10 million tons per year in the united states and 1 million tons per year in france . for most of the applications , the salt has to be transported from production to storage locations . because of the hygroscopic properties of the salt and with environmental fluctuations of temperature and relative humidity , the salt grains have the tendency to lump together . in order to prevent this , additives known as anticaking agents , mostly ferrocyanides , are added to the salt in order to prevent crystals from growing together and agglomerating . there is still a large research effort going into the development of new anticaking agents that do not pose any risk to the environment and our health . deicing salts on the roads inevitably end up dissolving in water when the snow and ice melt , and by capillary action invade the groundwater and subsequently end up in civil engineering structures ( i.e. , bridges and buildings ) and outdoor artworks . their presence there can produce damage ; both mass loss and weakening have been reported . as an example , chloride corrosion is a major issue for bridges ; the salt solution which penetrates through existing cracks in the concrete can induce the corrosion of the steel that is used for the reinforcement . subsequently , the expansive forces produced from the corroded steel will induce the spalling of the concrete . furthermore , with temperature and relative humidity fluctuations , through evaporation of the water , the dissolved salt can again crystallize in the porous materials . direct consequences of crystallization in such porous media are the reduction of the permeability , a major problem in oil recovery and soil mechanics , and the weathering of the mechanical and physical properties of civil engineering structures and artworks . it has been reported that salt crystallization in the porous network ( i.e. , confinement ) can lead to the development of a crystallization pressure exerted by the growing crystal onto the pore walls , that eventually causes disintegration of the stones . a strategy that has received considerable attention is again the use of additives that alter the crystallization phenomenon in such a way that it may prevent the development of the crystallization pressure . among these , surfactant molecules are an important class of additives that can modify the crystal growth process when present at very small concentrations , by adsorbing preferentially onto different crystalline faces . surfactants or amphiphilic compounds are composed of a hydrophilic ( polar ) head and a hydrophobic ( nonpolar ) hydrocarbon chain . this configuration enables them to adsorb at the interface between two immiscible liquids such as oil / water and water / air . surfactants can also adsorb at solid liquid interfaces and may even adsorb differently on different crystal facets , which has a significant influence on crystal nucleation and growth . most studies report that the presence of surfactants inhibits crystallization , but there are also known exceptions in which to the contrary they increase the nucleation rate . for instance , the presence of sodium dodecyl sulfate was observed to increase the nucleation rate of mgso4 . these contradictory examples suggest that the crystal impurity interactions are highly specific and need to be treated individually . even after numerous studies , no conclusive theory has been developed to predict the influence of surfactants on salt nucleation that allows one to tailor the surfactants for achieving specific effects on the formation of salt crystals . in this paper , we study the influence of different surfactant additives on nacl crystallization in confinement during evaporation of the ternary solutions . to be able to discuss these issues , we first need to consider the counter - effect of high salt concentrations on the properties of surfactant solutions themselves . usually , the properties of ionic ( charged ) surfactants are tuned by adding small amounts of salt ; in our case , however , we deal with very concentrated salt solutions close to the solubility limit of the salts for which not a lot of data can be found in the literature . we find that only cationic and nonionic surfactants are stable in solution at these high electrolyte concentrations . the presence of these surfactants is subsequently shown to inhibit the precipitation of the salt . this , in turn , leads to high supersaturation , which changes the dynamics of crystal growth . another important finding is that the evaporation of water from the salt and surfactant solutions is not influenced by the presence of surfactants , contrary to what is sometimes claimed in the literature . low concentrations of soluble salts are commonly used to tailor surfactants properties such as the critical micellar concentration ( cmc ) , the aggregation number in the micelles , and the equilibrium surface tension . the presence of the latter can alter the electrostatic repulsion between the headgroups ( for charged surfactants ) and the debye layer around the micelles , leading to changes in micelle size and shape . however , there is only a limited number of studies on micelle formation at concentrations of sodium chloride close to saturation , which is relevant for crystallization . these studies also discuss the miscibility of salts within the surfactant micelles and the adsorbed films in order to assess the modified characteristics of micellar systems by the addition of salt . in principle , the thermodynamic analysis of the surfactant behavior in the presence of salts can also be used to deduce any possible change in the composition of micelles , but this is beyond the scope of the present paper , which focuses on the effect of surfactants on the crystallization of the salt . in order to choose our surfactants for the crystallization experiments , we first tested different type of surfactants : cationic , anionic , and nonionic for their solution stability ( table 1 ) . ternary solutions of nacl and surfactant in water are prepared by first preparing surfactant solutions at their critical micellar concentration ( cmc ) followed by adding nacl ( sigma - aldrich purity 99% ) to a concentration of 5.5 m ( molal or mol / kg of water ) which is slightly below the saturation concentration ( cs = 6.14 m ) . all the anionic surfactants precipitated upon the addition of salt ; the cationic surfactants ctab and ctac remain stable and form transparent solutions . the origin of this interesting asymmetry between cationic and anionic surfactants is unclear . among the nonionic surfactants , tween 80 is the only one that remains stable in the presence of high nacl concentration . the sensitivity to the salt concentration is found to be different for the anionic and nonionic surfactants . for example , the threshold concentration for the anionic one , sds , is around 0.5 m of nacl , whereas the nonionic surfactant triton - x-100 can stay stable up to 4 m of salt ; precipitation is observed rapidly above these concentrations . consequently , for the crystallization experiments in the presence of surfactants only ctab ( sigma - aldrich purity 99% ) and tween 80 ( ameresco , proteomics grade purity ) which make transparent ternary solutions were chosen ( figure 1 ) . the physicochemical properties of ctab and tween 80 ( figure 2 ) solutions were characterized prior to the crystallization experiments . the transparent solutions of ( a ) ctab and ( b ) tween 80 and precipitation with ( c ) sds and ( d ) triton - x-100 . the surfactant adsorption at the liquid / air interface was characterized by measuring the surface tension lv and the cmc of the ternary solutions using a kruss k100 tensiometer based on the du noy ring method . the latter is based on measuring the force to slowly raise a platinum ring , dipped in the liquid , from the liquid s surface . the surface tension is then calculated from the diameter of the ring and the tear - off force . the results as a function of the concentration were obtained by performing automatic dilutions ; we start at high surfactant concentration at 5.5 m nacl and dilute with salt solution at 5.5 m. in this way , the instrument allows us to measure the surface tension values for very small concentration steps of the surfactant . as far as equilibration of the solutions is concerned , for each dilution step , the solution is stirred for 30 s , and the value of the measured sft is registered only when the standard deviation between five consecutive measurements is within 0.1 mn / m . consequently , the average total time taken for one measurement is around 10 min ( depending upon how fast the system equilibrates ) which is normally long enough for the equilibration of most the surfactants . the adsorption at the solid / liquid interface of glass of both surfactants in the salt solution was studied by performing sum frequency generation ( sfg ) experiments . we have probed the ch vibrations of the surfactants and the oh vibrations of the water molecules at the solid interface . in this method , a broadband infrared laser beam exciting molecular vibrations and a narrowband near visible laser beam the resulting sum - frequency signal is strongly enhanced if the infrared is resonant with a molecular vibration . because of its selection rules , this process is forbidden in centrosymmetric media , like bulk water , therefore giving the ir spectrum of only interfacial molecules . the signal intensity is a measure of the amount of order present at the interface . the primary nucleation and growth of nacl crystals was studied in confined geometry by controlled evaporation of aqueous solutions under the microscope . the concentrations of ctab and tween 80 used were 9 10 m and 7.6 10 m , respectively , both of which are above their respective cmcs and the nacl concentration used was 5.5 m which is slightly below the saturation concentration . in order to observe the influence of surfactants on crystallization , the results are compared with the evaporation and crystallization of pure nacl solution at 5.5 m. schematic of the experimental setup used for following the evaporation of ternary solutions ( initial volume v0 ) and the crystallization in microcapillary ( d = 100 m ) . c is the controlled water vapor concentration of the climatic chamber and ci(t ) the water vapor concentration just above the meniscus at time t ; (t ) is the distance over which water vapor diffusion takes place during the evaporation from the open side of the capillary . a small volume of salt solutions with and without surfactants were introduced by capillary action in cylindrical borosilicate glass microcapillaries ( vitrocom , d = 100 m ) . after closing one of the sides of the microcapillary using epoxy glue , the capillary was placed in a controlled miniclimatic chamber held at a constant relative humidity ( rh = 55 2% ) and room temperature ( t = 22 1 c ) . this whole construction is placed under a leica dm irm inverted microscope and the subsequent displacement of the meniscus resulting from evaporation is followed with 5 and 10 magnification objectives from which the change in volume is calculated by analyzing the images taken by a ccd camera . the latter has a resolution of 800 600 pixels and a 8-bit sensitivity ; the time interval between the images was set to 5 s. the volume variation also allows us to determine the evolution of the concentration and calculate the supersaturation of the solution prior to crystallization . once crystallization happens , the shape of the crystal and its growth rate are followed during each experiment . we have measured the surface tension of ctab and tween 80 solutions as a function of concentration of the surfactants in the absence and presence of nacl in the solution ( figure 4 ) . two salt concentrations were used : a relatively small concentration 0.01 m and a high salt concentration 5.5 m ; the latter is used subsequently as the initial concentration in the crystallization experiments . the cmc values obtained for ctab in water and in brine solution at 0.01 m are seen to be in good agreement with the literature data ; the addition of nacl to the ctab solution drastically shifts the cmc of ctab from 9 10 m without salt to 2 10 m at high nacl concentration ( 5.5 m ) . in addition to the decrease of the cmc , another important observation here was that the equilibrium surface tension after the cmc was seen to be lower cmc 32 mn / m at 5.5 m nacl compared to that of the pure solution of ctab without salt ( cmc 37 mn / m ) . interestingly , the slope below the cmc tells us ( using the gibbs adsorption equation ) that without salt , there is more ctab present on the interface . the lower surface tension at the cmc that is observed when salt is added must then be due to the change in chemical potential of the surfactant molecules in the bulk due to the presence of salt . surface tension as a function of ( a ) ctab and ( b ) tween 80 in water and salt solutions at different concentrations . in the case of nonionic surfactants also , studies have reported that the addition of electrolytes decreases the cmc value , but surprisingly , for the case of tween 80 , we do not observe a significant change in the cmc ( 1.5 10 m ) even in the presence of very high salt concentrations . also unlike the case of ctab , there is no observable change of the surface tension at the cmc upon the addition of salt , lv - cmc 42 mn / m . the different behavior of ionic and nonionic surfactants in terms of decrease in cmc can be attributed to the fact that addition of salts in ionic surfactants decreases the electrostatic repulsion between the head groups , thereby favoring their aggregation . however , in the case of the nonionic surfactant tween 80 , the repulsion between the head groups is not a limiting factor for micellization , which is the reason we do not see any change in cmc upon addition of salt . therefore , we start our crystallization experiments with ternary solutions of ctab and tween 80 that have similar cmc , whereas without salt the cmc of the ionic surfactant is much higher . for the crystallization experiments , the volume change during the evaporation of the solutions inside the microcapillaries is subsequently followed by recording the displacement of the meniscus while simultaneously visualizing the onset of crystal growth in the solution directly under the microscope coupled to a ccd camera . in this way , the evolution of the concentration with the evaporation up to the crystallization point can be determined ( figure 5 ) . the first important parameter examined to account for the influence of surfactants on the crystallization is the supersaturation , s = c / cs , at which nucleation takes place ( figure 5 ) . the supersaturation is defined as the ratio of the concentration at the moment of crystallization c to the saturation concentration cs ( 6.14 m ) ; thus , the value of the supersaturation gives an indication whether the nucleation is inhibited or promoted compared to the crystallization of pure nacl solution under the same experimental conditions . ( table 2 ) summarizes the mean value of the supersaturation obtained over 20 experiments for each solution . for a pure sodium chloride solution , the supersaturation at which the spontaneous nucleation and growth occurs was found to be s = 1.57 0.1 , in agreement with earlier experiments . in the presence of ctab and moreover , in the presence of surfactants , the variability in the supersaturation in different experiments becomes smaller . the increase of the supersaturation in the presence of surfactants suggests that the crystallization is inhibited by both ctab as well as tween 80 in the solution . these very high supersaturations lead to a rapid growth of the crystal which favors the formation of only a single crystal in small volume ( both with and without surfactant ) with a peculiar hoppered ( skeletal ) shape ( figure 6a ) . the hopper growth is only observed at early times in the growth process ; at later times due to the rapid decrease of the local supersaturation , the extremities of the hopper crystal will continue a slower growth as a cubic crystal eating up the smaller cubes attached to it . first appearance of crystals ( 5 s time - window ) in ( a ) pure nacl solutions , ( b ) the multibranched hopper formed in the nacl solution with ctab , and ( c ) the solution with tween 80 ; when the hopper crystal grows rapidly and reaches the wall of the capillary , a change in the direction of the branches can be seen . ( d , e ) illustration of hopper crystal with one branch and ( e ) star - like hopper crystal ( multibranched ) formed in bulk in the presence of ctab and tween 80 . another important observation here is that in the presence of surfactants the nucleation happens in the bulk of the solution , in contrast to the pure nacl solution where the growth of the crystal takes place mostly at the liquid this can explain the higher supersaturation reached when surfactants are added ; the nucleation at the interface is heterogeneous , and the energy barrier for heterogeneous nucleation near a surface is smaller than that for homogeneous nucleation . in general , nucleation is easier in contact with a surface ; because of geometrical reasons the size of the critical nucleus is smaller . this reduction in surface area of the nucleus reduces the height of the energy barrier that needs to be overcome in order to form the surface of the growing nucleus . air interface , i.e. , passivation of this interface when surfactants are present leads to a higher supersaturation before nucleation to takes place . figure 7 shows sfg spectra for the glass surface in contact with different salt solutions with and without surfactants . for the case of water and the pure salt solution there is no ch vibration visible in the range between 2800 and 3000 cm . between 3000 and 3300 cm the water signal is very low for the salt case , because the salt screens the negatively charged sio2 surface and thereby reduces the order of the water molecules and thus the sfg signal . in the cases of tween 80 + nacl and ctab + nacl , weak but clear peaks are visible in the range of 2900 to 2970 cm . this is a clear indication that both the nonionic tween 80 as well as the cationic ctab are present at the solid surface . sfg spectra for different salt solutions in contact with a glass ( sio2 ) window . the water curve has been multiplied by 0.2 to make the intensity comparable to that of the other curves . moreover , our results show that surfactant molecules or micelles in the solution do not act as nucleation sites in these experiments . if this was really the case , it should make nucleation more favorable , and consequently spontaneous precipitation and growth should be seen at lower supersaturation compared to pure nacl . we observe , to the contrary , that with surfactants the nucleation is rather inhibited ( higher supersaturation are reached prior to nucleation ) . star - like hopper crystals because of the precipitation of new cubic crystals on the edge of the pre - existing cubic nucleus in the bulk . because the rapid growth of the hopper crystal is unchanged in the presence of tween 80 and ctab , their adsorption to the supercritical nuclei limiting the growth is excluded by our observations . it is well - known that in salt solutions the evaporation rate could be another key factor determining the supersaturation at which nucleation takes place . if the nucleation happens at the liquid meniscus , such as for the pure nacl solution , it could be that the local supersaturation is higher in this region as the evaporation takes place at the liquid / air interface . if the evaporation is faster than the ion transport in the solution , a concentration gradient can develop close to the meniscus with a higher local concentration that can lead to an increase of the nucleation rate . we therefore examine the influence of ctab and tween 80 on the evaporation rate of nacl solution . it has been suggested by some authors that surfactants , by virtue of their partitioning at the interface , can block the evaporative surface , thereby leading to slower evaporation rates in cases of droplets , microcapillaries , as well as flat surfaces . the variation in volume of the solution in the cylindrical capillary is followed throughout the evaporation process ; the evaporation rate dv / dt ( m / s ) is shown in figure 8 . however , no change is observed in the evaporation rate when ctab surfactant is added to water and to the salt solution . this is in fact not so surprising if one notes that in general for the evaporation of water the diffusion of water molecules through the vapor is the rate - limiting step:1where e is the evaporation flux density ; g the vapor density ; d the diffusion coefficient of water vapor through the gas ; c the controlled water vapor concentration of the climatic chamber ( 55% ) and ci the water vapor concentration just above the meniscus ; is the distance over which diffusion takes place ( figure 3 ) . we conclude that the surfactants do not have any significant influence on the evaporation rate . it is clear from eq 1and figure 3 that the drying rate is controlled by and ( ci the equilibrium vapor pressure above the meniscus remains constant and only will change over time with the displacement of the meniscus . this is the only reason for the decrease of the evaporation rate in time in the case of water . however , for salt solutions , apart from increasing , the second reason responsible for the decrease in the evaporation rate is the increase of salt concentration in time , which also changes the equilibrium vapor pressure above the meniscus ci(t ) and the density of the liquid l(t ) . subsequently , eq 2 written in terms of time gives2where is the evaporation rate ; g the density of the gas phase ; l the density of liquid phase ; d the diffusion coefficient of water through the vapor ; ci the water vapor mass fraction above the interface ; c the water vapor mass fraction outside the capillary ; 0 the initial vapor diffusion distance of meniscus ; a the cross - sectional area r ( r is the radius of the capillary ) . as can be seen in figure 8 , the model based on such diffusive transport describes the data very well , and for salt solution with and without surfactant , the decrease of the evaporation rate in time is therefore due to the effects of both and ( ci c ) . also , in agreement with this idea , the small characteristic dimension of our samples and the very slow evaporation rates ensures the homogeneity of solutions . quantitatively , the heterogeneity in ion distribution in a solution can be inferred from the peclet number , which is defined as the ratio of the convective and diffusive transport of ions in the solution . since the peclet number depends on the concentration of ions and the size of the system , in evaporating systems , it is time - dependent and changes as the evaporation progresses:3where the diffusion time tdiff = z / dnacl and the convection time ts = z/(dz / dt ) ; z is the length of the solution cylinder at time t and dnacl is the diffusion coefficient . for pure nacl solution as well as with ctab and tween 80 , the values of peclet number were found to be on the order of 10 to 10 at the point of crystallization . microcapillary experiments ( at t = 21 c , rh = 55% 2 ) : evolution of the evaporation rate for water and brine with and without surfactants ( ctab and tween 80 ) . the continuous line corresponds to the model based on diffusive transport ( eq 2 ) . the surfactant has no impact on the evaporation rate . the slight differences are mainly due to slight fluctuations of the experimental conditions in different microcapillaries ( notably the distance between the meniscus and the outlet of the capillary and the relative humidity ( 2% ) . finally , we have determined with a simple method whether besides their role as nucleation inhibitor the micelles are incorporated within the crystal structure of sodium chloride because of its rapid growth . it has been shown by afm experiments that for calcite crystals , micelles can be entrapped during growth because they bind specifically to steps , enabling successive steps to close around them . it is reported that such incorporation can even increase at higher growth rates because of the greater rate of step generation with increasing supersaturation . to see whether this is also the case in our experiments , we perform bulk evaporation experiments in the same evaporation conditions , to have a larger quantity of crystal than in the microcapillary experiments . in the bulk experiment , we observe the same trend : ctab and tween 80 inhibit the nucleation process ; i.e. , crystal precipitation occurs sooner in a pure salt solution . the ctab / nacl solution is the next one to crystallize and tween 80 the final one . subsequently , the precipitated crystals ( figure 9 ) were collected , rinsed with a saturated sodium chloride solution , weighed , and dissolved in water to obtain nacl solutions at concentration of 5.5 m. the rinsing with salt solution allows removal of physisorbed surfactant molecules from the crystal surface . the chemistry of the nacl crystal surface and the functional groups at the hydrophilic head of the surfactant molecules makes it very unlikely for any chemisorption to take place . the pendant drop method employing a kruss easy - drop instrument is used then to measure the surface tension of the resulting solutions obtained by the dissolution of the crystals . if surfactants are incorporated in the crystal , they will redissolve and lower the surface tension ; if not , the surface tension will be that of the 5.5 m salt solution . dynamic surface tension measurements using pendant drop technique of salt solutions ( 5.5 m ) made by the dissolution of crystals : a , b , and c in water . the precipitated crystals were collected , rinsed with saturated sodium chloride solution , weighed , and dissolved in water to obtain a solution at concentration of 5.5 m. the measurements were done at 75% relative humidity in order to avoid any evaporation process and concentration change , which could affect the value of the surface tension during the measurement time . our results show that the surface tension of salt solution made by the crystal collected from ctab solution , within the experimental resolution , is identical to the surface tension of pure nacl solution at 5.5 m ( 80 mn / m ) indicating the absence of any ctab in the collected crystal . however , in the case of the solution made by the crystal collected from the tween 80 solution , a lower surface tension of 64 mn / m is measured which clearly reveals the presence of tween 80 molecules in the solution after the dissolution of the crystal ( figure 9 ) . the surface tension measurement by the pendant drop technique is therefore an easy way to obtain information on the interaction / incorporation of surfactants in salt crystals . in the context of crystallization damage , such incorporation can alter the mechanical properties of the crystallized salt . in the context of biomineralization , differences between different surfactants such as the one observed here can perhaps be useful to elucidate the conditions under which incorporation occurs . in this paper , we present results on the evaporation of ternary solutions of surfactants and nacl at high salt concentrations until crystallization occurred . the anionic surfactants tested in this study are found to be incompatible with highly concentrated nacl solutions : they precipitate . on the other hand , the cationic surfactants ctab ( and ctac ) and the nonionic surfactant tween 80 form homogeneous ternary solutions . the high electrolyte concentration reduces the cmc value and the surface tension at cmc of the cationic surfactant ; no change is observed for the nonionic . our results show that the evaporation rate is not affected by the presence of surfactants as the evaporation rate is controlled by diffusive transport through the gas phase . the presence of both surfactants in the salt solution delay the crystal nucleation by a significant amount leading to a high supersaturation prior to precipitation during evaporation . this inhibitor role seems to be due to a passivation of nucleation sites , i.e. , the availability of the liquid / air and solid / liquid interface . it follows from thermodynamic considerations that the free energy for the nucleation process mediated at a surface can be roughly half of that required to nucleate in the bulk , which provides a plausible explanation for the higher supersaturations reached when nucleation at the solution / air or solid / liquid interface is prevented by the presence of surfactants there . the high supersaturation has the consequence that in the first seconds of precipitation the rapid growth of one star - like hopper crystal is observed in the solution . because the nucleation occurs in the bulk , the higher the supersaturation is , the more branched the hopper crystal appears . even though the growth of the nucleus is very fast , the incorporation of surfactant micelles in the crystalline structure depends on the nature of the surfactant . surface tension measurements have been used as a simple and accurate method to get information on the interaction / incorporation of surfactants in the crystalline structure . our results show that the nonionic tween 80 surfactant is incorporated in nacl crystals . the cationic surfactant is not , which could be due to some electrostatic repulsion between the negatively charged surfaces of the nacl crystal and the cationic micelles surrounded by chlorine ions . the incorporation of copolymer micelles in calcium carbonate crystals has been used as a simple model to understand biomineral formation ; a mechanism of occlusion within single salt crystals has been proposed recently to describe this . the results reported in this paper bring some new insights on the role of surfactants on the kinetics of salt precipitation at high electrolyte concentration and their interaction with the crystalline structure .

we study the influence of different surfactants on nacl crystallization during evaporation of aqueous salt solutions . we found that at concentrations of sodium chloride close to saturation , only the cationic surfactant ctab and the nonionic surfactant tween 80 remain stable . for the nonionic surfactant , the high concentration of salt does not significantly change either the critical micellar concentration ( cmc ) or the surface tension at the cmc ; for the cationic surfactant , the cmc is reduced by roughly 2 orders of magnitude upon adding the salt . the presence of both types of surfactants in the salt solution delays the crystallization of sodium chloride with evaporation . this , in turn , leads to high supersaturation which induces the rapid precipitation of a hopper crystal in the bulk . the crystallization inhibitor role of these surfactants is shown to be mainly due to the passivation of nucleation sites at both liquid / air and solid / liquid interfaces rather than a change in the evaporation rate which is found not to be affected by the presence of the surfactants . the adsorption of surfactants at the liquid / air interface prevents the crystallization at this location which is generally the place where the precipitation of sodium chloride is observed . moreover , sum frequency generation spectroscopy measurements show that the surfactants are also present at the solid / liquid interface . the incorporation of the surfactants into the salt crystals is investigated using a novel , but simple , method based on surface tension measurements . our results show that the nonionic surfactant tween 80 is incorporated in the nacl crystals but the cationic surfactant ctab is not . taken together , these results therefore allow us to establish the effect of the presence of surfactants on sodium chloride crystallization .

Introduction Experimental Section Results and Discussion Conclusion

as an example , chloride corrosion is a major issue for bridges ; the salt solution which penetrates through existing cracks in the concrete can induce the corrosion of the steel that is used for the reinforcement . , confinement ) can lead to the development of a crystallization pressure exerted by the growing crystal onto the pore walls , that eventually causes disintegration of the stones . most studies report that the presence of surfactants inhibits crystallization , but there are also known exceptions in which to the contrary they increase the nucleation rate . for instance , the presence of sodium dodecyl sulfate was observed to increase the nucleation rate of mgso4 . even after numerous studies , no conclusive theory has been developed to predict the influence of surfactants on salt nucleation that allows one to tailor the surfactants for achieving specific effects on the formation of salt crystals . in this paper , we study the influence of different surfactant additives on nacl crystallization in confinement during evaporation of the ternary solutions . usually , the properties of ionic ( charged ) surfactants are tuned by adding small amounts of salt ; in our case , however , we deal with very concentrated salt solutions close to the solubility limit of the salts for which not a lot of data can be found in the literature . the presence of these surfactants is subsequently shown to inhibit the precipitation of the salt . this , in turn , leads to high supersaturation , which changes the dynamics of crystal growth . another important finding is that the evaporation of water from the salt and surfactant solutions is not influenced by the presence of surfactants , contrary to what is sometimes claimed in the literature . low concentrations of soluble salts are commonly used to tailor surfactants properties such as the critical micellar concentration ( cmc ) , the aggregation number in the micelles , and the equilibrium surface tension . however , there is only a limited number of studies on micelle formation at concentrations of sodium chloride close to saturation , which is relevant for crystallization . in principle , the thermodynamic analysis of the surfactant behavior in the presence of salts can also be used to deduce any possible change in the composition of micelles , but this is beyond the scope of the present paper , which focuses on the effect of surfactants on the crystallization of the salt . ternary solutions of nacl and surfactant in water are prepared by first preparing surfactant solutions at their critical micellar concentration ( cmc ) followed by adding nacl ( sigma - aldrich purity 99% ) to a concentration of 5.5 m ( molal or mol / kg of water ) which is slightly below the saturation concentration ( cs = 6.14 m ) . all the anionic surfactants precipitated upon the addition of salt ; the cationic surfactants ctab and ctac remain stable and form transparent solutions . among the nonionic surfactants , tween 80 is the only one that remains stable in the presence of high nacl concentration . the sensitivity to the salt concentration is found to be different for the anionic and nonionic surfactants . for example , the threshold concentration for the anionic one , sds , is around 0.5 m of nacl , whereas the nonionic surfactant triton - x-100 can stay stable up to 4 m of salt ; precipitation is observed rapidly above these concentrations . consequently , for the crystallization experiments in the presence of surfactants only ctab ( sigma - aldrich purity 99% ) and tween 80 ( ameresco , proteomics grade purity ) which make transparent ternary solutions were chosen ( figure 1 ) . the surfactant adsorption at the liquid / air interface was characterized by measuring the surface tension lv and the cmc of the ternary solutions using a kruss k100 tensiometer based on the du noy ring method . the results as a function of the concentration were obtained by performing automatic dilutions ; we start at high surfactant concentration at 5.5 m nacl and dilute with salt solution at 5.5 m. in this way , the instrument allows us to measure the surface tension values for very small concentration steps of the surfactant . the adsorption at the solid / liquid interface of glass of both surfactants in the salt solution was studied by performing sum frequency generation ( sfg ) experiments . we have probed the ch vibrations of the surfactants and the oh vibrations of the water molecules at the solid interface . the concentrations of ctab and tween 80 used were 9 10 m and 7.6 10 m , respectively , both of which are above their respective cmcs and the nacl concentration used was 5.5 m which is slightly below the saturation concentration . in order to observe the influence of surfactants on crystallization , the results are compared with the evaporation and crystallization of pure nacl solution at 5.5 m. schematic of the experimental setup used for following the evaporation of ternary solutions ( initial volume v0 ) and the crystallization in microcapillary ( d = 100 m ) . we have measured the surface tension of ctab and tween 80 solutions as a function of concentration of the surfactants in the absence and presence of nacl in the solution ( figure 4 ) . the cmc values obtained for ctab in water and in brine solution at 0.01 m are seen to be in good agreement with the literature data ; the addition of nacl to the ctab solution drastically shifts the cmc of ctab from 9 10 m without salt to 2 10 m at high nacl concentration ( 5.5 m ) . in addition to the decrease of the cmc , another important observation here was that the equilibrium surface tension after the cmc was seen to be lower cmc 32 mn / m at 5.5 m nacl compared to that of the pure solution of ctab without salt ( cmc 37 mn / m ) . the lower surface tension at the cmc that is observed when salt is added must then be due to the change in chemical potential of the surfactant molecules in the bulk due to the presence of salt . surface tension as a function of ( a ) ctab and ( b ) tween 80 in water and salt solutions at different concentrations . in the case of nonionic surfactants also , studies have reported that the addition of electrolytes decreases the cmc value , but surprisingly , for the case of tween 80 , we do not observe a significant change in the cmc ( 1.5 10 m ) even in the presence of very high salt concentrations . also unlike the case of ctab , there is no observable change of the surface tension at the cmc upon the addition of salt , lv - cmc 42 mn / m . however , in the case of the nonionic surfactant tween 80 , the repulsion between the head groups is not a limiting factor for micellization , which is the reason we do not see any change in cmc upon addition of salt . therefore , we start our crystallization experiments with ternary solutions of ctab and tween 80 that have similar cmc , whereas without salt the cmc of the ionic surfactant is much higher . for the crystallization experiments , the volume change during the evaporation of the solutions inside the microcapillaries is subsequently followed by recording the displacement of the meniscus while simultaneously visualizing the onset of crystal growth in the solution directly under the microscope coupled to a ccd camera . in this way , the evolution of the concentration with the evaporation up to the crystallization point can be determined ( figure 5 ) . the first important parameter examined to account for the influence of surfactants on the crystallization is the supersaturation , s = c / cs , at which nucleation takes place ( figure 5 ) . the supersaturation is defined as the ratio of the concentration at the moment of crystallization c to the saturation concentration cs ( 6.14 m ) ; thus , the value of the supersaturation gives an indication whether the nucleation is inhibited or promoted compared to the crystallization of pure nacl solution under the same experimental conditions . in the presence of ctab and moreover , in the presence of surfactants , the variability in the supersaturation in different experiments becomes smaller . the increase of the supersaturation in the presence of surfactants suggests that the crystallization is inhibited by both ctab as well as tween 80 in the solution . the hopper growth is only observed at early times in the growth process ; at later times due to the rapid decrease of the local supersaturation , the extremities of the hopper crystal will continue a slower growth as a cubic crystal eating up the smaller cubes attached to it . first appearance of crystals ( 5 s time - window ) in ( a ) pure nacl solutions , ( b ) the multibranched hopper formed in the nacl solution with ctab , and ( c ) the solution with tween 80 ; when the hopper crystal grows rapidly and reaches the wall of the capillary , a change in the direction of the branches can be seen . ( d , e ) illustration of hopper crystal with one branch and ( e ) star - like hopper crystal ( multibranched ) formed in bulk in the presence of ctab and tween 80 . another important observation here is that in the presence of surfactants the nucleation happens in the bulk of the solution , in contrast to the pure nacl solution where the growth of the crystal takes place mostly at the liquid this can explain the higher supersaturation reached when surfactants are added ; the nucleation at the interface is heterogeneous , and the energy barrier for heterogeneous nucleation near a surface is smaller than that for homogeneous nucleation . this is a clear indication that both the nonionic tween 80 as well as the cationic ctab are present at the solid surface . moreover , our results show that surfactant molecules or micelles in the solution do not act as nucleation sites in these experiments . star - like hopper crystals because of the precipitation of new cubic crystals on the edge of the pre - existing cubic nucleus in the bulk . because the rapid growth of the hopper crystal is unchanged in the presence of tween 80 and ctab , their adsorption to the supercritical nuclei limiting the growth is excluded by our observations . if the nucleation happens at the liquid meniscus , such as for the pure nacl solution , it could be that the local supersaturation is higher in this region as the evaporation takes place at the liquid / air interface . if the evaporation is faster than the ion transport in the solution , a concentration gradient can develop close to the meniscus with a higher local concentration that can lead to an increase of the nucleation rate . we therefore examine the influence of ctab and tween 80 on the evaporation rate of nacl solution . the variation in volume of the solution in the cylindrical capillary is followed throughout the evaporation process ; the evaporation rate dv / dt ( m / s ) is shown in figure 8 . however , no change is observed in the evaporation rate when ctab surfactant is added to water and to the salt solution . this is in fact not so surprising if one notes that in general for the evaporation of water the diffusion of water molecules through the vapor is the rate - limiting step:1where e is the evaporation flux density ; g the vapor density ; d the diffusion coefficient of water vapor through the gas ; c the controlled water vapor concentration of the climatic chamber ( 55% ) and ci the water vapor concentration just above the meniscus ; is the distance over which diffusion takes place ( figure 3 ) . we conclude that the surfactants do not have any significant influence on the evaporation rate . this is the only reason for the decrease of the evaporation rate in time in the case of water . however , for salt solutions , apart from increasing , the second reason responsible for the decrease in the evaporation rate is the increase of salt concentration in time , which also changes the equilibrium vapor pressure above the meniscus ci(t ) and the density of the liquid l(t ) . as can be seen in figure 8 , the model based on such diffusive transport describes the data very well , and for salt solution with and without surfactant , the decrease of the evaporation rate in time is therefore due to the effects of both and ( ci c ) . quantitatively , the heterogeneity in ion distribution in a solution can be inferred from the peclet number , which is defined as the ratio of the convective and diffusive transport of ions in the solution . since the peclet number depends on the concentration of ions and the size of the system , in evaporating systems , it is time - dependent and changes as the evaporation progresses:3where the diffusion time tdiff = z / dnacl and the convection time ts = z/(dz / dt ) ; z is the length of the solution cylinder at time t and dnacl is the diffusion coefficient . for pure nacl solution as well as with ctab and tween 80 , the values of peclet number were found to be on the order of 10 to 10 at the point of crystallization . microcapillary experiments ( at t = 21 c , rh = 55% 2 ) : evolution of the evaporation rate for water and brine with and without surfactants ( ctab and tween 80 ) . the slight differences are mainly due to slight fluctuations of the experimental conditions in different microcapillaries ( notably the distance between the meniscus and the outlet of the capillary and the relative humidity ( 2% ) . the chemistry of the nacl crystal surface and the functional groups at the hydrophilic head of the surfactant molecules makes it very unlikely for any chemisorption to take place . if surfactants are incorporated in the crystal , they will redissolve and lower the surface tension ; if not , the surface tension will be that of the 5.5 m salt solution . dynamic surface tension measurements using pendant drop technique of salt solutions ( 5.5 m ) made by the dissolution of crystals : a , b , and c in water . the precipitated crystals were collected , rinsed with saturated sodium chloride solution , weighed , and dissolved in water to obtain a solution at concentration of 5.5 m. the measurements were done at 75% relative humidity in order to avoid any evaporation process and concentration change , which could affect the value of the surface tension during the measurement time . our results show that the surface tension of salt solution made by the crystal collected from ctab solution , within the experimental resolution , is identical to the surface tension of pure nacl solution at 5.5 m ( 80 mn / m ) indicating the absence of any ctab in the collected crystal . however , in the case of the solution made by the crystal collected from the tween 80 solution , a lower surface tension of 64 mn / m is measured which clearly reveals the presence of tween 80 molecules in the solution after the dissolution of the crystal ( figure 9 ) . the surface tension measurement by the pendant drop technique is therefore an easy way to obtain information on the interaction / incorporation of surfactants in salt crystals . on the other hand , the cationic surfactants ctab ( and ctac ) and the nonionic surfactant tween 80 form homogeneous ternary solutions . the high electrolyte concentration reduces the cmc value and the surface tension at cmc of the cationic surfactant ; no change is observed for the nonionic . our results show that the evaporation rate is not affected by the presence of surfactants as the evaporation rate is controlled by diffusive transport through the gas phase . the presence of both surfactants in the salt solution delay the crystal nucleation by a significant amount leading to a high supersaturation prior to precipitation during evaporation . this inhibitor role seems to be due to a passivation of nucleation sites , i.e. , the availability of the liquid / air and solid / liquid interface . it follows from thermodynamic considerations that the free energy for the nucleation process mediated at a surface can be roughly half of that required to nucleate in the bulk , which provides a plausible explanation for the higher supersaturations reached when nucleation at the solution / air or solid / liquid interface is prevented by the presence of surfactants there . the high supersaturation has the consequence that in the first seconds of precipitation the rapid growth of one star - like hopper crystal is observed in the solution . even though the growth of the nucleus is very fast , the incorporation of surfactant micelles in the crystalline structure depends on the nature of the surfactant . surface tension measurements have been used as a simple and accurate method to get information on the interaction / incorporation of surfactants in the crystalline structure . our results show that the nonionic tween 80 surfactant is incorporated in nacl crystals . the cationic surfactant is not , which could be due to some electrostatic repulsion between the negatively charged surfaces of the nacl crystal and the cationic micelles surrounded by chlorine ions .

table 1 summarizes the patients ' demographic and clinical characteristics of the discovery and validation sets . all serum samples were collected from the ongoing cohort of patients being followed in the climb study ( comprehensive longitudinal investigation of ms at brigham and women 's hospital ) in which participants are followed with comprehensive clinical and imaging assessments to monitor disease progression and response to therapy on a yearly basis . samples were collected within ( mean sd ) 5.0 3.2 months of mri acquisition . patients were free of relapses or changes in disease - modifying therapy during the interval between blood collection and mri . this was a consecutive sample meeting the following criteria : ( 1 ) age 18 to 55 years ; ( 2 ) diagnosis of relapsing - remitting ms ; ( 3 ) absence of other major medical , neurologic , or neuropsychiatric disorders ; ( 4 ) lack of any relapse or corticosteroid use in the 4 weeks before mri or start of disease - modifying therapy 6 months before mri ( to reduce confounding effects on mri ) ; and ( 5 ) no history of smoking or substance abuse . the majority of patients were receiving disease - modifying treatment at the time of mri . within 3 months of mri , each patient received an examination by an ms specialist - neurologist , including evaluation of neurologic disability on the expanded disability status scale and a timed 25-foot walk . demographic , clinical , and brain mri data our study received approval from the ethical standards committee on human experimentation at our institution ( the partners health care institutional review board ) . all participants in the discovery set underwent mri on the same scanner ( 3 t signa ; general electric healthcare , milwaukee , wi ) using a receive - only phase array head coil with the same mri protocol . contiguous slices covering the whole brain were acquired in high - resolution protocols using 3-dimensional modified driven equilibrium fourier transform and t2-weighted fast fluid - attenuated inversion recovery sequences . patients in the validation set underwent brain mri on a 1.5 t scanner ( ge signa ) including a 2-dimensional axial conventional spin - echo dual - echo t2-weighted series ( voxel sizes 0.94 0.94 3 mm ) . analysis of these scans was performed by operators who were unaware of clinical and biomarker information . in the discovery set , we obtained normalized compartment - specific global brain volumes as surrogates of atrophy , including gm fraction ( gmf ) , wm fraction ( wmf ) , and total brain parenchymal fraction ( bpf ) , using statistical parametric mapping version 8 ( wellcome department of cognitive neurology , london , uk , http://www.fil.ion.ucl.ac.uk/spm ) , after manual correction of ( 1 ) misclassifications of tissue compartments due to ms lesion , and ( 2 ) ineffective contouring of the deep gm structures . in the validation set , bpf and gmf were obtained in statistical parametric mapping version 8 from the dual - echo images . because the source images did not show effective contrast for segmentation of the deep gray structures , we performed manual masking to derive only the cerebral cortical gmf . quantification of total brain t2 hyperintense lesion volume ( t2lv ) was performed using jim ( xinapse systems ltd . , west bergholt , uk , http://www.xinapse.com ) by the consensus of 2 experienced observers from the fluid - attenuated inversion recovery ( discovery set ) or dual - echo ( validation set ) images using a semiautomated technique . for the measurement of these atrophy and lesion surrogates from mri scans , our methods are well established regarding their operational procedures , validity , and reliability . peptides were synthesized at the biopolymers facility of the department of biological chemistry and molecular pharmacology of harvard medical school . recombinant proteins and lipids were purchased from sigma ( st . louis , mo ) , abnova ( taipei city , taiwan ) , matreya llc ( pleasant gap , pa ) , avanti polar lipids ( alabaster , al ) , calbiochem ( san diego , ca ) , chemicon ( temecula , ca ) , genetex ( san antonio , tx ) , novus biologicals ( littleton , co ) , assay designs ( ann arbor , mi ) , prosci inc . ( poway , ca ) , emd biosciences ( san diego , ca ) , cayman chemical ( ann arbor , mi ) , hytest ( turku , finland ) , meridian life science ( memphis , tn ) , and biodesign international ( saco , me ) . the antigens used in the construction of antigen microarrays are listed in table e-1 at neurology.org/nn . the antigens listed in table e-1 were spotted in replicates of 6 on superepoxy 2 slides ( telechem , sunnyvale , ca ) using an arrayit nanoprint 2 lm210 microarray printer ( arrayit corporation , sunnyvale , ca ) and optimized spotting conditions as described . the microarrays were hybridized using an hs 4800pro hybridization station ( tecan , mnnedorf , switzerland ) , in which they were blocked with 1% bovine serum albumin for 1 hour and incubated for 2 hours at 37c with the test serum at a 1:100 dilution in blocking buffer . the arrays were then washed and incubated for 45 minutes with a 1:500 dilution of goat anti - human immunoglobulin g ( igg ) cy3-conjugated and goat anti - human igm cy5-conjugated detection antibodies ( jackson immunoresearch labs , west grove , pa ) . the arrays were scanned with a tecan powerscanner . repeated measurements indicate that our antigen microarray technique is reproducible , exhibiting a coefficient of variation of 13.3 1.2 . background signal was subtracted and raw data were normalized and analyzed using the genespring software ( silicon genetics , redwood city , ca ) . antigen reactivity was defined by the mean intensity of binding to the replicates of that antigen on the microarray and expressed as relative fluorescence units . pearson product - moment correlation coefficients were calculated between the mri measures ( bpf , gmf , wmf , t2lv ) and the weighted average of a group of statistically significant lipids . the weighted average of significant lipids was calculated using the formula ( wiai/wi ) ( wi = 1 , i = 1 , 2 , 3 ) , where wi is the proportion of intensity of antigen , ai , ai is the observed intensity of an antigen , and wi is the sum of the weights . table 1 summarizes the patients ' demographic and clinical characteristics of the discovery and validation sets . all serum samples were collected from the ongoing cohort of patients being followed in the climb study ( comprehensive longitudinal investigation of ms at brigham and women 's hospital ) in which participants are followed with comprehensive clinical and imaging assessments to monitor disease progression and response to therapy on a yearly basis . samples were collected within ( mean sd ) 5.0 3.2 months of mri acquisition . patients were free of relapses or changes in disease - modifying therapy during the interval between blood collection and mri . this was a consecutive sample meeting the following criteria : ( 1 ) age 18 to 55 years ; ( 2 ) diagnosis of relapsing - remitting ms ; ( 3 ) absence of other major medical , neurologic , or neuropsychiatric disorders ; ( 4 ) lack of any relapse or corticosteroid use in the 4 weeks before mri or start of disease - modifying therapy 6 months before mri ( to reduce confounding effects on mri ) ; and ( 5 ) no history of smoking or substance abuse . the majority of patients were receiving disease - modifying treatment at the time of mri . within 3 months of mri , each patient received an examination by an ms specialist - neurologist , including evaluation of neurologic disability on the expanded disability status scale and a timed 25-foot walk . our study received approval from the ethical standards committee on human experimentation at our institution ( the partners health care institutional review board ) . all participants in the discovery set underwent mri on the same scanner ( 3 t signa ; general electric healthcare , milwaukee , wi ) using a receive - only phase array head coil with the same mri protocol . contiguous slices covering the whole brain were acquired in high - resolution protocols using 3-dimensional modified driven equilibrium fourier transform and t2-weighted fast fluid - attenuated inversion recovery sequences . patients in the validation set underwent brain mri on a 1.5 t scanner ( ge signa ) including a 2-dimensional axial conventional spin - echo dual - echo t2-weighted series ( voxel sizes 0.94 0.94 3 mm ) . analysis of these scans was performed by operators who were unaware of clinical and biomarker information . in the discovery set , we obtained normalized compartment - specific global brain volumes as surrogates of atrophy , including gm fraction ( gmf ) , wm fraction ( wmf ) , and total brain parenchymal fraction ( bpf ) , using statistical parametric mapping version 8 ( wellcome department of cognitive neurology , london , uk , http://www.fil.ion.ucl.ac.uk/spm ) , after manual correction of ( 1 ) misclassifications of tissue compartments due to ms lesion , and ( 2 ) ineffective contouring of the deep gm structures . in the validation set , bpf and gmf were obtained in statistical parametric mapping version 8 from the dual - echo images . because the source images did not show effective contrast for segmentation of the deep gray structures , we performed manual masking to derive only the cerebral cortical gmf . quantification of total brain t2 hyperintense lesion volume ( t2lv ) was performed using jim ( xinapse systems ltd . , west bergholt , uk , http://www.xinapse.com ) by the consensus of 2 experienced observers from the fluid - attenuated inversion recovery ( discovery set ) or dual - echo ( validation set ) images using a semiautomated technique . for the measurement of these atrophy and lesion surrogates from mri scans , our methods are well established regarding their operational procedures , validity , and reliability . peptides were synthesized at the biopolymers facility of the department of biological chemistry and molecular pharmacology of harvard medical school . recombinant proteins and lipids were purchased from sigma ( st . louis , mo ) , abnova ( taipei city , taiwan ) , matreya llc ( pleasant gap , pa ) , avanti polar lipids ( alabaster , al ) , calbiochem ( san diego , ca ) , chemicon ( temecula , ca ) , genetex ( san antonio , tx ) , novus biologicals ( littleton , co ) , assay designs ( ann arbor , mi ) , prosci inc . ( poway , ca ) , emd biosciences ( san diego , ca ) , cayman chemical ( ann arbor , mi ) , hytest ( turku , finland ) , meridian life science ( memphis , tn ) , and biodesign international ( saco , me ) . the antigens used in the construction of antigen microarrays are listed in table e-1 at neurology.org/nn . the antigens listed in table e-1 were spotted in replicates of 6 on superepoxy 2 slides ( telechem , sunnyvale , ca ) using an arrayit nanoprint 2 lm210 microarray printer ( arrayit corporation , sunnyvale , ca ) and optimized spotting conditions as described . the microarrays were hybridized using an hs 4800pro hybridization station ( tecan , mnnedorf , switzerland ) , in which they were blocked with 1% bovine serum albumin for 1 hour and incubated for 2 hours at 37c with the test serum at a 1:100 dilution in blocking buffer . the arrays were then washed and incubated for 45 minutes with a 1:500 dilution of goat anti - human immunoglobulin g ( igg ) cy3-conjugated and goat anti - human igm cy5-conjugated detection antibodies ( jackson immunoresearch labs , west grove , pa ) . repeated measurements indicate that our antigen microarray technique is reproducible , exhibiting a coefficient of variation of 13.3 1.2 . background signal was subtracted and raw data were normalized and analyzed using the genespring software ( silicon genetics , redwood city , ca ) . antigen reactivity was defined by the mean intensity of binding to the replicates of that antigen on the microarray and expressed as relative fluorescence units . pearson product - moment correlation coefficients were calculated between the mri measures ( bpf , gmf , wmf , t2lv ) and the weighted average of a group of statistically significant lipids . the weighted average of significant lipids was calculated using the formula ( wiai/wi ) ( wi = 1 , i = 1 , 2 , 3 ) , where wi is the proportion of intensity of antigen , ai , ai is the observed intensity of an antigen , and wi is the sum of the weights . to study the relationship between the peripheral immune response and mri measures of disease severity , we analyzed serum antibody reactivity in those ms samples . we analyzed igg serum antibodies using a panel of antigens including cns antigens , heat shock proteins , and lipids . the association between the antibody reactivity against each antigen and 4 mri measures of disease ( t2lv , bpf , wmf , and gmf ) was investigated using spearman correlation tests . we found significant associations between each mri measure of disease and different sets of igg antibody reactivity , which are shown in table 2 . similar patterns of antibody reactivity were linked to bpf and gmf , consistent with the known dominant contribution of gm atrophy to whole brain atrophy measures . strikingly , there was little overlap between the antibody reactivity associated with gmf and wmf , suggesting that different immunopathologic processes contribute to tissue degeneration in these areas of the brain . similarly , these profiles of antibody reactivity were also different from those associated with t2lv . serum immunoglobulin gs associated with mri measures of disease severity in evaluation of mri measures and their link to disease , bpf , gmf , and wmf decrease with disease progression while t2lv increases with disease progression . accordingly , we analyzed the linkage between each mri measure and the significant antibody reactivities shown in table 2 . we identified igg antibody reactivities associated with increased tissue destruction , as evidenced by decreased bpf values ( figure 1 ) . strikingly , we found that antibodies linked to disease pathology as measured by bpf were enriched for reactivity against lipids . furthermore , a selective increase in lipid - reactive antibodies linked to disease severity was also observed when we analyzed all available mri measures ( figure 2 ) . of note , we did not detect an enrichment in lipid - reactive igm in the group of antibodies associated with increased disease pathogenesis ( not shown ) . heatmap in which each column represents the mean immunoglobulin g antibody reactivity in a serum sample from a patient with multiple sclerosis , sorted according to the whole brain parenchymal fraction ( bpf ) ( indicated at the top a lower bpf indicates more brain atrophy ) , and each row represents the antibody reactivity to an antigen according to the colorimetric scale shown . ( a ) heatmap in which each column represents an mri measure of either atrophy ( bpf , gmf , wmf ) or lesions ( t2lv ) , and each row represents the correlation to igg serum antibody reactivity according to a colorimetric scale . ( b ) frequency of lipid - reactive antibodies linked to higher or lower mri disease severity . bpf = whole brain parenchymal fraction ; gmf = global cerebral gray matter fraction ; igg = immunoglobulin g ; t2lv = cerebral t2 ( fluid - attenuated inversion recovery ) hyperintense lesion volume ; wmf = global cerebral white matter fraction . to further investigate the association between serum igg reactivity to lipids and mri measures of disease severity , we calculated an igg anti - lipid index for each patient corresponding to the information on each lipid - specific antibody listed in table 2 , normalized by the strength of its correlation with that mri measure under investigation . the antibody reactivities to lipids used to calculate the lipid index are shown in figure 3a . with this approach , we calculated one index for each of the mri measures analyzed in this study . we found a significant correlation between each igg lipid antibody index and bpf , gmf , and t2lv ( figure 3b ) ; no significant correlation was found with wmf . moreover , no significant correlations were found between bpf , wmf , gmf , and t2lv measures and anti - lipid antibody indexes based on igm reactivity ( not shown ) . ( a ) heatmap in which each column represents an mri measure and each row represents the correlation to immunoglobulin g serum antibody reactivity to lipids according to a colorimetric scale . ( b ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the discovery set . ( c ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the validation set . bpf = whole brain parenchymal fraction ; gmf = cerebral gray matter fraction ( see methods section for details ) ; t2lv = cerebral t2 hyperintense lesion volume ; wmf = global cerebral white matter fraction . finally , we evaluated the performance of the lipid antibody indexes linked to bpf , gmf , and t2lv on an additional set of independent ms samples ( validation set , table 1 ) . we detected a similar trend to the one detected in the discovery set with regard to the correlation between bpf and gmf and their respective lipid indexes , and we validated the correlation between t2lv and its lipid index ( figure 3c ) . to study the relationship between the peripheral immune response and mri measures of disease severity , we analyzed serum antibody reactivity in those ms samples . we analyzed igg serum antibodies using a panel of antigens including cns antigens , heat shock proteins , and lipids . the association between the antibody reactivity against each antigen and 4 mri measures of disease ( t2lv , bpf , wmf , and gmf ) was investigated using spearman correlation tests . we found significant associations between each mri measure of disease and different sets of igg antibody reactivity , which are shown in table 2 . similar patterns of antibody reactivity were linked to bpf and gmf , consistent with the known dominant contribution of gm atrophy to whole brain atrophy measures . strikingly , there was little overlap between the antibody reactivity associated with gmf and wmf , suggesting that different immunopathologic processes contribute to tissue degeneration in these areas of the brain . similarly , these profiles of antibody reactivity were also different from those associated with t2lv . in evaluation of mri measures and their link to disease , bpf , gmf , and wmf decrease with disease progression while t2lv increases with disease progression . accordingly , we analyzed the linkage between each mri measure and the significant antibody reactivities shown in table 2 . we identified igg antibody reactivities associated with increased tissue destruction , as evidenced by decreased bpf values ( figure 1 ) . strikingly , we found that antibodies linked to disease pathology as measured by bpf were enriched for reactivity against lipids . furthermore , a selective increase in lipid - reactive antibodies linked to disease severity was also observed when we analyzed all available mri measures ( figure 2 ) . of note , we did not detect an enrichment in lipid - reactive igm in the group of antibodies associated with increased disease pathogenesis ( not shown ) . heatmap in which each column represents the mean immunoglobulin g antibody reactivity in a serum sample from a patient with multiple sclerosis , sorted according to the whole brain parenchymal fraction ( bpf ) ( indicated at the top a lower bpf indicates more brain atrophy ) , and each row represents the antibody reactivity to an antigen according to the colorimetric scale shown . ( a ) heatmap in which each column represents an mri measure of either atrophy ( bpf , gmf , wmf ) or lesions ( t2lv ) , and each row represents the correlation to igg serum antibody reactivity according to a colorimetric scale . ( b ) frequency of lipid - reactive antibodies linked to higher or lower mri disease severity . bpf = whole brain parenchymal fraction ; gmf = global cerebral gray matter fraction ; igg = immunoglobulin g ; t2lv = cerebral t2 ( fluid - attenuated inversion recovery ) hyperintense lesion volume ; wmf = global cerebral white matter fraction . to further investigate the association between serum igg reactivity to lipids and mri measures of disease severity , we calculated an igg anti - lipid index for each patient corresponding to the information on each lipid - specific antibody listed in table 2 , normalized by the strength of its correlation with that mri measure under investigation . the antibody reactivities to lipids used to calculate the lipid index are shown in figure 3a . with this approach , we calculated one index for each of the mri measures analyzed in this study . we found a significant correlation between each igg lipid antibody index and bpf , gmf , and t2lv ( figure 3b ) ; no significant correlation was found with wmf . moreover , no significant correlations were found between bpf , wmf , gmf , and t2lv measures and anti - lipid antibody indexes based on igm reactivity ( not shown ) . ( a ) heatmap in which each column represents an mri measure and each row represents the correlation to immunoglobulin g serum antibody reactivity to lipids according to a colorimetric scale . ( b ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the discovery set . ( c ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the validation set . bpf = whole brain parenchymal fraction ; gmf = cerebral gray matter fraction ( see methods section for details ) ; t2lv = cerebral t2 hyperintense lesion volume ; wmf = global cerebral white matter fraction . finally , we evaluated the performance of the lipid antibody indexes linked to bpf , gmf , and t2lv on an additional set of independent ms samples ( validation set , table 1 ) . we detected a similar trend to the one detected in the discovery set with regard to the correlation between bpf and gmf and their respective lipid indexes , and we validated the correlation between t2lv and its lipid index ( figure 3c ) . these studies have included cellular immune measures as well as oligoclonal bands or neurofilament - specific antibodies in csf . in the present study , we analyzed the association between serum antibody profiles detected with antigen microarrays and mri measures of disease , including cerebral atrophy and t2 hyperintense lesions . we found that specific antibody patterns are associated with different aspects of mri - defined disease pathology . our major finding was that increased reactivity to lipids is associated with different aspects of brain mri measures of disease severity . of note , the specific set of lipids associated with atrophy differed from those associated with lesions . taken together , these data suggest that anti - lipid antibodies in serum are related to mri measures of disease . our findings highlight the important role of lipid and lipid - specific immunity in the pathogenesis of ms . although a potential limitation of these studies is the relatively small number of samples analyzed , we have validated our findings in an independent validation cohort , strengthening their significance . in addition , further studies in large cohorts from patients affected by non - ms neurodegenerative diseases and healthy controls may indicate whether the antibody reactivities and lipid indexes identified in these studies are exclusive to ms , or are associated with additional biological processes . indeed , some of these antibody reactivities contributing to the immune signatures described in this work have been found in newborns . different sets of antibody reactivities were associated with gmf , wmf , bpf , and t2lv . the different antibody link between lesions and atrophy is in keeping with the long - held view that brain atrophy in patients with ms is multifactorial and is only weakly related to lesions . this has been underscored in recent studies showing the complementary information obtained by combining lesion and atrophy measures . among the cerebral atrophy measures , there was a striking overlap between those antibodies associated with gmf and bpf , which separated them from wmf results . these findings are in keeping with previous observations that whole brain atrophy is dominated by gm loss . in the early stages of ms , this gm atrophy selectively involves the deep gm nuclei . thus , the similarities observed in the autoantibodies linked to gmf and bpf might reflect the dominant contribution and colinearity of gm atrophy to total brain atrophy . the set of antibodies linked to gmf may be the most relevant given that mri studies have shown that gm volume is more closely linked to physical disability and cognitive impairment than are wm volume or lesion measures . using antigen microarrays , antibody reactivity to lipids has been detected in the csf and serum of patients with ms at different stages of the disease and such antibodies in the csf have been linked to disease progression and mri involvement . although several mechanisms are thought to drive brain inflammation and atrophy in ms , the connection between these mechanisms and the antibodies detected with our antigen microarrays is yet unknown . one possibility is that the serum autoantibodies identified in our studies are not pathogenic and reflect the result of immunization against self - antigens released from the cns during the course of the disease . indeed , neurofilament is released from damaged axons during the course of ms , and both neurofilament light chains and antibodies against it have been linked to mri measures of disease . moreover , heat shock proteins are upregulated in different cell types during inflammation and have been shown to have an important role as immunomodulators when released to the extracellular medium . thus , it is possible that heat shock protein reactive antibodies reflect changes in the production and release of these immunomodulators during the course of disease pathogenesis . similarly , bioactive lipids and their products are released from damaged myelin , and lipid - specific antibodies have been detected in patients with ms . lipids have an important role in the immune response , both as bioactive molecules with immunomodulatory properties and also as targets of the adaptive immune response . moreover , lipids have significant effects on the murine model of ms experimental autoimmune encephalomyelitis . indeed , we recently found that the glycolipid lactosylceramide activates a broad set of biological processes in astrocytes , promoting neurodegeneration and inflammation . thus , the lipid - reactive antibodies detected in this work may reflect the release of myelin lipids in the context of ms pathogenesis , and/or may directly contribute to immune - mediated damage in cns tissue . of note , lipid - reactive antibodies are highly cross - reactive . in addition , the lipid - reactive antibodies detected in this work as associated with mri measures of disease activity were of the igg class . these are important points to consider when evaluating a potential role of lipid - reactive antibodies in ms pathogenesis . further studies are warranted to investigate whether bioactive lipids offer new therapeutic targets in ms . we have found that unique patterns of immune reactivity determined with antibody arrays are associated with specific mri measures of disease severity . these patterns agree with the interpretation that different pathogenic mechanisms drive diverse disease processes reflected by these mri measures . these patterns also suggest a predominant role for lipids and lipid - specific immunity in ms pathology . further studies are warranted to determine whether the early appearance of anti - lipid antibodies predicts the subsequent development of clinical and mri - defined disease worsening . rohit bakshi : drafting / revising the manuscript , study concept or design , analysis or interpretation of data , acquisition of data , study supervision , obtaining funding . pia kiviskk : drafting / revising the manuscript , study concept or design , contribution of vital reagents / tools / patients , acquisition of data , study supervision . francisco j. quintana : drafting / revising the manuscript , study concept or design , contribution of vital reagents / tools / patients , study supervision , obtaining funding . from emd serono , rg4111a1 and jf2161-a-5 from the national multiple sclerosis society , pa0069 from the international progressive ms alliance , and by grants from the nih ( 5r01ns055083 - 04 ) and nmss ( rg3798a2 ) to r.b . quintana serves on the editorial board for systems biomedicine , inmunologia , american journal of clinical and experimental immunology , is an associate editor for immunology ( uk ) , is an advisory board member for seminars in immunopathology ; received research support from harvard medical school , baderc , nmss . s. tummala , r. rahbari , r. chu , and k. regev report no disclosures . r. bakshi is editor - in - chief for journal of neuroimaging ; received consulting fees from abbvie , alkermes , biogen , novartis , questcor ; received research support from biogen , emd - serono , novartis , sanofi - genzyme , teva ; his spouse holds stock in biogen , inc . h. weiner served on the advisory board for the guthy - jackson charitable foundation , teva pharmaceuticals industries ltd . , biogen idec , novartis , sanofi - aventis ; has consulted for therapix , bioven , novartis , serono , teva , sanofi ; received research support from national multiple sclerosis society .

objective : to determine whether peripheral immune responses as measured by serum antigen arrays are linked to cerebral mri measures of disease severity in multiple sclerosis ( ms).methods : in this cross - sectional study , serum samples were obtained from patients with relapsing - remitting ms ( n = 21 ) and assayed using antigen arrays that contained 420 antigens including cns - related autoantigens , lipids , and heat shock proteins . normalized compartment - specific global brain volumes were obtained from 3-tesla mri as surrogates of atrophy , including gray matter fraction ( gmf ) , white matter fraction ( wmf ) , and total brain parenchymal fraction ( bpf ) . total brain t2 hyperintense lesion volume ( t2lv ) was quantified from fluid - attenuated inversion recovery images.results:we found serum antibody patterns uniquely correlated with bpf , gmf , wmf , and t2lv . furthermore , we identified immune signatures linked to mri markers of neurodegeneration ( bpf , gmf , wmf ) that differentiated those linked to t2lv . each mri measure was correlated with a specific set of antibodies . strikingly , immunoglobulin g ( igg ) antibodies to lipids were linked to brain mri measures . based on the association between igg antibody reactivity and each unique mri measure , we developed a lipid index . this comprised the reactivity directed against all of the lipids associated with each specific mri measure . we validated these findings in an additional independent set of patients with ms ( n = 14 ) and detected a similar trend for the correlations between bpf , gmf , and t2lv vs their respective lipid indexes.conclusions:we propose serum antibody repertoires that are associated with mri measures of cerebral ms involvement . such antibodies may serve as biomarkers for monitoring disease pathology and progression .

METHODS Patients. Standard protocol approvals, registrations, and patient consents. MRI acquisition and analysis. Antigens. Antigen microarray production, development, and data analysis. RESULTS Serum IgG antibodies correlate with brain MRI measures of disease severity. Serum lipid-reactive IgGs are associated with increased disease pathogenesis determined by MRI. An index of serum IgG reactivity to lipids is associated with MRI measures of disease severity. DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

all serum samples were collected from the ongoing cohort of patients being followed in the climb study ( comprehensive longitudinal investigation of ms at brigham and women 's hospital ) in which participants are followed with comprehensive clinical and imaging assessments to monitor disease progression and response to therapy on a yearly basis . this was a consecutive sample meeting the following criteria : ( 1 ) age 18 to 55 years ; ( 2 ) diagnosis of relapsing - remitting ms ; ( 3 ) absence of other major medical , neurologic , or neuropsychiatric disorders ; ( 4 ) lack of any relapse or corticosteroid use in the 4 weeks before mri or start of disease - modifying therapy 6 months before mri ( to reduce confounding effects on mri ) ; and ( 5 ) no history of smoking or substance abuse . demographic , clinical , and brain mri data our study received approval from the ethical standards committee on human experimentation at our institution ( the partners health care institutional review board ) . contiguous slices covering the whole brain were acquired in high - resolution protocols using 3-dimensional modified driven equilibrium fourier transform and t2-weighted fast fluid - attenuated inversion recovery sequences . in the discovery set , we obtained normalized compartment - specific global brain volumes as surrogates of atrophy , including gm fraction ( gmf ) , wm fraction ( wmf ) , and total brain parenchymal fraction ( bpf ) , using statistical parametric mapping version 8 ( wellcome department of cognitive neurology , london , uk , http://www.fil.ion.ucl.ac.uk/spm ) , after manual correction of ( 1 ) misclassifications of tissue compartments due to ms lesion , and ( 2 ) ineffective contouring of the deep gm structures . because the source images did not show effective contrast for segmentation of the deep gray structures , we performed manual masking to derive only the cerebral cortical gmf . quantification of total brain t2 hyperintense lesion volume ( t2lv ) was performed using jim ( xinapse systems ltd . , west bergholt , uk , http://www.xinapse.com ) by the consensus of 2 experienced observers from the fluid - attenuated inversion recovery ( discovery set ) or dual - echo ( validation set ) images using a semiautomated technique . for the measurement of these atrophy and lesion surrogates from mri scans , our methods are well established regarding their operational procedures , validity , and reliability . the arrays were then washed and incubated for 45 minutes with a 1:500 dilution of goat anti - human immunoglobulin g ( igg ) cy3-conjugated and goat anti - human igm cy5-conjugated detection antibodies ( jackson immunoresearch labs , west grove , pa ) . pearson product - moment correlation coefficients were calculated between the mri measures ( bpf , gmf , wmf , t2lv ) and the weighted average of a group of statistically significant lipids . the weighted average of significant lipids was calculated using the formula ( wiai/wi ) ( wi = 1 , i = 1 , 2 , 3 ) , where wi is the proportion of intensity of antigen , ai , ai is the observed intensity of an antigen , and wi is the sum of the weights . all serum samples were collected from the ongoing cohort of patients being followed in the climb study ( comprehensive longitudinal investigation of ms at brigham and women 's hospital ) in which participants are followed with comprehensive clinical and imaging assessments to monitor disease progression and response to therapy on a yearly basis . this was a consecutive sample meeting the following criteria : ( 1 ) age 18 to 55 years ; ( 2 ) diagnosis of relapsing - remitting ms ; ( 3 ) absence of other major medical , neurologic , or neuropsychiatric disorders ; ( 4 ) lack of any relapse or corticosteroid use in the 4 weeks before mri or start of disease - modifying therapy 6 months before mri ( to reduce confounding effects on mri ) ; and ( 5 ) no history of smoking or substance abuse . within 3 months of mri , each patient received an examination by an ms specialist - neurologist , including evaluation of neurologic disability on the expanded disability status scale and a timed 25-foot walk . contiguous slices covering the whole brain were acquired in high - resolution protocols using 3-dimensional modified driven equilibrium fourier transform and t2-weighted fast fluid - attenuated inversion recovery sequences . in the discovery set , we obtained normalized compartment - specific global brain volumes as surrogates of atrophy , including gm fraction ( gmf ) , wm fraction ( wmf ) , and total brain parenchymal fraction ( bpf ) , using statistical parametric mapping version 8 ( wellcome department of cognitive neurology , london , uk , http://www.fil.ion.ucl.ac.uk/spm ) , after manual correction of ( 1 ) misclassifications of tissue compartments due to ms lesion , and ( 2 ) ineffective contouring of the deep gm structures . quantification of total brain t2 hyperintense lesion volume ( t2lv ) was performed using jim ( xinapse systems ltd . , west bergholt , uk , http://www.xinapse.com ) by the consensus of 2 experienced observers from the fluid - attenuated inversion recovery ( discovery set ) or dual - echo ( validation set ) images using a semiautomated technique . the arrays were then washed and incubated for 45 minutes with a 1:500 dilution of goat anti - human immunoglobulin g ( igg ) cy3-conjugated and goat anti - human igm cy5-conjugated detection antibodies ( jackson immunoresearch labs , west grove , pa ) . pearson product - moment correlation coefficients were calculated between the mri measures ( bpf , gmf , wmf , t2lv ) and the weighted average of a group of statistically significant lipids . the weighted average of significant lipids was calculated using the formula ( wiai/wi ) ( wi = 1 , i = 1 , 2 , 3 ) , where wi is the proportion of intensity of antigen , ai , ai is the observed intensity of an antigen , and wi is the sum of the weights . to study the relationship between the peripheral immune response and mri measures of disease severity , we analyzed serum antibody reactivity in those ms samples . we analyzed igg serum antibodies using a panel of antigens including cns antigens , heat shock proteins , and lipids . the association between the antibody reactivity against each antigen and 4 mri measures of disease ( t2lv , bpf , wmf , and gmf ) was investigated using spearman correlation tests . we found significant associations between each mri measure of disease and different sets of igg antibody reactivity , which are shown in table 2 . similar patterns of antibody reactivity were linked to bpf and gmf , consistent with the known dominant contribution of gm atrophy to whole brain atrophy measures . strikingly , there was little overlap between the antibody reactivity associated with gmf and wmf , suggesting that different immunopathologic processes contribute to tissue degeneration in these areas of the brain . similarly , these profiles of antibody reactivity were also different from those associated with t2lv . serum immunoglobulin gs associated with mri measures of disease severity in evaluation of mri measures and their link to disease , bpf , gmf , and wmf decrease with disease progression while t2lv increases with disease progression . accordingly , we analyzed the linkage between each mri measure and the significant antibody reactivities shown in table 2 . we identified igg antibody reactivities associated with increased tissue destruction , as evidenced by decreased bpf values ( figure 1 ) . strikingly , we found that antibodies linked to disease pathology as measured by bpf were enriched for reactivity against lipids . furthermore , a selective increase in lipid - reactive antibodies linked to disease severity was also observed when we analyzed all available mri measures ( figure 2 ) . of note , we did not detect an enrichment in lipid - reactive igm in the group of antibodies associated with increased disease pathogenesis ( not shown ) . heatmap in which each column represents the mean immunoglobulin g antibody reactivity in a serum sample from a patient with multiple sclerosis , sorted according to the whole brain parenchymal fraction ( bpf ) ( indicated at the top a lower bpf indicates more brain atrophy ) , and each row represents the antibody reactivity to an antigen according to the colorimetric scale shown . ( a ) heatmap in which each column represents an mri measure of either atrophy ( bpf , gmf , wmf ) or lesions ( t2lv ) , and each row represents the correlation to igg serum antibody reactivity according to a colorimetric scale . bpf = whole brain parenchymal fraction ; gmf = global cerebral gray matter fraction ; igg = immunoglobulin g ; t2lv = cerebral t2 ( fluid - attenuated inversion recovery ) hyperintense lesion volume ; wmf = global cerebral white matter fraction . to further investigate the association between serum igg reactivity to lipids and mri measures of disease severity , we calculated an igg anti - lipid index for each patient corresponding to the information on each lipid - specific antibody listed in table 2 , normalized by the strength of its correlation with that mri measure under investigation . with this approach , we calculated one index for each of the mri measures analyzed in this study . we found a significant correlation between each igg lipid antibody index and bpf , gmf , and t2lv ( figure 3b ) ; no significant correlation was found with wmf . moreover , no significant correlations were found between bpf , wmf , gmf , and t2lv measures and anti - lipid antibody indexes based on igm reactivity ( not shown ) . ( a ) heatmap in which each column represents an mri measure and each row represents the correlation to immunoglobulin g serum antibody reactivity to lipids according to a colorimetric scale . ( b ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the discovery set . ( c ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the validation set . bpf = whole brain parenchymal fraction ; gmf = cerebral gray matter fraction ( see methods section for details ) ; t2lv = cerebral t2 hyperintense lesion volume ; wmf = global cerebral white matter fraction . finally , we evaluated the performance of the lipid antibody indexes linked to bpf , gmf , and t2lv on an additional set of independent ms samples ( validation set , table 1 ) . we detected a similar trend to the one detected in the discovery set with regard to the correlation between bpf and gmf and their respective lipid indexes , and we validated the correlation between t2lv and its lipid index ( figure 3c ) . to study the relationship between the peripheral immune response and mri measures of disease severity , we analyzed serum antibody reactivity in those ms samples . we analyzed igg serum antibodies using a panel of antigens including cns antigens , heat shock proteins , and lipids . the association between the antibody reactivity against each antigen and 4 mri measures of disease ( t2lv , bpf , wmf , and gmf ) was investigated using spearman correlation tests . we found significant associations between each mri measure of disease and different sets of igg antibody reactivity , which are shown in table 2 . similar patterns of antibody reactivity were linked to bpf and gmf , consistent with the known dominant contribution of gm atrophy to whole brain atrophy measures . strikingly , there was little overlap between the antibody reactivity associated with gmf and wmf , suggesting that different immunopathologic processes contribute to tissue degeneration in these areas of the brain . in evaluation of mri measures and their link to disease , bpf , gmf , and wmf decrease with disease progression while t2lv increases with disease progression . accordingly , we analyzed the linkage between each mri measure and the significant antibody reactivities shown in table 2 . we identified igg antibody reactivities associated with increased tissue destruction , as evidenced by decreased bpf values ( figure 1 ) . strikingly , we found that antibodies linked to disease pathology as measured by bpf were enriched for reactivity against lipids . furthermore , a selective increase in lipid - reactive antibodies linked to disease severity was also observed when we analyzed all available mri measures ( figure 2 ) . of note , we did not detect an enrichment in lipid - reactive igm in the group of antibodies associated with increased disease pathogenesis ( not shown ) . heatmap in which each column represents the mean immunoglobulin g antibody reactivity in a serum sample from a patient with multiple sclerosis , sorted according to the whole brain parenchymal fraction ( bpf ) ( indicated at the top a lower bpf indicates more brain atrophy ) , and each row represents the antibody reactivity to an antigen according to the colorimetric scale shown . ( a ) heatmap in which each column represents an mri measure of either atrophy ( bpf , gmf , wmf ) or lesions ( t2lv ) , and each row represents the correlation to igg serum antibody reactivity according to a colorimetric scale . ( b ) frequency of lipid - reactive antibodies linked to higher or lower mri disease severity . bpf = whole brain parenchymal fraction ; gmf = global cerebral gray matter fraction ; igg = immunoglobulin g ; t2lv = cerebral t2 ( fluid - attenuated inversion recovery ) hyperintense lesion volume ; wmf = global cerebral white matter fraction . to further investigate the association between serum igg reactivity to lipids and mri measures of disease severity , we calculated an igg anti - lipid index for each patient corresponding to the information on each lipid - specific antibody listed in table 2 , normalized by the strength of its correlation with that mri measure under investigation . with this approach , we calculated one index for each of the mri measures analyzed in this study . we found a significant correlation between each igg lipid antibody index and bpf , gmf , and t2lv ( figure 3b ) ; no significant correlation was found with wmf . moreover , no significant correlations were found between bpf , wmf , gmf , and t2lv measures and anti - lipid antibody indexes based on igm reactivity ( not shown ) . ( a ) heatmap in which each column represents an mri measure and each row represents the correlation to immunoglobulin g serum antibody reactivity to lipids according to a colorimetric scale . ( b ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the discovery set . ( c ) scatter plots depicting the correlation between each lipid index and mri measures ( bpf , gmf , and t2lv ) in the validation set . bpf = whole brain parenchymal fraction ; gmf = cerebral gray matter fraction ( see methods section for details ) ; t2lv = cerebral t2 hyperintense lesion volume ; wmf = global cerebral white matter fraction . finally , we evaluated the performance of the lipid antibody indexes linked to bpf , gmf , and t2lv on an additional set of independent ms samples ( validation set , table 1 ) . we detected a similar trend to the one detected in the discovery set with regard to the correlation between bpf and gmf and their respective lipid indexes , and we validated the correlation between t2lv and its lipid index ( figure 3c ) . in the present study , we analyzed the association between serum antibody profiles detected with antigen microarrays and mri measures of disease , including cerebral atrophy and t2 hyperintense lesions . we found that specific antibody patterns are associated with different aspects of mri - defined disease pathology . our major finding was that increased reactivity to lipids is associated with different aspects of brain mri measures of disease severity . of note , the specific set of lipids associated with atrophy differed from those associated with lesions . taken together , these data suggest that anti - lipid antibodies in serum are related to mri measures of disease . although a potential limitation of these studies is the relatively small number of samples analyzed , we have validated our findings in an independent validation cohort , strengthening their significance . in addition , further studies in large cohorts from patients affected by non - ms neurodegenerative diseases and healthy controls may indicate whether the antibody reactivities and lipid indexes identified in these studies are exclusive to ms , or are associated with additional biological processes . indeed , some of these antibody reactivities contributing to the immune signatures described in this work have been found in newborns . different sets of antibody reactivities were associated with gmf , wmf , bpf , and t2lv . the different antibody link between lesions and atrophy is in keeping with the long - held view that brain atrophy in patients with ms is multifactorial and is only weakly related to lesions . thus , the similarities observed in the autoantibodies linked to gmf and bpf might reflect the dominant contribution and colinearity of gm atrophy to total brain atrophy . the set of antibodies linked to gmf may be the most relevant given that mri studies have shown that gm volume is more closely linked to physical disability and cognitive impairment than are wm volume or lesion measures . using antigen microarrays , antibody reactivity to lipids has been detected in the csf and serum of patients with ms at different stages of the disease and such antibodies in the csf have been linked to disease progression and mri involvement . indeed , neurofilament is released from damaged axons during the course of ms , and both neurofilament light chains and antibodies against it have been linked to mri measures of disease . moreover , heat shock proteins are upregulated in different cell types during inflammation and have been shown to have an important role as immunomodulators when released to the extracellular medium . similarly , bioactive lipids and their products are released from damaged myelin , and lipid - specific antibodies have been detected in patients with ms . in addition , the lipid - reactive antibodies detected in this work as associated with mri measures of disease activity were of the igg class . we have found that unique patterns of immune reactivity determined with antibody arrays are associated with specific mri measures of disease severity . further studies are warranted to determine whether the early appearance of anti - lipid antibodies predicts the subsequent development of clinical and mri - defined disease worsening . from emd serono , rg4111a1 and jf2161-a-5 from the national multiple sclerosis society , pa0069 from the international progressive ms alliance , and by grants from the nih ( 5r01ns055083 - 04 ) and nmss ( rg3798a2 ) to r.b .

bioactive glasses ( bgs ) and calcium phosphates ( cps ) have numerous applications in the repair and reconstruction of bone . but , as a bulk , they are brittle and relatively weak when compared with common implant metals and alloys and high strength ceramics . bioactive materials ( cps and bgs ) have osteoconductive properties an ability to serve as a scaffold or template to guide the newly forming bone along its surfaces . osteoconductive materials allow bone cell attachment , proliferation , migration and phenotypic expression , leading to the formation of new bone in direct apposition to the biomaterial , thus creating a uniquely strong interface . metal implants , primarily titanium ( ti ) or ti alloy , are not bioactive and therefore do not bond directly to bone . plasma - sprayed hydroxyapatite ( ha ) coating was developed to combine the strength of the metal and the bioactivity of the ha . however , the plasma - spray methods involve very high temperatures causing the partial transformation of the ha to amorphous calcium phosphate ( acp ) and ha ( untransformed ) . it is conceivable that a coating with high acp / ha could biodegrade prematurely and delaminate before the bone had the opportunity to attach to the implant , thereby causing loosening and eventual implant failure . in addition , because the plasma - spray method is a line of sight method , implants with complex geometry or porosity can not have a homogeneous coating . bgs are synthetic biocompatible osteoconductive bone substitutes , with bone bonding capacity and documented antibacterial and angiogenesis - promoting properties . our previous study showed that 58s ( 57.72 sio2 , 35.09 cao and 7.1 p2o5 by weight ) and 63s ( 62.17 sio2 , 28.47 cao and 9.25 p2o5 by weight ) bgs nanoparticles have antibacterial activities . especially , compared to the 63s bg nanoparticles , the 58s bg nanoparticles showed a stronger bactericidal effect on the studied pathogens with a lower minimum bactericidal concentration . in fact , the antibacterial activity of bgs has been suggested to be based on several factors including high ph levels and osmotic effects caused by the nonphysiological concentration of ions dissolved from glass . ph measurements revealed that the broth containing the 58s bg nanoparticles had higher ph levels as high as 9 when compared with the 63s and 72s bgs nanoparticles , which is the threshold concentration inducing antibacterial activity . the synergic effects of high calcium concentration and alkaline ph level may make the broth containing 58s a good antibacterial agent . polyesters based on poly ( lactic acid ) , poly ( glycolic acid ) and poly ( lactic - co - glycolic acid ) ( plga ) are found as the best biomaterial with regard to design and performance . biocompatibility of monomer is considered as the foundation for biocompatibility of degradable polymer systems , not the polymer itself . even though plga is extensively used and represents the gold standard of degradable polymers , increased local acidity due to its degradation can lead to irritation at the site of polymer implant . agrawal and athanasiou have introduced a technique in which basic salts are used to control the ph in the local environment of plga implant . the feasibility of lactide / glycolide polymers as excipients for the controlled release of bioactive agents is well - proven and they are the most widely investigated biodegradable polymers for drug delivery . furthermore , it has been proposed that the use of plga in the composite coating provides a locking mechanism between the coating and the juxtaposed bone with time . the composite coatings of ha and tio2 in plga is produced by sol - gel method . this study demonstrated increased adhesion of osteoblast - like cells on this coating compared with conventional plasma - spray techniques . our previous study showed excellent attachment and viability of human adipose - derived stem cells ( hascs ) on the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) nanocomposite coating . therefore , pbgha nanocomposite coating provides an ideal surface for the stem cells attachment , viability and proliferation . this study aimed at preparation and in vivo evaluation of novel bioactive and biodegradable pbgha and pbg nanocomposite coatings as candidates for dental and orthopedic implant applications . starting materials used in this preparation were of analytical grade tetraethyl orthosilicate ( teos ) , triethyl phosphate , ca(no3)2 . the composition of studied bg belongs to the sio2-cao - p2o5 system with 58s ( 57.72 sio2 , 35.09 cao and 7.1 p2o5 by weight ) composition . proper amounts of deionized water ( 15 ml ) , 2n hydrochloric acid ( 2.5 ml ) and teos ( 20.49 ml ) were dissolved in ethanol and stirred at room temperature for 30 min . the reaction mixture was transferred to a large teflon container , which was then placed in an oven for aging at 60c for 54 h. the aged gel was transferred into another teflon vessel which was placed inside an especially designed drying chamber ( cylindrical steel chamber capped with an aluminum foil with holes in it ) with a proper amount of water . in the next step , the whole chamber was placed in an oven at 130c for 54 h. finally , the dried gel nanopowders were calcined at 600c for 1 h. the solvent casting process was applied to coat the substrates . commercially pure ti ( grade 2 ) , was cut into pieces 20 mm 10 mm in size and used as the substrate . these substrates were ultrasonically cleaned first in acetone for 20 min , then in a 70% ethanol solution for 20 min and finally in distilled water for 15 min . ha nanoparticles ( aldrich , usa ) and the 58s bg nanoparticles were dispersed ultrasonically in 20 ml of chloroform ( merck , germany ) and added to plga ( 0.1 g / ml in chcl3 ) ( resomer rg 502h , plga ; 50/50 wt% poly ( lactic acid)/poly ( glycolic acid ) ; inherent viscosity = 0.20 dl / g ( 25c ; 0.1 in chcl3 ) ) with stirring . solutions with one ratio of the components ( plga : nanoparticles = 90:10 by wt% ) were prepared to coat the specimens by the dip - coating procedure . the substrates were soaked in the solutions at a speed of 2 cm / min . after each dip - coating step , samples were dried for 1 min and the procedure was repeated . solutions with the ratio of the components ( plga : nanoparticles 90:10 by wt% ) were prepared to coat the specimens by the dip - coating procedure . in the pbgha nanocomposite coating equal amounts of ha ( 5 wt% ) and bg ( 5 wt% ) nanoparticles were used . the solvent was then allowed to evaporate at the room temperature ( 21c ) for 24 h. scanning electron microscopy ( sem , phillips xl 30 ) and atomic force microscopy ( afm , bruker , nanos 1.1 , germany ) were used to study the microstructure , morphology and surface roughness of the nanocomposite coatings . x - ray diffraction ( xrd , philips x pert - mpd system with cu k wavelength of 1.5418 ) technique was utilized to determine the composition of the coatings . the assessment of in vitro bioactivity was carried out by soaking the samples in simulated body fluid ( sbf ) in sterilized polyethylene containers maintained at 37c . the samples were soaked in sbf for three different periods ( 7 , 14 and 30 days ) . in order to provide more favorable conditions for apatite deposition , hence , the exchange of sbf leads to better simulation of the in vivo conditions , making the assay more precise and reliable . next , the samples were removed from the solution , rinsed gently , first with pure ethanol , then with deionized water . finally , they were dried at the room temperature for 3 h. the formation and growth of apatite layer on the samples were verified by sem and xrd . in order to study the effect of bg and ha nanoparticles on the degradation of the nanocomposite coatings , in vitro degradation tests were carried out in phosphate buffer saline ( pbs ; ph 7.4 ) at 37c . each sample was placed in a vessel containing 50 ml of pbs and incubated for periods up to 60 days . the ratio of the sample mass to the pbs solution volume was selected 6 mg / ml . ( 14 , 30 and 60 days ) , samples were removed from the solution , washed with distilled water and air - dried overnight . changes in the surface morphologies of the coatings during in vitro degradation were evaluated by sem . in order to evaluate the effect of nanoparticles on the degradation of nanocomposite coatings , the degradation of plga coating was investigated , simultaneously . the experimental animal study was approved by the school of dentistry , isfahan university of medical sciences , isfahan , iran . titanium screws ( diameter 1.5 mm , length 6 mm ) were purchased from synthes , switzerland . 20 screws were coated with pbgha nanocomposite coating , 20 screws were coated with pbg nanocomposite coating ( plga : nanoparticles = 90:10 by wt% ) and 20 screws were used without coating . samples were sterilized by exposure to uv light for 2 h followed by soaking overnight in 70% ethanol according to standard techniques . the screws were implanted in the tibia of 16 white mature male new zealand rabbits ( razi vaccine & serum research institute ) . the rabbit 's ages were ranged from 8 to 10 months and the weights were ranged from 3 to 3.5 kg . the animals were kept in separate stainless steel cages that allowed some movement , therefore their legs were load bearing before and after the surgical placement of implants . the animals were anesthetized with an intramuscular injection of ketamine ( 10 mg / kg ) . the local nerve supplies of the internal surface of the tibia were further blocked with 0.5 ml of 2% lidocaine . for each of the right and left tibia , an incision was made at the medial side of the tibia . bone preparations of 6 mm depth were done under profuse sterile physiological saline cooling using careful drilling with low rotary speed ( 500 rpm ) . the fascia and muscles were sutured by a resorbable suture and the skin was sutured by a silk suture . after surgery , animals were injected subcutaneously with 3 generation cephalosporin antibiotic once per day for 5 days at a dose of 20 mg / kg body weight . in addition , analgesic diclofenac sodium was injected intra - muscularly once a day for 2 days at a dose of 5 mg / kg bodyweight . after 30 and 60 days , the animals were sacrificed by an intravenous overdose of pentobarbital . the implants , together with the surrounding bone and soft - tissues , were removed and were fixed in 10% buffered formaldehyde solution ( ph 7.4 ) at 4c for 1 day . the implants were removed and they were decalcified in a mixture of formic acid and sodium citrate at 4c for 6 days . then , the samples were embedded in paraffin , decalcified in nitric acid , cut into 5 m serial sections and stained with hematoxylin and eosin ( h and e ) . histologic evaluation include : type of bone ( trabecular , lamellar and woven bone ) , bone formation percentage ( histomorphic analysis by ihmm , ver.1 , sbmu , iran ) and the connective tissue ( fibrosis , granulation tissue , normal bone marrow [ bw ] ) formation . histological evaluations were made under a light microscope ( e400 , olympus , japan ) at 40 , 100 and 200 magnifications . twenty commercially available kirschner wires ( k - wires ) ( synthes , switzerland ) made of stainless steel with a diameter of 0.8 mm were coated with pbgha and pbg nanocomposite coatings . total coating mass was determined with an electronic micro - balance ( sartorius ag , gottingen , germany , readability 0.01 mg ) . then , without previous drilling , k - wires were incorporated proximally into rabbit tibiae ( white mature male new zealand rabbits . ) as intramedullary rods and they were immediately explanted . after explantation and removal of adherent bone and bw , the loss of coating mass ( denoted lcm ) was determined gravimetrically . the non - parametric paired friedman test was used to analyze differences between the amount of bone formation for the three samples ( pbgha and pbg coated implants and ti without nanocomposite coating ) on the same experimental day while the non - parametric kruskal - wallis test was used to compare the results between the different experimental days . for all tests , starting materials used in this preparation were of analytical grade tetraethyl orthosilicate ( teos ) , triethyl phosphate , ca(no3)2 . the composition of studied bg belongs to the sio2-cao - p2o5 system with 58s ( 57.72 sio2 , 35.09 cao and 7.1 p2o5 by weight ) composition . proper amounts of deionized water ( 15 ml ) , 2n hydrochloric acid ( 2.5 ml ) and teos ( 20.49 ml ) were dissolved in ethanol and stirred at room temperature for 30 min . the reaction mixture was transferred to a large teflon container , which was then placed in an oven for aging at 60c for 54 h. the aged gel was transferred into another teflon vessel which was placed inside an especially designed drying chamber ( cylindrical steel chamber capped with an aluminum foil with holes in it ) with a proper amount of water . in the next step , the whole chamber was placed in an oven at 130c for 54 h. finally , the dried gel nanopowders were calcined at 600c for 1 h. commercially pure ti ( grade 2 ) , was cut into pieces 20 mm 10 mm in size and used as the substrate . these substrates were ultrasonically cleaned first in acetone for 20 min , then in a 70% ethanol solution for 20 min and finally in distilled water for 15 min . ha nanoparticles ( aldrich , usa ) and the 58s bg nanoparticles were dispersed ultrasonically in 20 ml of chloroform ( merck , germany ) and added to plga ( 0.1 g / ml in chcl3 ) ( resomer rg 502h , plga ; 50/50 wt% poly ( lactic acid)/poly ( glycolic acid ) ; inherent viscosity = 0.20 dl / g ( 25c ; 0.1 in chcl3 ) ) with stirring . solutions with one ratio of the components ( plga : nanoparticles = 90:10 by wt% ) were prepared to coat the specimens by the dip - coating procedure . the substrates were soaked in the solutions at a speed of 2 cm / min . after each dip - coating step , samples were dried for 1 min and the procedure was repeated . solutions with the ratio of the components ( plga : nanoparticles 90:10 by wt% ) were prepared to coat the specimens by the dip - coating procedure . in the pbgha nanocomposite coating equal amounts of ha ( 5 wt% ) and bg ( 5 wt% ) nanoparticles were used . the solvent was then allowed to evaporate at the room temperature ( 21c ) for 24 h. scanning electron microscopy ( sem , phillips xl 30 ) and atomic force microscopy ( afm , bruker , nanos 1.1 , germany ) were used to study the microstructure , morphology and surface roughness of the nanocomposite coatings . x - ray diffraction ( xrd , philips x pert - mpd system with cu k wavelength of 1.5418 ) technique was utilized to determine the composition of the coatings . the assessment of in vitro bioactivity was carried out by soaking the samples in simulated body fluid ( sbf ) in sterilized polyethylene containers maintained at 37c . the samples were soaked in sbf for three different periods ( 7 , 14 and 30 days ) . in order to provide more favorable conditions for apatite deposition , hence , the exchange of sbf leads to better simulation of the in vivo conditions , making the assay more precise and reliable . next , the samples were removed from the solution , rinsed gently , first with pure ethanol , then with deionized water . finally , they were dried at the room temperature for 3 h. the formation and growth of apatite layer on the samples were verified by sem and xrd . in order to study the effect of bg and ha nanoparticles on the degradation of the nanocomposite coatings , in vitro degradation tests were carried out in phosphate buffer saline ( pbs ; ph 7.4 ) at 37c . each sample was placed in a vessel containing 50 ml of pbs and incubated for periods up to 60 days . the ratio of the sample mass to the pbs solution volume was selected 6 mg / ml . ( 14 , 30 and 60 days ) , samples were removed from the solution , washed with distilled water and air - dried overnight . changes in the surface morphologies of the coatings during in vitro degradation were evaluated by sem . in order to evaluate the effect of nanoparticles on the degradation of nanocomposite coatings , the experimental animal study was approved by the school of dentistry , isfahan university of medical sciences , isfahan , iran . titanium screws ( diameter 1.5 mm , length 6 mm ) were purchased from synthes , switzerland . 20 screws were coated with pbgha nanocomposite coating , 20 screws were coated with pbg nanocomposite coating ( plga : nanoparticles = 90:10 by wt% ) and 20 screws were used without coating . samples were sterilized by exposure to uv light for 2 h followed by soaking overnight in 70% ethanol according to standard techniques . the screws were implanted in the tibia of 16 white mature male new zealand rabbits ( razi vaccine & serum research institute ) . the rabbit 's ages were ranged from 8 to 10 months and the weights were ranged from 3 to 3.5 kg . the animals were kept in separate stainless steel cages that allowed some movement , therefore their legs were load bearing before and after the surgical placement of implants . the animals were anesthetized with an intramuscular injection of ketamine ( 10 mg / kg ) . the local nerve supplies of the internal surface of the tibia were further blocked with 0.5 ml of 2% lidocaine . all operations were performed in standard surgical sterile conditions . for each of the right and left tibia , an incision was made at the medial side of the tibia . bone preparations of 6 mm depth were done under profuse sterile physiological saline cooling using careful drilling with low rotary speed ( 500 rpm ) . the fascia and muscles were sutured by a resorbable suture and the skin was sutured by a silk suture . after surgery , animals were injected subcutaneously with 3 generation cephalosporin antibiotic once per day for 5 days at a dose of 20 mg / kg body weight . in addition , analgesic diclofenac sodium was injected intra - muscularly once a day for 2 days at a dose of 5 mg / kg bodyweight . after 30 and 60 days , the animals were sacrificed by an intravenous overdose of pentobarbital . the implants , together with the surrounding bone and soft - tissues , were removed and were fixed in 10% buffered formaldehyde solution ( ph 7.4 ) at 4c for 1 day . the implants were removed and they were decalcified in a mixture of formic acid and sodium citrate at 4c for 6 days . then , the samples were embedded in paraffin , decalcified in nitric acid , cut into 5 m serial sections and stained with hematoxylin and eosin ( h and e ) . histologic evaluation include : type of bone ( trabecular , lamellar and woven bone ) , bone formation percentage ( histomorphic analysis by ihmm , ver.1 , sbmu , iran ) and the connective tissue ( fibrosis , granulation tissue , normal bone marrow [ bw ] ) formation . histological evaluations were made under a light microscope ( e400 , olympus , japan ) at 40 , 100 and 200 magnifications . twenty commercially available kirschner wires ( k - wires ) ( synthes , switzerland ) made of stainless steel with a diameter of 0.8 mm were coated with pbgha and pbg nanocomposite coatings . total coating mass was determined with an electronic micro - balance ( sartorius ag , gottingen , germany , readability 0.01 mg ) . then , without previous drilling , k - wires were incorporated proximally into rabbit tibiae ( white mature male new zealand rabbits . ) as intramedullary rods and they were immediately explanted . after explantation and removal of adherent bone and bw , the loss of coating mass ( denoted lcm ) was determined gravimetrically . the non - parametric paired friedman test was used to analyze differences between the amount of bone formation for the three samples ( pbgha and pbg coated implants and ti without nanocomposite coating ) on the same experimental day while the non - parametric kruskal - wallis test was used to compare the results between the different experimental days . for all tests , the level of significance was p < 0.05 . nanoparticles were exposed on the surface after sterilization because the plga was decomposed during sterilization in ethanol . xrd was used to provide clues to phases that existed in the nanocomposite coatings [ figure 3 ] . xrd pattern of the prepared pbgha nanocomposite coating showed the peaks belonging to ti and ha and an amorphous pattern is related to the plga and bg . xrd pattern of the pbg nanocomposite coating showed the peaks belonging to ti and an amorphous pattern is related to the plga and bg . quantitative measurements of root mean square ( rms ) roughness and surface area obtained using afm [ table 1 and figure 4 ] showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( a ) pbgha ( b ) pbg scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings after sterilization . ( a ) pbgha ( b ) pbg x - ray diffraction pattern of the poly ( lactideco - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( 10 wt% nanoparticles ) . . an amorphous pattern belongs to the plga and bioactive glass ( bg ) ( b ) pbg nanocomposite coating : the peaks belong to titanium . an amorphous pattern belongs to the plga and bg surface roughness / area of substrates and nanocomposite coatings afm analysis of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactideco - glycolide)/bioactive glass ( pbg ) nanocomposite coating . quantitative measurements of root mean square roughness and surface area showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization sem analysis shows the effect of pbgha and pbg nanocomposite coatings on ha deposition during increasing immersion times in sbf . it was observed that pbgha and pbg nanocomposite coatings were able to nucleate more ha formation on their surfaces compared with ti substrate . after 7 and 14 days , apatite deposits were observed and detected by xrd on the nanocomposite coatings but was not detected on the ti substrates . after 30 days , ha deposits covered all the surface of pbgha and pbg nanocomposite coatings [ figure 5a and b ] . xrd patterns of the pbgha and pbg nanocomposite coatings surfaces before and after 7 days immersion in sbf are shown in figure 6a and b. as it was observed , after 7 days immersion in sbf , the diffraction peaks of apatite deposits formed on the surface of the prepared nanocomposite coatings were sharp . as it was observed , ha deposits on pbgha nanocomposite coating has finer than the ha deposits on pbg nanocomposite coating [ figure 5a and b ] . scanning electron microscopy micrographs of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coating ( 10 wt% nanoparticles ) after immersion for 30 days in simulated body fluid . after 30 days , hydroxyapatite deposits covered all the surface of pbgha and pbg nanocomposite coatings x - ray diffraction of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite and ( b ) poly ( lactide - co - glycolide)/bioactive glass nanocomposite coatings ( 10 wt% nanoparticles ) before and after immersion in simulated body fluid for 7 days figure 7 shows morphological changes of the pbgha and pbg coatings before and after degradation in pbs . results showed that the morphology of the sterilized coatings before degradation was porous . after degrading for 14 days pores were formed by plga degradation . during degradation , the dissolution of nanoparticles and their aggregates could create pores of similar dimension to them and their aggregates and some of the nanoparticles were exposed on the surface . nanoparticle aggregates dissolution made pores with greater dimensions than pores formed by pure plga degradation . after 30 days , the plga coating showed more pore formation and it was observed that it swelled in some parts and lost its adhesion to the substrate . pbgha nanocomposite coating considerably degraded after about 60 days immersion in pbs . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite nanocomposite coatins ( 10 wt% nanoparticles ) ( a , b , c and d ) and poly ( lactide - co - glycolide)/bioactive glass nanocomposite coating ( e , f , g and h ) after degradation for 7 days ( a , e ) , 14 days ( b , f ) , 30 days ( c , g ) and 60 days ( d , h ) , bar = 50 m after 30 days , pbg nanocomposite coating induces more bone formation than pbgha nanocomposite coating [ table 2 ] . newly formed woven bone tissue was observed in the periosteal and endosteal region as indicated by deep red immature bone . the implants coated with pbgha and pbg nanocomposite coatings showed more bone formation in the medullary cavity compared with that of the ti implants without nanocomposite coating ( p < 0.05 ) [ figure 8 ] . after 60 days , the healing process had proceeded . pbg nanocomposite coating showed more bone formation than pbgha nanocomposite coating ( p < 0.05 ) . titanium screws without coatings showed considerably less bone formation than pbgha and pbg nanocomposite coatings ( p < 0.05 ) [ table 2 ] . in all sections , osteoblasts synthesized lamellar bone on woven bone surfaces and then built up tissue deposition circumferentially around and toward the central vessel . histomorphometric analysis histological analysis of titanium ( ti ) screws coated with poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( a ) and poly ( lactide - co - glycolide)/bioactive glass ( b ) nanocomposite coatings and ti screws without nanocomposite coating ( c ) implanted in rabbits tibia after implantation for 30 days . newly formed woven bone , lamellar bone segments , osteoblastic rim , bone marrow and osteocyte lacunae can be observed . samples were stained with h and e , ( 40 ) after implantation , lcm averaged 3.88 0.21% for pbgha nanocomposite - coated k - wires and 3.55 0.32% for pbg nanocomposite coating . thus , about 96% of the both nanocomposite coatings mass remained attached to the k - wires during intramedullary implantation . no adhesive failure between the coating and the substrate was observed and lcm was mainly due to abrasion with cohesive failure within the nanocomposite coating itself . nanoparticles were exposed on the surface after sterilization because the plga was decomposed during sterilization in ethanol . xrd was used to provide clues to phases that existed in the nanocomposite coatings [ figure 3 ] . xrd pattern of the prepared pbgha nanocomposite coating showed the peaks belonging to ti and ha and an amorphous pattern is related to the plga and bg . xrd pattern of the pbg nanocomposite coating showed the peaks belonging to ti and an amorphous pattern is related to the plga and bg . quantitative measurements of root mean square ( rms ) roughness and surface area obtained using afm [ table 1 and figure 4 ] showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( a ) pbgha ( b ) pbg scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings after sterilization . ( a ) pbgha ( b ) pbg x - ray diffraction pattern of the poly ( lactideco - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( 10 wt% nanoparticles ) . . an amorphous pattern belongs to the plga and bioactive glass ( bg ) ( b ) pbg nanocomposite coating : the peaks belong to titanium . an amorphous pattern belongs to the plga and bg surface roughness / area of substrates and nanocomposite coatings afm analysis of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactideco - glycolide)/bioactive glass ( pbg ) nanocomposite coating . quantitative measurements of root mean square roughness and surface area showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization sem analysis shows the effect of pbgha and pbg nanocomposite coatings on ha deposition during increasing immersion times in sbf . it was observed that pbgha and pbg nanocomposite coatings were able to nucleate more ha formation on their surfaces compared with ti substrate . after 7 and 14 days , apatite deposits were observed and detected by xrd on the nanocomposite coatings but was not detected on the ti substrates . after 30 days , ha deposits covered all the surface of pbgha and pbg nanocomposite coatings [ figure 5a and b ] . xrd patterns of the pbgha and pbg nanocomposite coatings surfaces before and after 7 days immersion in sbf are shown in figure 6a and b. as it was observed , after 7 days immersion in sbf , the diffraction peaks of apatite deposits formed on the surface of the prepared nanocomposite coatings were sharp . as it was observed , ha deposits on pbgha nanocomposite coating has finer than the ha deposits on pbg nanocomposite coating [ figure 5a and b ] . scanning electron microscopy micrographs of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coating ( 10 wt% nanoparticles ) after immersion for 30 days in simulated body fluid . after 30 days , hydroxyapatite deposits covered all the surface of pbgha and pbg nanocomposite coatings x - ray diffraction of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite and ( b ) poly ( lactide - co - glycolide)/bioactive glass nanocomposite coatings ( 10 wt% nanoparticles ) before and after immersion in simulated body fluid for 7 days figure 7 shows morphological changes of the pbgha and pbg coatings before and after degradation in pbs . results showed that the morphology of the sterilized coatings before degradation was porous . after degrading for 14 days pores were formed by plga degradation . during degradation , the dissolution of nanoparticles and their aggregates could create pores of similar dimension to them and their aggregates and some of the nanoparticles were exposed on the surface . nanoparticle aggregates dissolution made pores with greater dimensions than pores formed by pure plga degradation . after 30 days , the plga coating showed more pore formation and it was observed that it swelled in some parts and lost its adhesion to the substrate . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite nanocomposite coatins ( 10 wt% nanoparticles ) ( a , b , c and d ) and poly ( lactide - co - glycolide)/bioactive glass nanocomposite coating ( e , f , g and h ) after degradation for 7 days ( a , e ) , 14 days ( b , f ) , 30 days ( c , g ) and 60 days ( d , h ) , bar = 50 m after 30 days , pbg nanocomposite coating induces more bone formation than pbgha nanocomposite coating [ table 2 ] . newly formed woven bone tissue was observed in the periosteal and endosteal region as indicated by deep red immature bone . osteoblasts were lining the woven bone trabeculae and some lamellar segments were presented . the implants coated with pbgha and pbg nanocomposite coatings showed more bone formation in the medullary cavity compared with that of the ti implants without nanocomposite coating ( p < 0.05 ) [ figure 8 ] . after 60 days , the healing process had proceeded . pbg nanocomposite coating showed more bone formation than pbgha nanocomposite coating ( p < 0.05 ) . titanium screws without coatings showed considerably less bone formation than pbgha and pbg nanocomposite coatings ( p < 0.05 ) [ table 2 ] . in all sections , osteoblasts synthesized lamellar bone on woven bone surfaces and then built up tissue deposition circumferentially around and toward the central vessel . histomorphometric analysis histological analysis of titanium ( ti ) screws coated with poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( a ) and poly ( lactide - co - glycolide)/bioactive glass ( b ) nanocomposite coatings and ti screws without nanocomposite coating ( c ) implanted in rabbits tibia after implantation for 30 days . newly formed woven bone , lamellar bone segments , osteoblastic rim , bone marrow and osteocyte lacunae can be observed . after implantation , lcm averaged 3.88 0.21% for pbgha nanocomposite - coated k - wires and 3.55 0.32% for pbg nanocomposite coating . thus , about 96% of the both nanocomposite coatings mass remained attached to the k - wires during intramedullary implantation . no adhesive failure between the coating and the substrate was observed and lcm was mainly due to abrasion with cohesive failure within the nanocomposite coating itself . due to the aging of the population the need of orthopedic and oral bone - anchored implants increases almost considerably every year . therefore , different techniques have been used to improve surface wettability , bulk composition and surface topography . since the discovery of the osteointegration by brnemark et al . and schroeder et al . and its applicability presented by adell et al . , implants have been created with many new designs and different surfaces in the hope of developing and facilitating both technique and results . it is known that osteointegration has to do with the close contact of the newly formed bone with the implant surface . the attempts to increase the osteointegration and osteogenesis range from the improvement in the implant material and design to the application of ceramic coatings in its surface . this technique is used to explore the ceramic osteoconductive properties , taking necessary care , during the surgical procedures for installation of the implants . the search for biocompatible materials with osteoconductive properties to be used as titanium is the best biocompatible material with its remarkable corrosion resistance to make dental implants . it was confirmed that the cp ceramics coatings make the implant surfaces more bioactive and accelerating the appositional bone growth . however , these coating treatments at high temperatures seem not to be favorable for the bone biological repair . a study by sato et al . showed enhanced osteoblast adhesion on hydrothermally treated ha / titania / plga sol - gel ti coatings . coatings can modify the surface properties of surgical - grade biomaterials to achieve improvements in performance , reliability and biocompatibility . for example , sol - gel derived ha coating with pores could be beneficial on the load bearing implants . in another study , it was showed that the alkaline phosphatase activity of the osteoblast - like cells on the ha / tio2 double layer was expressed to a higher degree than that on the tio2 single coating and pure ti surfaces . the deposition of nanoparticles onto the ti surface was performed to impart nanofeatures to a ti dental implant . sol - gel transformation techniques achieve deposition of nanometer - scale cp accretions to the implant surface . owing to their resultant atomicscale interactions , the accretions display strong physical interactions . in a modified approach , nishimura and colleagues recently demonstrated a directed approach to assembly of capo4 nanofeatures on dual acid - etched cpti implant surfaces . the deposition of discrete 20 - 40 nm nanoparticles on an acid - etched ti surface led to increased mechanical interlocking with bone and the early healing of bone at the endosseous implant surface in a rat model . in the present study , the pbgha and pbg nanocomposite coatings were prepared on the ti substrates ( plates and screws ) . our hypothesis suggested that the presence of the 58s bg and ha nanoparticles could be effective in enhancing the biological behavior of the surface . the presence of the bg and ha nanoparticles in the nanocomposite coatings could have a synergistic effect for increasing the rate of bone formation . therefore , both bg and ha nanoparticles were used in the pbgha nanocomposite coating preparation . furthermore , pbg nanocomposite coating was prepared to evaluate the effect of bg nanoparticles on the bone formation in vivo . previous study showed that nanoparticles content in the nanocomposite coatings could not exceed 10 wt% due to the formation of non - uniform coating on the substrates . for nanoparticle contents of 15 wt% and especially 20 wt% , many particle aggregates were observed throughout the specimen . bg and ha nanoparticles were exposed on the surface after the nanocomposite coatings sterilization process because the plga decomposed during sterilization in ethanol . quantitative measurements of rms roughness and surface area obtained using afm [ table 1 and figure 4 ] showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization . porous structure of the nanocomposite coatings could provide a suitable surface topography for cell adhesion . actually , porous structure of the nanocomposite coating surfaces is capable of accommodating tissue ingrowth . pbgha and pbg biodegradable nanocomposite coatings could be effective especially at the early stages to induce bone formation . in fact , a biodegradable coating will degrade as new bone grows and sometime after surgery it will degrade entirely . in degradation studies furthermore , degradation of plga could lead to expose more nanoparticles and their aggregates on the surface and consequently , increase the size of the pores in the nanocomposite coating . during degradation , the dissolution of nanoparticles could create pores of similar dimension to the original nanoparticles , which will facilitate oligomer diffusion in the composite . this enhancement in oligomer diffusion through the sample surface , together with the buffering effect of dissolution compounds , could reduce the autocatalysis degradation of plga . the swelling of the nanocomposite coating could become more homogeneous without the formation of a surface shell found in plga coating alone . our previous study showed that the 58s bg nanoparticle was effective at buffering ; producing a higher ca ion concentration and ph value in the dissolution medium . furthermore , nanoparticles degradation made some pores in the nanocomposite coating which could reduce the autocatalytic mechanism of degradation by diffusing the acidic degradation products out of the coatings . observation of the samples during the degradation process confirmed that the presence of bg nanoparticles in the plga matrix provides a uniform degradation without any swell formation . bioactivity test showed that pbgha and pbg nanocomposite coatings provides a suitable surface for bone - like apatite precipitation formation . furthermore , it was showed that after 7 days , samples with pure plga coating induced the deposition of bone - like apatite . in our previous research , we used hascs to evaluate their attachment and viability on the prepared pbgha and pbg ( not published ) nanocomposite coatings containing 10 wt% nanoparticles . the results showed excellent attachment and viability of hascs on the pbgha and pbg nanocomposite coatings . afm analysis of the nanocomposite coatings showed more roughness of pbgha nanocomposite coating than pbg nanocomposite coating . however , there were more cells attached and proliferated on the pbg nanocomposite coating than pbgha nanocomposite coating . in fact , it was showed that bg nanoparticles have a positive effect for cell capture . animal experiments revealed a superior response to bg particles with small grain size range ( 300 - 355 m ) compared to ha granules . osteoconductive bone formation starting from the wall of the defects was observed around the bg particles more than around ha particles . it was reported that the existence of bg particles could enhance cell attachment and spreading . the composites with bg particles were hydrophilic , which could induce a more wettable surface than pure poly ( l - lactic acid ) . however , these in vitro tests mainly focus on single stresses such as bending forces and tensile , shear , or compression loads without mimicking the in vivo situation , where forces act in a complicated manner . astm c-633 - 01 , for instance , is a standard test to evaluate adhesion and cohesion strength of inorganic coatings . for inorganic coating , bonding agents with epoxy resins and organic solvents are applied to fix the substrate coating to the loading fixture . penetrating bonding agents may interact with biodegradable polymer coatings such as pbgha and pbg nanocomposite coatings , invalidating mechanical stability or even dissolving the polymer . therefore , stability testing is therefore recommended for every specific application . in this research , stability of the pbgha and pbg nanocomposite coatings pbgha and pbg nanocomposite coatings proved great stability on k - wires during intramedullary implantation ( lcm of pbgha and pbg nanocomposite coatings was determined 3.88 and 3.55 present , respectively ) . about 96% of the pbgha and pbg nanocomposite coatings mass remained attached to the k - wires which presented high stability and adhesion on k - wires . after 30 days of implantation in rabbit tibiae , the pbgha nanocomposite coating showed less bone formation ( 41.18% ) than the pbg nanocomposite coating ( 62.69% ) . interfacial characteristics of biomaterials , such as wettability , chemical composition , electric charges , surface roughness and porosity play a vital role in tissue reactions . in this study , the main role of chemical composition on tissue response was confirmed . after 60 days of implantation , more than 85% bone formation was observed for both nanocomposite coatings ( 86.92 and 90.9% for pbgha and pbg nanocomposite coatings , respectively ) . therefore , such nanocomposite coatings especially pbg nanocomposite coating could provide an optimum surface for bone formation . bioactive and biodegradable pbgha and pbg nanocomposite coatings on ti implants can provide an ideal surface for bone formation . this in vivo study confirmed that bg nanoparticles induce more bone formation than ha nanoparticles . although pbg nanocomposite coating has less roughness than pbgha nanocomposite coating , bone regeneration was occurred more rapidly on the pbg nanocomposite coating .

background : the aim of this study was to evaluate the interaction of bioactive and biodegradable poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings with bone.materials and methods : sol - gel derived 58s bioactive glass nanoparticles , 50/50 wt% poly ( lactic acid)/poly ( glycolic acid ) and hydroxyapatite nanoparticles were used to prepare the coatings . the nanocomposite coatings were characterized by scanning electron microscopy , x - ray diffraction and atomic force microscopy . mechanical stability of the prepared nanocomposite coatings was studied during intramedullary implantation of coated kirschner wires ( k - wires ) into rabbit tibia . titanium mini - screws coated with nanocomposite coatings and without coating were implanted intramedullary in rabbit tibia . bone tissue interaction with the prepared nanocomposite coatings was evaluated 30 and 60 days after surgery . the non - parametric paired friedman and kruskal - wallis tests were used to compare the samples . for all tests , the level of significance was p < 0.05.results:the results showed that nanocomposite coatings remained stable on the k - wires with a minimum of 96% of the original coating mass . tissue around the coated implants showed no adverse reactions to the coatings . woven and trabecular bone formation were observed around the coated samples with a minimum inflammatory reaction . pbg nanocomposite coating induced more rapid bone healing than pbgha nanocomposite coating and titanium without coating ( p < 0.05).conclusion : it was concluded that pbg nanocomposite coating provides an ideal surface for bone formation and it could be used as a candidate for coating dental and orthopedic implants .

INTRODUCTION MATERIALS AND METHODS 58S BG nanoparticles synthesis Preparation of the nanocomposite coatings for bioactivity and degradation studies None The coatings degradation studies Animal implantation test RESULTS Characterization of the nanocomposite coatings None Degradation studies Histological findings PBGHA and PBG nanocomposite coatings adhesion to K-wires DISCUSSION CONCLUSION

especially , compared to the 63s bg nanoparticles , the 58s bg nanoparticles showed a stronger bactericidal effect on the studied pathogens with a lower minimum bactericidal concentration . polyesters based on poly ( lactic acid ) , poly ( glycolic acid ) and poly ( lactic - co - glycolic acid ) ( plga ) are found as the best biomaterial with regard to design and performance . our previous study showed excellent attachment and viability of human adipose - derived stem cells ( hascs ) on the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) nanocomposite coating . therefore , pbgha nanocomposite coating provides an ideal surface for the stem cells attachment , viability and proliferation . this study aimed at preparation and in vivo evaluation of novel bioactive and biodegradable pbgha and pbg nanocomposite coatings as candidates for dental and orthopedic implant applications . ha nanoparticles ( aldrich , usa ) and the 58s bg nanoparticles were dispersed ultrasonically in 20 ml of chloroform ( merck , germany ) and added to plga ( 0.1 g / ml in chcl3 ) ( resomer rg 502h , plga ; 50/50 wt% poly ( lactic acid)/poly ( glycolic acid ) ; inherent viscosity = 0.20 dl / g ( 25c ; 0.1 in chcl3 ) ) with stirring . the solvent was then allowed to evaporate at the room temperature ( 21c ) for 24 h. scanning electron microscopy ( sem , phillips xl 30 ) and atomic force microscopy ( afm , bruker , nanos 1.1 , germany ) were used to study the microstructure , morphology and surface roughness of the nanocomposite coatings . x - ray diffraction ( xrd , philips x pert - mpd system with cu k wavelength of 1.5418 ) technique was utilized to determine the composition of the coatings . in order to study the effect of bg and ha nanoparticles on the degradation of the nanocomposite coatings , in vitro degradation tests were carried out in phosphate buffer saline ( pbs ; ph 7.4 ) at 37c . 20 screws were coated with pbgha nanocomposite coating , 20 screws were coated with pbg nanocomposite coating ( plga : nanoparticles = 90:10 by wt% ) and 20 screws were used without coating . twenty commercially available kirschner wires ( k - wires ) ( synthes , switzerland ) made of stainless steel with a diameter of 0.8 mm were coated with pbgha and pbg nanocomposite coatings . the non - parametric paired friedman test was used to analyze differences between the amount of bone formation for the three samples ( pbgha and pbg coated implants and ti without nanocomposite coating ) on the same experimental day while the non - parametric kruskal - wallis test was used to compare the results between the different experimental days . ha nanoparticles ( aldrich , usa ) and the 58s bg nanoparticles were dispersed ultrasonically in 20 ml of chloroform ( merck , germany ) and added to plga ( 0.1 g / ml in chcl3 ) ( resomer rg 502h , plga ; 50/50 wt% poly ( lactic acid)/poly ( glycolic acid ) ; inherent viscosity = 0.20 dl / g ( 25c ; 0.1 in chcl3 ) ) with stirring . in the pbgha nanocomposite coating equal amounts of ha ( 5 wt% ) and bg ( 5 wt% ) nanoparticles were used . the solvent was then allowed to evaporate at the room temperature ( 21c ) for 24 h. scanning electron microscopy ( sem , phillips xl 30 ) and atomic force microscopy ( afm , bruker , nanos 1.1 , germany ) were used to study the microstructure , morphology and surface roughness of the nanocomposite coatings . in order to study the effect of bg and ha nanoparticles on the degradation of the nanocomposite coatings , in vitro degradation tests were carried out in phosphate buffer saline ( pbs ; ph 7.4 ) at 37c . in order to evaluate the effect of nanoparticles on the degradation of nanocomposite coatings , the experimental animal study was approved by the school of dentistry , isfahan university of medical sciences , isfahan , iran . 20 screws were coated with pbgha nanocomposite coating , 20 screws were coated with pbg nanocomposite coating ( plga : nanoparticles = 90:10 by wt% ) and 20 screws were used without coating . twenty commercially available kirschner wires ( k - wires ) ( synthes , switzerland ) made of stainless steel with a diameter of 0.8 mm were coated with pbgha and pbg nanocomposite coatings . the non - parametric paired friedman test was used to analyze differences between the amount of bone formation for the three samples ( pbgha and pbg coated implants and ti without nanocomposite coating ) on the same experimental day while the non - parametric kruskal - wallis test was used to compare the results between the different experimental days . for all tests , the level of significance was p < 0.05 . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( a ) pbgha ( b ) pbg scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings after sterilization . ( a ) pbgha ( b ) pbg x - ray diffraction pattern of the poly ( lactideco - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( 10 wt% nanoparticles ) . an amorphous pattern belongs to the plga and bioactive glass ( bg ) ( b ) pbg nanocomposite coating : the peaks belong to titanium . an amorphous pattern belongs to the plga and bg surface roughness / area of substrates and nanocomposite coatings afm analysis of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactideco - glycolide)/bioactive glass ( pbg ) nanocomposite coating . quantitative measurements of root mean square roughness and surface area showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization sem analysis shows the effect of pbgha and pbg nanocomposite coatings on ha deposition during increasing immersion times in sbf . xrd patterns of the pbgha and pbg nanocomposite coatings surfaces before and after 7 days immersion in sbf are shown in figure 6a and b. as it was observed , after 7 days immersion in sbf , the diffraction peaks of apatite deposits formed on the surface of the prepared nanocomposite coatings were sharp . scanning electron microscopy micrographs of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coating ( 10 wt% nanoparticles ) after immersion for 30 days in simulated body fluid . after 30 days , hydroxyapatite deposits covered all the surface of pbgha and pbg nanocomposite coatings x - ray diffraction of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite and ( b ) poly ( lactide - co - glycolide)/bioactive glass nanocomposite coatings ( 10 wt% nanoparticles ) before and after immersion in simulated body fluid for 7 days figure 7 shows morphological changes of the pbgha and pbg coatings before and after degradation in pbs . after 30 days , the plga coating showed more pore formation and it was observed that it swelled in some parts and lost its adhesion to the substrate . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite nanocomposite coatins ( 10 wt% nanoparticles ) ( a , b , c and d ) and poly ( lactide - co - glycolide)/bioactive glass nanocomposite coating ( e , f , g and h ) after degradation for 7 days ( a , e ) , 14 days ( b , f ) , 30 days ( c , g ) and 60 days ( d , h ) , bar = 50 m after 30 days , pbg nanocomposite coating induces more bone formation than pbgha nanocomposite coating [ table 2 ] . the implants coated with pbgha and pbg nanocomposite coatings showed more bone formation in the medullary cavity compared with that of the ti implants without nanocomposite coating ( p < 0.05 ) [ figure 8 ] . pbg nanocomposite coating showed more bone formation than pbgha nanocomposite coating ( p < 0.05 ) . titanium screws without coatings showed considerably less bone formation than pbgha and pbg nanocomposite coatings ( p < 0.05 ) [ table 2 ] . histomorphometric analysis histological analysis of titanium ( ti ) screws coated with poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( a ) and poly ( lactide - co - glycolide)/bioactive glass ( b ) nanocomposite coatings and ti screws without nanocomposite coating ( c ) implanted in rabbits tibia after implantation for 30 days . samples were stained with h and e , ( 40 ) after implantation , lcm averaged 3.88 0.21% for pbgha nanocomposite - coated k - wires and 3.55 0.32% for pbg nanocomposite coating . thus , about 96% of the both nanocomposite coatings mass remained attached to the k - wires during intramedullary implantation . xrd pattern of the prepared pbgha nanocomposite coating showed the peaks belonging to ti and ha and an amorphous pattern is related to the plga and bg . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( a ) pbgha ( b ) pbg scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings after sterilization . ( a ) pbgha ( b ) pbg x - ray diffraction pattern of the poly ( lactideco - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coatings ( 10 wt% nanoparticles ) . an amorphous pattern belongs to the plga and bioactive glass ( bg ) ( b ) pbg nanocomposite coating : the peaks belong to titanium . an amorphous pattern belongs to the plga and bg surface roughness / area of substrates and nanocomposite coatings afm analysis of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactideco - glycolide)/bioactive glass ( pbg ) nanocomposite coating . quantitative measurements of root mean square roughness and surface area showed more surface roughness of the pbgha nanocomposite coating than the pbg nanocomposite coating after sterilization sem analysis shows the effect of pbgha and pbg nanocomposite coatings on ha deposition during increasing immersion times in sbf . xrd patterns of the pbgha and pbg nanocomposite coatings surfaces before and after 7 days immersion in sbf are shown in figure 6a and b. as it was observed , after 7 days immersion in sbf , the diffraction peaks of apatite deposits formed on the surface of the prepared nanocomposite coatings were sharp . scanning electron microscopy micrographs of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( pbgha ) and ( b ) poly ( lactide - co - glycolide)/bioactive glass ( pbg ) nanocomposite coating ( 10 wt% nanoparticles ) after immersion for 30 days in simulated body fluid . after 30 days , hydroxyapatite deposits covered all the surface of pbgha and pbg nanocomposite coatings x - ray diffraction of the ( a ) poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite and ( b ) poly ( lactide - co - glycolide)/bioactive glass nanocomposite coatings ( 10 wt% nanoparticles ) before and after immersion in simulated body fluid for 7 days figure 7 shows morphological changes of the pbgha and pbg coatings before and after degradation in pbs . after 30 days , the plga coating showed more pore formation and it was observed that it swelled in some parts and lost its adhesion to the substrate . scanning electron microscopy micrographs of the poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite nanocomposite coatins ( 10 wt% nanoparticles ) ( a , b , c and d ) and poly ( lactide - co - glycolide)/bioactive glass nanocomposite coating ( e , f , g and h ) after degradation for 7 days ( a , e ) , 14 days ( b , f ) , 30 days ( c , g ) and 60 days ( d , h ) , bar = 50 m after 30 days , pbg nanocomposite coating induces more bone formation than pbgha nanocomposite coating [ table 2 ] . the implants coated with pbgha and pbg nanocomposite coatings showed more bone formation in the medullary cavity compared with that of the ti implants without nanocomposite coating ( p < 0.05 ) [ figure 8 ] . pbg nanocomposite coating showed more bone formation than pbgha nanocomposite coating ( p < 0.05 ) . titanium screws without coatings showed considerably less bone formation than pbgha and pbg nanocomposite coatings ( p < 0.05 ) [ table 2 ] . histomorphometric analysis histological analysis of titanium ( ti ) screws coated with poly ( lactide - co - glycolide)/bioactive glass / hydroxyapatite ( a ) and poly ( lactide - co - glycolide)/bioactive glass ( b ) nanocomposite coatings and ti screws without nanocomposite coating ( c ) implanted in rabbits tibia after implantation for 30 days . after implantation , lcm averaged 3.88 0.21% for pbgha nanocomposite - coated k - wires and 3.55 0.32% for pbg nanocomposite coating . thus , about 96% of the both nanocomposite coatings mass remained attached to the k - wires during intramedullary implantation . for example , sol - gel derived ha coating with pores could be beneficial on the load bearing implants . in another study , it was showed that the alkaline phosphatase activity of the osteoblast - like cells on the ha / tio2 double layer was expressed to a higher degree than that on the tio2 single coating and pure ti surfaces . in the present study , the pbgha and pbg nanocomposite coatings were prepared on the ti substrates ( plates and screws ) . furthermore , pbg nanocomposite coating was prepared to evaluate the effect of bg nanoparticles on the bone formation in vivo . previous study showed that nanoparticles content in the nanocomposite coatings could not exceed 10 wt% due to the formation of non - uniform coating on the substrates . bioactivity test showed that pbgha and pbg nanocomposite coatings provides a suitable surface for bone - like apatite precipitation formation . in our previous research , we used hascs to evaluate their attachment and viability on the prepared pbgha and pbg ( not published ) nanocomposite coatings containing 10 wt% nanoparticles . afm analysis of the nanocomposite coatings showed more roughness of pbgha nanocomposite coating than pbg nanocomposite coating . however , there were more cells attached and proliferated on the pbg nanocomposite coating than pbgha nanocomposite coating . in this research , stability of the pbgha and pbg nanocomposite coatings pbgha and pbg nanocomposite coatings proved great stability on k - wires during intramedullary implantation ( lcm of pbgha and pbg nanocomposite coatings was determined 3.88 and 3.55 present , respectively ) . about 96% of the pbgha and pbg nanocomposite coatings mass remained attached to the k - wires which presented high stability and adhesion on k - wires . after 30 days of implantation in rabbit tibiae , the pbgha nanocomposite coating showed less bone formation ( 41.18% ) than the pbg nanocomposite coating ( 62.69% ) . therefore , such nanocomposite coatings especially pbg nanocomposite coating could provide an optimum surface for bone formation . bioactive and biodegradable pbgha and pbg nanocomposite coatings on ti implants can provide an ideal surface for bone formation . although pbg nanocomposite coating has less roughness than pbgha nanocomposite coating , bone regeneration was occurred more rapidly on the pbg nanocomposite coating .

the incidence of early preterm delivery ( < 34 gestational weeks ' ) is 13.6% [ 1 , 2 ] . preterm delivery is associated with a high prevalence of severe neurological deficits and developmental disabilities and is a leading cause of infant and neonatal mortality . preterm neonates are at increased risk of developing respiratory distress syndrome , bronchopulmonary dysplasia , sepsis , intraventricular hemorrhage , patent ductus arteriosus , necrotizing enterocolitis , and disorders related to gestational age at birth [ 8 , 9 ] . risk factors for preterm delivery include demographic characteristics , behavioral factors , and aspects of obstetric history such as previous preterm birth . demographic factors for preterm labor include black race , extremes of maternal age ( < 18 or > 35 ) , low socioeconomic status , and low prepregnancy weight . preterm labor and birth can be associated with stressful life situations ( e.g. , domestic violence , close family death , work and home environment ) either indirectly by associated risk behaviors or directly by mechanisms not completely understood . the exact mechanism of preterm labor is largely unknown but is believed to include decidual hemorrhage ( e.g. , abruption , mechanical factors such as uterine overdistension from multiple gestation or polyhydramnios ) , cervical incompetence ( e.g. , cone biopsy ) , mllerian duct abnormalities , fibroid uterus , cervical inflammation ( e.g. , resulting from bacterial vaginosis , trichomonas ) , maternal inflammation and fever ( e.g. , urinary tract infection ) , hormonal changes ( e.g. , mediated by maternal or fetal stress ) , and uteroplacental insufficiency ( e.g. , hypertension , insulin - dependent diabetes , drug abuse , smoking , alcohol consumption ) . each of these underlying causes can initiate the cascade of events that ultimately lead to uterine activity and cervical dilation . thus , a reduction in the spontaneous ptd rate may require not only accurate identification of patients at risk for preterm delivery but also effective treatment strategies aimed at correcting the underlying causes of preterm labor [ 13 , 1016 ] . methods used for predicting preterm birth include risk scoring system , assessments of salivary estriol , fetal fibronectin ( ffn ) , maternal serum alpha fetoprotein ( ms - afp ) , cervicovaginal intracellular adhesion molecule-1 ( icam-1 ) , phosphorylated insulin - like growth factor binding protein-1 ( phigfbp-1 ) , cervicovaginal beta - human chorionic gonadotropin ( -hcg ) , and the cervical morphology and biometry . while hospital tocodynamometry has been effective for monitoring uterine contractions to evaluate preterm labor , home uterine activity monitoring ( huam ) has not been proven valuable in detecting or preventing preterm birth and is not currently recommended for use [ 17 , 18 ] . fetal fibronectin ( ffn ) is a glycoprotein produced by fetal membranes and trophoblasts which form a biological glue that adheres the fetal membranes and placenta to the decidua . before approximately 20 gestational weeks it thereafter it is a pathological finding and a marker of choriodecidual disruption [ 1921 ] . initially , lockwood et al . reported that the presence of cervicovaginal fetal fibronectin in the second and third trimesters of pregnancy identifies a subgroup of women who are at high risk for preterm delivery . they showed that ffn had a sensitivity of 81.7% and specificity of 82.5% for detecting ptd at 37 weeks of gestation in asymptomatic patients . the systematic review by honest et al . demonstrated that in asymptomatic women the best summary likelihood ratio for positive ffn results was 4.01 ( 95% confidence interval 2.93 to 5.49 ) for predicting birth before 34 weeks ' gestation , with corresponding summary likelihood ratio for negative ffn results of 0.78 ( 0.72 to 0.84 ) . among symptomatic women the best summary likelihood ratio for positive results for ffn was 5.42 ( 4.36 to 6.74 ) for predicting birth within 710 days of testing , with corresponding ratio for negative ffn results of 0.25 ( 0.20 to 0.31 ) . recently , cervical or vaginal fetal fibronectin is the most powerful biochemical prediction marker of sptd due to the high negative predictive values [ 23 , 24 ] . investigated a sequential test with fetal fibronectin detection after ultrasound measurement of cervical length to predict preterm delivery in women with preterm labor . they reported that the sensitivity , specificity , and positive and negative predictive values of fetal fibronectin positiveness were 75 , 71 , 17 , and 97% for delivery within 14 days ; those of cervical length inferior or equal to 20 mm were 75 , 52 , 21 , and 92% for delivery before 34 weeks . the efficiency of the sequential test was similar with excellent negative predictive value : sensitivity , specificity , and positive and negative predictive values of 75 , 63 , 26 , and 93.5% for prediction of preterm delivery before 34 entire weeks . the maternal salivary e3 level seems to correlate well with the serum level and it has been shown that elevated maternal serum e3 levels are associated with increased risk of preterm birth in asymptomatic and symptomatic women presenting for symptoms of preterm labor . it also has low sensitivity and is currently mainly used in clinical settings due to its negative predictive value ( i.e. , women who test negative are at very low risk of preterm birth and no interventions are necessary ) [ 17 , 27 ] . previous research has showed an association with elevated afp and adverse pregnancy outcomes , including spontaneous preterm birth . cut - off value of 1.8 mom for marking the higher - risk group was used for marking the high - risk group . women with equal or higher values of afp were 3.8 times more likely to have premature delivery than those with lower afp values ( 95% ci : 2.2 ; 6.3 ) . sensitivity of 25% and specificity of 92% were proven . at a patient - level meta - analysis of 24 studies by yuan et al . , there was no association with preterm birth ( or = 1.80 , 95% ci : 0.922.68 ) at women in whom afp was elevated in isolation . their findings suggest that maternal afp levels are strongly related to preterm birth , but only in the context of other abnormal pregnancy markers . reported that measurement of sicam-1 in cervicovaginal fluid has potential as a predictor of preterm delivery in women with symptoms of preterm labor . elevated sicam-1 concentrations predicted short intervals to delivery ( area under receiver operator characteristic ( roc ) curves , 0.700.72 for delivery within 3 , 7 , and 10 days ) , with high specificity . characteristics for delivery within 3 days at a 3 ng / ml threshold for a positive test were sensitivity of 33.3% , specificity of 98.9% , and positive and negative predictive values of 75.0% and 93.9% , respectively . . showed that icam-1 gene k469e polymorphism may be a candidate region and useful predictor of susceptibility to ptd . the recent studies showed that a new cervicovaginal test to detect phosphorylated insulin - like growth factor binding protein-1 ( phigfbp-1 ) may improve the accuracy of predicting preterm delivery . the phigfbp-1 is mainly secreted by maternal decidual cells and may be an indicator of tissue damage of the choriodecidual interface . in the first trimester , 24.5% of women , and in the mid - second trimester , 20.2% of women , had an elevated cervical fluid phigfbp-1 level [ 32 , 33 ] . they found that the rates of spontaneous ptd before 32 and before 37 weeks of gestation were higher in women with an elevated cervical fluid phigfbp-1 level , compared with women who had cervical phigfbp-1 of < 10 micrograms / l ( 1.1% versus 0.3% and 5.7% versus 3.2% , resp . ) . an elevated phigfbp-1 level in the first trimester was an independent predictor for ptd before 32 and before 37 weeks of gestation , with odds ratios of 3.0 ( 95% ci 1.37.0 ) and 1.6 ( 95% ci 1.22.3 ) , respectively . cervical phigfbp-1 levels of 10 micrograms / l or more in the first trimester predicted ptd before 32 and before 37 weeks of gestation , with sensitivities of 53.8% and 37.0% , respectively . they showed that elevated cervical fluid phigfbp-1 levels in the first trimester were associated with an increased risk of spontaneous ptd . in another study , rahkonen et al . investigated that short cervix ( < 25 mm ) , positive phigfbp-1 test , combination of both , and clinician 's judgment were all associated with preterm delivery < or = 34 weeks or within 14 days in a total of 246 women between 22 and 34 weeks of gestation . the negative predictive values for delivery < or = 34 weeks were 97.4 , 97.6 , 97.1 , and 98.7% , respectively , and within 14 days 98.7 , 99.0 , 98.3 , and 99.6% , respectively . the corresponding positive lrs for delivery < or = 34 weeks were 6.8 , 3.8 , 75.0 , and 14.9 , and within 14 days 9.7 , 5.5 , 107.3 , and 17.1 . the negative lrs were 0.6 , 0.6 , 0.7 , and 0.3 and 0.5 , 0.3 , 0.6 , and 0.2 . they showed that the rapid phigfbp-1-test has a high negative predictive value for preterm delivery , comparable to that of ultrasonographic cervical length measurement . paternoster et al . assessed phigfbp-1 in cervical secretions and the sonographic measurement of cervical length in 210 symptomatic patients . they found that 26 mm was the best cut - off value for cervical length in terms of predicting preterm delivery ( lr+ , 3.69 ; lr , 0.22 ) , with a sensitivity of 86.4% , specificity of 71.9% , positive predictive value ( ppv ) of 34.5% , and negative predicting value ( npv ) of 96.8% . they also found that the sensitivity , specificity , ppv , and npv of phigfbp-1 of a positive phigfbp-1 test were 52.9% , 89.2% , 48.7% , and 90.8% , respectively , in predicting birth before 37 weeks ' gestation with an or of 9.3 ( 95% ci , 4.0521.3 ) , an lr+ of 4.9 , and an lr of 0.5 and that their combination had an npv of 90% , greater specificity , and a better ppv ( 64.3% ) than either method alone for preterm delivery . found that measuring cervical length at 2224 weeks ' gestation and phigfbp-1 at 30 weeks ' gestation improved the prediction of preterm delivery over either method used alone . reported that igfbp-1 screening did not predict preterm delivery and ffn screening provided the best predictive capacity . a policy of contingent use of testing for ffn after cl measurement or contingent use of cl measurement after ffn screening ( depending on available resources ) is a promising approach to limit use of resources . studied the relationship between levels of insulin - like growth factors 1 and 2 ( igf-1 , igf-2 ) and insulin - like growth factor binding protein 3 ( igfbp-3 ) in antenatal maternal serum and gestational age at delivery . they reported that there was no significant association between maternal igf-1 or igf-2 and preterm birth ( ptb ) . maternal mean igfbp-3 levels are significantly reduced in cases complicated by delivery < 32 completed weeks . cervicovaginal beta - hcg measurement in patients with preterm labor may be used as a predictive test . studied with a group of 100 women with a singleton pregnancy with preterm labour between 2636 weeks ' gestation . cervicovaginal secretions were collected for hcg assay and cervical length was measured by transvaginal sonography ( tvs ) . the preterm delivery rate was 55% ; 24% delivered within 48 h and 11% within 7 days of admission . the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive values ( npv ) of cervical length less than or equal to 2.5 cm to predict delivery within 48 h and 7 days of admission were 62.5% , 89.5% , 65.2% , and 88.3% and 60.0% , 96.9% , 91.3% , and 81.8% , respectively ; and those of qualitative hcg were 87.5% , 80.3% , 58.3% , and 95.3% and 77.1% , 86.2% , 75% , and 87.5% , respectively . hcg value of > or = 45 miu / ml was the optimal cut - off , with a sensitivity , specificity , ppv , and npv for predicting delivery within 48 h and 7 days to be 95.8% , 73.7% , 53.5% , and 98.2% and 85.7% , 80% , 69.8% , and 91.2% , respectively . combining either qualitative or quantitative hcg assay with cervical length significantly increased the sensitivity and npv of cervical length alone for prediction of preterm delivery both within 48 h and 7 days . it was concluded that increased cervicovaginal hcg and reduced cervical length predicted an increased risk of preterm delivery in women with preterm labour . qualitative cervicovaginal hcg assay may be used as a bedside test to predict preterm delivery within 48 h or within 7 days . investigated prediction of the risk of preterm birth ( < 37 weeks ) or early preterm birth ( < 34 weeks ) by cervicovaginal hcg and cervical length measured between 24 and 28 weeks of gestation in asymptomatic women at high risk for preterm birth . they reported that to predict delivery < 37 weeks , cervical length < 2.95 cm had a sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 75% , 80.1% , 71.4% , and 90.7% respectively , and cervicovaginal hcg > 4.75 miu / ml had a sensitivity , specificity , ppv , and npv of 70% , 61.81% , 40% , and 85% , respectively . to predict delivery < 34 weeks , cervical length < 2.65 cm had a sensitivity , specificity , ppv , and npv of 50% , 85.50% , 23.08% , and 95.16% , respectively ; and cervicovaginal hcg > 14 miu / ml had a sensitivity , specificity , ppv , and npv of 83.3% , 85.5% , 33.3% , and 98.3% , respectively . cervical length was superior to predict delivery < 37 weeks , whereas hcg was superior to predict delivery < 34 weeks . their combination was superior to predict preterm birth both < 37 weeks or < 34 weeks , than either parameter used alone . combined marker evaluation could be used as a sensitive parameter for identifying women at risk of spontaneous preterm delivery but it is not possible to obtain biomarkers in most of the clinics . cervix can be evaluated by transabdominal , translabial , and transvaginal ultrasound ( tvu ) . each technique has its costs and benefits ; however , a review of the current literature will show that the transvaginal method of cervical assessment is the most reliable . tvu is objective , reproducible , and acceptable to patients . at the transabdominal approach , the cervix may not be visualised in up to 50% of cases unless the bladder is full , but bladder filling significantly increases the length of the cervix . the transperineal route is limited by both the inconsistency in correlation between transvaginal and transperineal measurements and the inadequate visualisation of the cervix in up to 25% of cases . cervical changes such as dilatation of the internal cervical os with funneling ( beaking ) of the membranes can be easily appreciated by tvu , but not by digital examination [ 4244 ] . the ultrasound images were analyzed to assess changes in the cervix that are associated with spontaneous prematurity and to evaluate ultrasonography as an indicator of the risk of preterm delivery . before the evaluation of the cervix with transvaginal ultrasonography , first of all , the patient should have an empty bladder and be placed in dorsal lithotomy position . a distended bladder can alter the shape of the cervix and compass the cervical canal in some cases preventing the detection of cervical incompetence [ 44 , 46 ] . if the probe is pressed too hard against the cervix , it can obscure cervical incompetence . cervical length ( cl ) is defined as the distance between the internal to external os along the endocervical canal ( figure 1 ) . if the cervical canal is curved , the cl can be measured either as the sum of two straight lines that essentially follow the curve or by a straight line between internal and external os . a short cl is usually straight , and the presence of curved cervix generally signifies a cl greater than 25 mm and , therefore , is a reassuring finding [ 47 , 48 ] . if the cervical canal is closed , cl is probably the only parameter that needs to be measured . if a normal appearing internal os can not be visualized , the cervix should be assessed further to determine whether funneling ( the internal os width is greater than 5 mm ) is present ( figure 2 ) . if funneling is present , the shape can be recorded [ 49 , 50 ] . a continuous process of funneling has been described , going from a normal t shape to y , then v , and finally a u shape . it appears that u shape is more likely to be associated with ptd , compared with a v - shaped funnel [ 51 , 52 ] . cl during pregnancy can range from 25 to 70 mm and ultrasound width of the cervical canal ranges from 2 to 4 mm [ 47 , 48 , 53 , 54 ] . percentile values for cl between 17 and 32 weeks of gestation are indicated in table 1 ( unpublished data ) . before 14 weeks therefore , the measurement of the true cervical length is very difficult before 14 weeks . there is agreement that the best time to examine patients with this method to estimate their preterm birth risk is between 18 and 24 gestational weeks . several studies reported that the measurement of cervical length in the first trimester is not predictive of preterm delivery [ 55 , 56 ] . finally , greco et al . have recently reported that the endocervical length at 11 to 13 weeks is shorter in pregnancies resulting in spontaneous delivery before 34 weeks than in those delivering after 34 weeks . many parameters other than cl and presence or absence of a funnel have been studied including funnel width , funnel length , endocervical canal dilation , cervical index ( funnel length + 1/functional length ) , anterior and posterior cervical width , cervical angle , cervical canal contour , and cervical gland area ( cga ) [ 5860 ] . the length of the cervix may be useful in predicting the risk of premature delivery , with a shorter cervix predicting a higher risk . a short cl is a better predictor of early ptd than later ptd [ 47 , 6163 ] . in a prospective multicenter study , iams et al . performed tvs of the cervix in low - risk women with a singleton pregnancy at 24 weeks ( n = 2915 ) and 28 weeks ( n = 2531 ) of gestation . at 24 weeks , a cervical length of 25 mm had a sensitivity of 37% , a specificity of 92% , a positive predictive value 18% , and a negative predictive value % 97 in predicting spontaneous preterm birth at < 35 weeks ' gestation . the rr of preterm birth before 35 weeks of gestation was about sixfold higher ( 95% ci : 3.849.97 ) among women whose cervical length was less than 25 mm than that among women with a cervical length above 40 mm . to et al . conducted a population - based prospective multicentre study in 39 284 women with singleton pregnancies attending for routine hospital antenatal care in london , uk . the detection rate of spontaneous delivery before 32 weeks by measuring cervical length was 55% , with 10% false - positive rate . measured the cl by tvs at 1622 weeks in 760 singleton pregnancies in unselected women attending routine antenatal care . relative risks ( 95% ci ) for spontaneous preterm delivery before 37 weeks were 3.8 ( 2.6 , 5.6 ) , 5.4 ( 3.3 , 9.0 ) , and 6.3 ( 3.0 , 13.0 ) for the tenth ( 30 mm ) , fifth ( 27 mm ) , and two and a half ( 22 mm ) percentiles , respectively ; rrs for before 35 weeks were 4.5 ( 2.9 , 6.9 ) , 7.5 ( 4.5 , 12.5 ) , and 7.8 ( 3.6 , 16.7 ) . sensitivity ranged from 13 to 44% , specificity 9099% , positive predictive value 1547% , and negative predictive value 8098% for prediction of preterm birth before 35 weeks . a study of cervical length in low - risk women found an eightfold ( 95% ci 319 ) increased risk of preterm birth when the cervix was less than 30 mm at 18 to 22 weeks of gestation , but the sensitivity and positive predictive values were low : 19 and 6 percent , respectively . although low sensitivity and low positive predictive value limit its usefulness , it has high negative predictive values and it can be used in screening of low - risk obstetric populations ( table 2 ) . a number of studies have assessed the predictive value of tvs cl in women with some of the most important of these risk factors including a prior ptd [ 50 , 66 , 67 ] , a history of excisional cervical procedures ( cone biopsy , leep ) [ 68 , 69 ] , mullerian anomaly , and two or more voluntary termination ( table 3 ) . in a prospective study of 705 high - risk women , the risk of spontaneous ptb before 35 weeks decreased by approximately 6% for each additional millimeter of cl ( or : 0.94 , 95% ci : 0.920.95 ) and by approximately 5% for each additional week of pregnancy during which the cl was measured ( or : 0.95 , 95% ci : 0.920.98 ) . they conclude that gestational age at which transvaginal ultrasound cervical length is measured significantly affects the calculation of risk of spontaneous preterm birth . the spontaneous preterm birth risk increases as the length of the cervix declines and as the gestational age decreases . funneling comprising 4050% of the total cervical length or a persistently shortened cervix ( < 2530 mm ) has , in several studies , been associated with an increased risk of preterm birth [ 17 , 47 , 49 , 65 ] . to et al . measured cervical length among 6334 women with singleton pregnancies at 2224 weeks and looked for the presence of funneling to evaluate its possible additional risk . funneling of the internal os was present in about 4% of pregnancies and the prevalence decreased with increasing cervical length from 98% when the length was 15 mm to about 25% for lengths of 1630 mm and less than 1% at lengths of > 30 mm . the rate of preterm delivery was 6.9% in those with funneling compared to 0.7% in those without funneling . however , logistic regression analysis demonstrated that funneling did not provide a significant additional contribution to cervical length in the prediction of spontaneous delivery before 33 weeks ( or , for short cervix = 24.9 p < 0.0001 ; or , for funneling = 1.8 , p = 0.40 ) . as an independent finding , funneling does not add appreciably to the risk of early gestational age at delivery associated with a shortened cervical length . so , women with a long cervix and funneling are not at increased risk of preterm delivery [ 50 , 51 , 72 ] . in the past few years publications have also highlighted the importance of another morphological ultrasonographic marker for ptd , named as the cervical gland area ( cga ) . the cga is defined as the sonographically hipoechoic or hiperechoic zone surrounding the endocervical canal . if the cga around the endocervical canal is not detected , it is defined as absent [ 58 , 59 ] . fukami et al . reported that the absence of cga at second trimester ultrasonography appeared to be new and powerful predictor of ptd before 32 weeks gestation , similar to that reported by pires et al . reported that short cl ( < 20 mm ) with absent cga represents an independent predictor for ptd . the absence of the cga as a new marker for the risk of ptd has to be confirmed by further investigations . many interventions have been proposed in an attempt to prevent ptd in women at high risk . bed rest and hydration are often recommended in an attempt to prevent ptd in women at high risk , but there is no consistent evidence that they are able to delay delivery . a cochrane meta - analysis from 2005 showed that intramuscular progesterone is associated with a reduction in risk of preterm birth before 37 gestational weeks . fonseca et al . in a multicenter , randomized trial proposed using daily vaginal micronized progesterone ( 200 mg ) to women with cl 15 mm or less , irrespective of other risk factors . they showed a significant reduction in preterm birth at < 34 weeks with intravaginal progesterone in patients treated based on premature cervical shortening as the indication for therapy . they found that the progesterone - treated patients had significantly less cervical shortening than the placebo group . a significant difference was also observed between groups for categorical outcomes including the frequency of cervical length progression to 25 mm and a 50% reduction in cervical length from baseline in this subpopulation . further research is necessary and several randomized trials are underway to clarify the efficacy and fetal safety of progesterone treat . cervical cerclage is an old , easily performed procedure for treatment of true cervical incompetence . studied 80 women whose primary physician determined that a prophylactic ( n = 50 ) or urgent cerclage ( n = 30 ) was indicated and had transvaginal ultrasonographic evaluation before and after cerclage . they found that the mean standard deviation precerclage cervical length was 27.2 10.3 mm and after cerclage was 34.1 9.9 mm ( n = 80 , p < 0.001 ) . the increase in cervical length after cerclage is not predictive of term delivery . until recently , cerclage was the only intervention studied to prevent ptb in asymptomatic women with short cl . randomized 113 women with cl < 25 mm or 25% funneling measured between 16 and 24 weeks to either modified bed rest or cerclage . no significant differences between two groups regarding risk of ptb < 34 weeks or perinatal death were noted . it is important that women were included based on an incidental finding of a short cervix without taking into account other risk factors in the maternal history . therefore , the majority of women were considered low risk before the sonographic findings . to et al . also sampled 47,123 asymptomatic women and identified a cervix of 15 mm or less in 470 , of whom 253 ( 54% ) were randomized to either cervical cerclage ( n = 127 ) or expectant management ( n = 126 ) . no significant differences in the rate of ptb <33 weeks or in perinatal or maternal morbidity or mortality were noted . subgroup analysis of the utility of cerclage in the high - risk population , based on maternal history , was not performed . cerclage is not indicated in low - risk patients . a case - controlled study by incerti et al . found that cerclage does not reduce ptb in low - risk women with short cl compared with rest alone . however , this is not the case in women with previous ptd . a recent multicenter randomized trial included 302 women with at least one prior ptb 32 weeks and tvu cl < 25 mm between 16 and 22 weeks randomized to either cerclage or no cerclage . ptb < 35 weeks was similar in both groups , but the benefit was most pronounced when cl was < 15 mm , suggesting the presence of a more significant , and treatable , component of cervical insufficiency . has shown that cerclage , when performed in women with a singleton gestation , previous preterm birth , and cervical length < 25 mm , seems to have a similar effect regardless of the degree of cervical shortening , including cl 1624 mm , as well as cl 5.9 mm . weekly intramuscular 17-alpha - hydroxyprogesterone caproate ( 17 p ) was compared with mcdonald cerclage in women with short cl 25 mm at between 16 and 24 weeks ' gestation . the study was terminated , however , when the interim analysis showed no difference in ptd < 35 weeks between treatment groups . however , cerclage may be more effective in preventing spontaneous ptd in women with cl 15 mm . in another study , berghella et al . showed that 17 p had no additional benefit for prevention of ptd in women who had prior sptd and got ultrasound - indicated cerclage for cl < 25 mm . in women who did not get cerclage , 17 p reduced previable birth and perinatal mortality . evaluation of the cervical morphology and biometry with transvaginal ultrasonography at 1624 weeks of gestation is a useful tool to predict the risk of preterm birth in low- and high - risk singleton pregnancies . for instance , a sonographic cervical length > 30 mm and present cga have a 96 - 97% negative predictive value for preterm delivery at < 37 weeks . transvaginal evaluation of cervix during routine fetal morphological examination helps identify asymptomatic low- and high - risk women for prediction of preterm delivery . available evidence supports the use of progesterone to women with cervical length 25 mm , irrespective of other risk factors . in women with prior spontaneous ptd with asymptomatic cervical shortening ( cl 25 mm ) there should be prophylactic cerclage procedure and weekly to every two weeks follow - up .

preterm delivery ( ptd ) , defined as birth before 37 completed weeks of gestation , is the leading cause of perinatal morbidity and mortality . evaluation of the cervical morphology and biometry with transvaginal ultrasonography at 1624 weeks of gestation is a useful tool to predict the risk of preterm birth in low- and high - risk singleton pregnancies . for instance , a sonographic cervical length ( cl ) > 30 mm and present cervical gland area have a 96 - 97% negative predictive value for preterm delivery at < 37 weeks . available evidence supports the use of progesterone to women with cervical length 25 mm , irrespective of other risk factors . in women with prior spontaneous ptd with asymptomatic cervical shortening ( cl 25 mm ) , prophylactic cerclage procedure must be performed and weekly to every two weeks follow - up is essential . this article reviews the evidence in support of the clinical introduction of transvaginal sonography for both the prediction and management of spontaneous preterm labour .

1. Introduction 2. Biomarkers of Preterm Birth 3. Cervical Assessment by Ultrasonography 4. The Cervical Morphology and Biometry for the Prediction of Preterm Birth 5. Management Options for Short Cervix

preterm delivery is associated with a high prevalence of severe neurological deficits and developmental disabilities and is a leading cause of infant and neonatal mortality . risk factors for preterm delivery include demographic characteristics , behavioral factors , and aspects of obstetric history such as previous preterm birth . thus , a reduction in the spontaneous ptd rate may require not only accurate identification of patients at risk for preterm delivery but also effective treatment strategies aimed at correcting the underlying causes of preterm labor [ 13 , 1016 ] . methods used for predicting preterm birth include risk scoring system , assessments of salivary estriol , fetal fibronectin ( ffn ) , maternal serum alpha fetoprotein ( ms - afp ) , cervicovaginal intracellular adhesion molecule-1 ( icam-1 ) , phosphorylated insulin - like growth factor binding protein-1 ( phigfbp-1 ) , cervicovaginal beta - human chorionic gonadotropin ( -hcg ) , and the cervical morphology and biometry . reported that the presence of cervicovaginal fetal fibronectin in the second and third trimesters of pregnancy identifies a subgroup of women who are at high risk for preterm delivery . they showed that ffn had a sensitivity of 81.7% and specificity of 82.5% for detecting ptd at 37 weeks of gestation in asymptomatic patients . recently , cervical or vaginal fetal fibronectin is the most powerful biochemical prediction marker of sptd due to the high negative predictive values [ 23 , 24 ] . investigated a sequential test with fetal fibronectin detection after ultrasound measurement of cervical length to predict preterm delivery in women with preterm labor . they reported that the sensitivity , specificity , and positive and negative predictive values of fetal fibronectin positiveness were 75 , 71 , 17 , and 97% for delivery within 14 days ; those of cervical length inferior or equal to 20 mm were 75 , 52 , 21 , and 92% for delivery before 34 weeks . the efficiency of the sequential test was similar with excellent negative predictive value : sensitivity , specificity , and positive and negative predictive values of 75 , 63 , 26 , and 93.5% for prediction of preterm delivery before 34 entire weeks . the maternal salivary e3 level seems to correlate well with the serum level and it has been shown that elevated maternal serum e3 levels are associated with increased risk of preterm birth in asymptomatic and symptomatic women presenting for symptoms of preterm labor . it also has low sensitivity and is currently mainly used in clinical settings due to its negative predictive value ( i.e. , women who test negative are at very low risk of preterm birth and no interventions are necessary ) [ 17 , 27 ] . previous research has showed an association with elevated afp and adverse pregnancy outcomes , including spontaneous preterm birth . reported that measurement of sicam-1 in cervicovaginal fluid has potential as a predictor of preterm delivery in women with symptoms of preterm labor . elevated sicam-1 concentrations predicted short intervals to delivery ( area under receiver operator characteristic ( roc ) curves , 0.700.72 for delivery within 3 , 7 , and 10 days ) , with high specificity . they found that the rates of spontaneous ptd before 32 and before 37 weeks of gestation were higher in women with an elevated cervical fluid phigfbp-1 level , compared with women who had cervical phigfbp-1 of < 10 micrograms / l ( 1.1% versus 0.3% and 5.7% versus 3.2% , resp . ) an elevated phigfbp-1 level in the first trimester was an independent predictor for ptd before 32 and before 37 weeks of gestation , with odds ratios of 3.0 ( 95% ci 1.37.0 ) and 1.6 ( 95% ci 1.22.3 ) , respectively . cervical phigfbp-1 levels of 10 micrograms / l or more in the first trimester predicted ptd before 32 and before 37 weeks of gestation , with sensitivities of 53.8% and 37.0% , respectively . they showed that elevated cervical fluid phigfbp-1 levels in the first trimester were associated with an increased risk of spontaneous ptd . investigated that short cervix ( < 25 mm ) , positive phigfbp-1 test , combination of both , and clinician 's judgment were all associated with preterm delivery < or = 34 weeks or within 14 days in a total of 246 women between 22 and 34 weeks of gestation . they showed that the rapid phigfbp-1-test has a high negative predictive value for preterm delivery , comparable to that of ultrasonographic cervical length measurement . they found that 26 mm was the best cut - off value for cervical length in terms of predicting preterm delivery ( lr+ , 3.69 ; lr , 0.22 ) , with a sensitivity of 86.4% , specificity of 71.9% , positive predictive value ( ppv ) of 34.5% , and negative predicting value ( npv ) of 96.8% . they also found that the sensitivity , specificity , ppv , and npv of phigfbp-1 of a positive phigfbp-1 test were 52.9% , 89.2% , 48.7% , and 90.8% , respectively , in predicting birth before 37 weeks ' gestation with an or of 9.3 ( 95% ci , 4.0521.3 ) , an lr+ of 4.9 , and an lr of 0.5 and that their combination had an npv of 90% , greater specificity , and a better ppv ( 64.3% ) than either method alone for preterm delivery . found that measuring cervical length at 2224 weeks ' gestation and phigfbp-1 at 30 weeks ' gestation improved the prediction of preterm delivery over either method used alone . a policy of contingent use of testing for ffn after cl measurement or contingent use of cl measurement after ffn screening ( depending on available resources ) is a promising approach to limit use of resources . studied with a group of 100 women with a singleton pregnancy with preterm labour between 2636 weeks ' gestation . cervicovaginal secretions were collected for hcg assay and cervical length was measured by transvaginal sonography ( tvs ) . the sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive values ( npv ) of cervical length less than or equal to 2.5 cm to predict delivery within 48 h and 7 days of admission were 62.5% , 89.5% , 65.2% , and 88.3% and 60.0% , 96.9% , 91.3% , and 81.8% , respectively ; and those of qualitative hcg were 87.5% , 80.3% , 58.3% , and 95.3% and 77.1% , 86.2% , 75% , and 87.5% , respectively . combining either qualitative or quantitative hcg assay with cervical length significantly increased the sensitivity and npv of cervical length alone for prediction of preterm delivery both within 48 h and 7 days . it was concluded that increased cervicovaginal hcg and reduced cervical length predicted an increased risk of preterm delivery in women with preterm labour . qualitative cervicovaginal hcg assay may be used as a bedside test to predict preterm delivery within 48 h or within 7 days . investigated prediction of the risk of preterm birth ( < 37 weeks ) or early preterm birth ( < 34 weeks ) by cervicovaginal hcg and cervical length measured between 24 and 28 weeks of gestation in asymptomatic women at high risk for preterm birth . they reported that to predict delivery < 37 weeks , cervical length < 2.95 cm had a sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of 75% , 80.1% , 71.4% , and 90.7% respectively , and cervicovaginal hcg > 4.75 miu / ml had a sensitivity , specificity , ppv , and npv of 70% , 61.81% , 40% , and 85% , respectively . to predict delivery < 34 weeks , cervical length < 2.65 cm had a sensitivity , specificity , ppv , and npv of 50% , 85.50% , 23.08% , and 95.16% , respectively ; and cervicovaginal hcg > 14 miu / ml had a sensitivity , specificity , ppv , and npv of 83.3% , 85.5% , 33.3% , and 98.3% , respectively . cervical length was superior to predict delivery < 37 weeks , whereas hcg was superior to predict delivery < 34 weeks . their combination was superior to predict preterm birth both < 37 weeks or < 34 weeks , than either parameter used alone . combined marker evaluation could be used as a sensitive parameter for identifying women at risk of spontaneous preterm delivery but it is not possible to obtain biomarkers in most of the clinics . each technique has its costs and benefits ; however , a review of the current literature will show that the transvaginal method of cervical assessment is the most reliable . the transperineal route is limited by both the inconsistency in correlation between transvaginal and transperineal measurements and the inadequate visualisation of the cervix in up to 25% of cases . the ultrasound images were analyzed to assess changes in the cervix that are associated with spontaneous prematurity and to evaluate ultrasonography as an indicator of the risk of preterm delivery . before the evaluation of the cervix with transvaginal ultrasonography , first of all , the patient should have an empty bladder and be placed in dorsal lithotomy position . a distended bladder can alter the shape of the cervix and compass the cervical canal in some cases preventing the detection of cervical incompetence [ 44 , 46 ] . cervical length ( cl ) is defined as the distance between the internal to external os along the endocervical canal ( figure 1 ) . a short cl is usually straight , and the presence of curved cervix generally signifies a cl greater than 25 mm and , therefore , is a reassuring finding [ 47 , 48 ] . cl during pregnancy can range from 25 to 70 mm and ultrasound width of the cervical canal ranges from 2 to 4 mm [ 47 , 48 , 53 , 54 ] . several studies reported that the measurement of cervical length in the first trimester is not predictive of preterm delivery [ 55 , 56 ] . many parameters other than cl and presence or absence of a funnel have been studied including funnel width , funnel length , endocervical canal dilation , cervical index ( funnel length + 1/functional length ) , anterior and posterior cervical width , cervical angle , cervical canal contour , and cervical gland area ( cga ) [ 5860 ] . the length of the cervix may be useful in predicting the risk of premature delivery , with a shorter cervix predicting a higher risk . performed tvs of the cervix in low - risk women with a singleton pregnancy at 24 weeks ( n = 2915 ) and 28 weeks ( n = 2531 ) of gestation . at 24 weeks , a cervical length of 25 mm had a sensitivity of 37% , a specificity of 92% , a positive predictive value 18% , and a negative predictive value % 97 in predicting spontaneous preterm birth at < 35 weeks ' gestation . the rr of preterm birth before 35 weeks of gestation was about sixfold higher ( 95% ci : 3.849.97 ) among women whose cervical length was less than 25 mm than that among women with a cervical length above 40 mm . conducted a population - based prospective multicentre study in 39 284 women with singleton pregnancies attending for routine hospital antenatal care in london , uk . the detection rate of spontaneous delivery before 32 weeks by measuring cervical length was 55% , with 10% false - positive rate . relative risks ( 95% ci ) for spontaneous preterm delivery before 37 weeks were 3.8 ( 2.6 , 5.6 ) , 5.4 ( 3.3 , 9.0 ) , and 6.3 ( 3.0 , 13.0 ) for the tenth ( 30 mm ) , fifth ( 27 mm ) , and two and a half ( 22 mm ) percentiles , respectively ; rrs for before 35 weeks were 4.5 ( 2.9 , 6.9 ) , 7.5 ( 4.5 , 12.5 ) , and 7.8 ( 3.6 , 16.7 ) . sensitivity ranged from 13 to 44% , specificity 9099% , positive predictive value 1547% , and negative predictive value 8098% for prediction of preterm birth before 35 weeks . a study of cervical length in low - risk women found an eightfold ( 95% ci 319 ) increased risk of preterm birth when the cervix was less than 30 mm at 18 to 22 weeks of gestation , but the sensitivity and positive predictive values were low : 19 and 6 percent , respectively . although low sensitivity and low positive predictive value limit its usefulness , it has high negative predictive values and it can be used in screening of low - risk obstetric populations ( table 2 ) . a number of studies have assessed the predictive value of tvs cl in women with some of the most important of these risk factors including a prior ptd [ 50 , 66 , 67 ] , a history of excisional cervical procedures ( cone biopsy , leep ) [ 68 , 69 ] , mullerian anomaly , and two or more voluntary termination ( table 3 ) . in a prospective study of 705 high - risk women , the risk of spontaneous ptb before 35 weeks decreased by approximately 6% for each additional millimeter of cl ( or : 0.94 , 95% ci : 0.920.95 ) and by approximately 5% for each additional week of pregnancy during which the cl was measured ( or : 0.95 , 95% ci : 0.920.98 ) . they conclude that gestational age at which transvaginal ultrasound cervical length is measured significantly affects the calculation of risk of spontaneous preterm birth . the spontaneous preterm birth risk increases as the length of the cervix declines and as the gestational age decreases . funneling comprising 4050% of the total cervical length or a persistently shortened cervix ( < 2530 mm ) has , in several studies , been associated with an increased risk of preterm birth [ 17 , 47 , 49 , 65 ] . measured cervical length among 6334 women with singleton pregnancies at 2224 weeks and looked for the presence of funneling to evaluate its possible additional risk . funneling of the internal os was present in about 4% of pregnancies and the prevalence decreased with increasing cervical length from 98% when the length was 15 mm to about 25% for lengths of 1630 mm and less than 1% at lengths of > 30 mm . the rate of preterm delivery was 6.9% in those with funneling compared to 0.7% in those without funneling . however , logistic regression analysis demonstrated that funneling did not provide a significant additional contribution to cervical length in the prediction of spontaneous delivery before 33 weeks ( or , for short cervix = 24.9 p < 0.0001 ; or , for funneling = 1.8 , p = 0.40 ) . as an independent finding , funneling does not add appreciably to the risk of early gestational age at delivery associated with a shortened cervical length . so , women with a long cervix and funneling are not at increased risk of preterm delivery [ 50 , 51 , 72 ] . in the past few years publications have also highlighted the importance of another morphological ultrasonographic marker for ptd , named as the cervical gland area ( cga ) . the absence of the cga as a new marker for the risk of ptd has to be confirmed by further investigations . a cochrane meta - analysis from 2005 showed that intramuscular progesterone is associated with a reduction in risk of preterm birth before 37 gestational weeks . in a multicenter , randomized trial proposed using daily vaginal micronized progesterone ( 200 mg ) to women with cl 15 mm or less , irrespective of other risk factors . they showed a significant reduction in preterm birth at < 34 weeks with intravaginal progesterone in patients treated based on premature cervical shortening as the indication for therapy . a significant difference was also observed between groups for categorical outcomes including the frequency of cervical length progression to 25 mm and a 50% reduction in cervical length from baseline in this subpopulation . they found that the mean standard deviation precerclage cervical length was 27.2 10.3 mm and after cerclage was 34.1 9.9 mm ( n = 80 , p < 0.001 ) . randomized 113 women with cl < 25 mm or 25% funneling measured between 16 and 24 weeks to either modified bed rest or cerclage . it is important that women were included based on an incidental finding of a short cervix without taking into account other risk factors in the maternal history . subgroup analysis of the utility of cerclage in the high - risk population , based on maternal history , was not performed . found that cerclage does not reduce ptb in low - risk women with short cl compared with rest alone . a recent multicenter randomized trial included 302 women with at least one prior ptb 32 weeks and tvu cl < 25 mm between 16 and 22 weeks randomized to either cerclage or no cerclage . has shown that cerclage , when performed in women with a singleton gestation , previous preterm birth , and cervical length < 25 mm , seems to have a similar effect regardless of the degree of cervical shortening , including cl 1624 mm , as well as cl 5.9 mm . weekly intramuscular 17-alpha - hydroxyprogesterone caproate ( 17 p ) was compared with mcdonald cerclage in women with short cl 25 mm at between 16 and 24 weeks ' gestation . however , cerclage may be more effective in preventing spontaneous ptd in women with cl 15 mm . showed that 17 p had no additional benefit for prevention of ptd in women who had prior sptd and got ultrasound - indicated cerclage for cl < 25 mm . evaluation of the cervical morphology and biometry with transvaginal ultrasonography at 1624 weeks of gestation is a useful tool to predict the risk of preterm birth in low- and high - risk singleton pregnancies . for instance , a sonographic cervical length > 30 mm and present cga have a 96 - 97% negative predictive value for preterm delivery at < 37 weeks . transvaginal evaluation of cervix during routine fetal morphological examination helps identify asymptomatic low- and high - risk women for prediction of preterm delivery . available evidence supports the use of progesterone to women with cervical length 25 mm , irrespective of other risk factors . in women with prior spontaneous ptd with asymptomatic cervical shortening ( cl 25 mm ) there should be prophylactic cerclage procedure and weekly to every two weeks follow - up .

diabetic nephropathy ( dn ) is one of the common microvascular complications of diabetes , and it is characterized by expansion of the mesangial matrix , thickening of the glomerular basement membrane ( gbm ) , persistent proteinuria , and progressive renal dysfunction in diabetic patients with hyperglycemia ; it has been estimated that approximately 20%30% of type 1 and type 2 diabetic patients develop dn . it is considered the leading cause of end - stage renal disease ( esrd ) in many developed countries , and , in china , it accounts for more than 25% of all cases of esrd . the occurrence and development of dn seriously affect the quality of life , prognosis , and even survival of diabetic patients [ 4 , 5 ] . components of the high glucose milieu in diabetes are the main causes of renal injury in dn ; therefore , controlling the plasma glucose level is considered the most crucial goal during treatment . in addition to patients with type 1 diabetes mellitus , those with type 2 diabetes might also eventually require insulin therapy . an increasing number of studies have found that although diabetic patients start taking medication or insulin at the early disease stage , most of them still develop progressive dn , indicating that appropriate therapeutic targets and additional treatments are urgently needed . in the past few decades , with a general understanding of diabetes mellitus , various therapeutic protocols have been proposed to treat diabetes mellitus or its late complications , including vascularized , whole - pancreas transplantation and islet cell transplantation . recent studies have suggested that pancreas or islet transplantation can ameliorate the clinical signs of diabetes mellitus , stabilize further progression , and even reverse the related renal injury in early dn [ 911 ] , primarily because it is more suitable for use under physiological conditions in vivo . nevertheless , the complexity of the vascularized whole - pancreas transplantation procedure may , along with the preexisting diseases present in many diabetic patients , increase the risks of operative and postoperative complications . recently , with gradual advances in methods of islet isolation , islet cell transplantation has progressed from the laboratory to the clinical setting . compared with whole - pancreas transplantation , it is a simpler operation with lower morbidity , and it has become an attractive alternative therapeutic method to conventional insulin injection or pancreas transplantation for treating diabetes and its complications . the purpose of this study was to investigate the efficacy of islet transplantation for reversing early dn and to establish whether this treatment is superior to insulin therapy for the regression of dn . furthermore , commonly measured parameters were assessed , as described in previous studies , including the blood glucose level , body weight , and the urine protein level . podocyte abnormalities are crucial components of progressive renal injury and are known as good predictors of early dn . studies of the role of podocytes in renal injury have highlighted their importance in recent years . in this study , we have elaborated upon the significance of the recovery of podocyte structure and function during the treatment of diabetic nephropathy . additionally , the roles of profibrogenic factors and interstitial cytokines , including tgf-1 , hgf , ctgf , -sma , and mcp-1 , during the progression of renal fibrosis were assessed in early dn . the links between these fibrotic factors and the possible mechanisms of podocyte injury in early dn are also discussed in this study . a total of 50 male sprague - dawley ( sd ) rats weighing 200220 g were provided by the experimental animal center of wenzhou medical university , and some of the rats ( n = 12 ) were used as islet donors . all rats were housed with a 12 h light / dark cycle at 24c 1c and provided with food ad libitum for a week before initiation of the study . all animal experiments were approved by the wenzhou medical university management committee for medical laboratory animal sciences . a rat diabetic model was induced by a single peritoneal injection of streptozotocin ( stz ) ( 55 mg / kg of body weight ) in sodium citrate buffer ( ph 4.5 ) after fasting overnight . the plasma glucose concentration was measured in a drop of tail vein blood using an accu - chek glucometer ( roche diagnostics , indianapolis , in ) . one week later , when the nonfasting blood glucose concentration was 16.67 mmol / l for 3 days , the experimental diabetic rat model was established . after 12 weeks , the urine acr and protein - to - creatinine ratio were determined , and transmission electron microscopy ( tem ) was performed to assess whether the early dn rat model had been successfully established . next , the rats were randomly divided into three different groups : in the untreated group ( dn group , n = 8) , the rats were left untreated and studied 4 weeks later ; in the islet - transplanted group ( it group , n = 8) , the rats underwent islet transplantation under the kidney capsule and were studied 4 weeks later ; and , in the insulin treatment group ( in group , n = 8) , the rats were given glargine insulin ( wanbang pharmaceuticals , jiangsu , china ) by subcutaneous injection at 9 a.m. and 9 p.m. every day for the following 4 weeks ( 3 u each ) . the normal control rats were regarded as the control group ( nc group , n = 8) . briefly , the rats were anesthetized by intraperitoneal injection of chloral hydrate , and laparotomy was performed to expose the pancreas . the entrance of the common bile duct into the intestine was located and ligated , and 8 ml collagenase v ( 0.8 mg / ml , dissolved with hanks solution ) was injected into the common bile duct by retrograde intubation . when the pancreas was fully inflated , it was separated from the surrounding tissues with surgical tweezers , transferred to a 50 ml centrifuge tube , and digested for 1015 min at 37 0.5c . then , the islets were purified by density gradient centrifugation ( histopaque -1119 and histopaque -1077 ) at 2000 rpm for 5 min . the supernatant was poured into a new centrifuge tube and transferred to a black glass culture dish for manual selection of islets . the final purified islets were cultured in rpmi-1640 ( gibco , carlsbad , ca , usa ) containing 10% fetal bovine serum ( fbs ; gibco , invitrogen , inc . , the purified islets were adjusted to an appropriate concentration in the culture medium and transferred to a small culture dish with a 2 mm lattice for their quantification under a microscope . based on the methods of lembert and others , the cell clusters were counted , and the diameters were measured with a microscope eyepiece scale . a single aliquot of 100 freshly isolated islets was aspirated into a 200 l pipette tip and transferred to a small culture dish . the activity of the islets was evaluated by fluorescein diacetate - propidium iodide ( fda - pi ) staining under an inverted fluorescence microscope . the activity ratio was determined using 100 islet cell clusters to estimate that of the total final purified islets . , the final purified islets of approximately 8001000 ieq were aspirated into a 1 ml syringe connected to p50 polyethylene tubing , and the islet was transferred to the head end . the recipient rat was anesthetized by intraperitoneal injection of chloral hydrate , the left flank was shaved , and the kidney was exposed through a small lumbar incision . capsulotomy of the kidney was performed on the caudal outer surface , and the tip of the polyethylene tubing was inserted and advanced gently under the kidney capsule . the islet in the tubing was pushed out slowly and carefully , and the tube was removed after complete transfer of the islet into the capsule . the nonfasting blood glucose levels and body weights of the rats in each group were measured once per week . individual rat metabolic cages were used to collect random and 24 h urine samples twice per week . the urine protein concentration was measured using the sulfosalicylic acid precipitation method , and the creatinine concentration was determined with a creatinine assay kit ( bioassay systems , hayward , ca ) . albuminuria was determined using a rat albumin - specific elisa kit ( exocell laboratories , philadelphia , pa ) . the small pieces of renal cortex obtained from the kidney tissue were cut into small cubes and fixed in 2.5% glutaraldehyde . the remaining tissue was fixed overnight in 4% paraformaldehyde for histopathological staining . after gradient alcohol dehydration and incubation with xylene transparent agent , the kidney tissue was embedded in paraffin and cut into 5 m sections using a microtome . following the ihc staining protocol described by pichaiwong et al . , the paraffin tissue sections were incubated overnight with primary antibodies to tgf-1 , hgf , ctgf , -sma , mcp-1 , and synaptopodin at 4c . then , they were incubated with secondary antibodies and visualized with diaminobenzidine ( dab ; brown color ) and hematoxylin counterstaining under a microscope . the intensity of positive staining was determined according to the iod / area value with image - pro plus 6.0 image analysis software ( media cybernetics , silver spring , md ) . more than 10 fields for each section in each group were evaluated , and the mean value was used . immunofluorescence staining was performed using a rabbit anti - insulin polyclonal antibody to measure the activity of transplanted islets under the kidney capsule . the secondary antibody used for this analysis was changed to goat anti - rabbit igg - fitc , and the nucleus was stained with hoechst staining solution . small pieces of the renal cortex tissue block were fixed in 2.5% glutaraldehyde and washed with pbs ( 0.01 m ) . then , they were postfixed with 1% osmium tetroxide , dehydrated with an acetone gradient , and embedded in araldite m ( sigma aldrich ) . ultrathin sections were counterstained with uranyl acetate and lead citrate and examined with a transmission electron microscope ( h-7700 , hitachi , japan ) . the thickness of the gbm was measured with image - pro plus 6.0 image analysis software . the proteins were extracted , and the total protein concentration was determined by protein bca assay ( beyotime , jiangsu , china ) . after blocking with 5% skim milk , the membranes were incubated overnight at 4c with the primary antibodies . then , they were incubated with goat anti - rabbit igg ( 1 : 5000 ) conjugated to horseradish peroxidase ( hrp ) and visualized with an ecl western blotting detection system ( amersham , arlington heights , il , usa ) . all statistical data were analyzed using spss version 19.0 statistical software ( spss , chicago , il usa ) , and the values are expressed as the mean standard deviation . multiple comparisons between groups were performed by one - way anova , and post hoc analyses were conducted using the least significant difference test . as shown in figure 1 , the urine microalbumin - to - creatinine ratio ( acr ) and protein - to - creatinine ratio were determined to assess renal injury , and transmission electron microscopy ( tem ) detection of the kidney tissue was performed to identify pathological changes . as local gbm thickening , podocyte depletion with fusion of foot processes , mesangial expansion , disordered endothelial cell arrangement , and glomerular filtration barrier structure abnormalities were evident , and the mean acr ( 2.73 0.58 mg / mmol ) and urine protein - to - creatinine ratio ( 66.14 7.25 mg / mmol ) were significantly higher compared to those in the nc group ( 0.28 0.06 mg / mmol and 14.37 1.17 mg / mmol , resp . ( figures 1(c ) and 1(d ) ) , p < 0.05 ) ; the early dn rat model was considered to have been established successfully , as described previously . thus , this rat model was considered suitable for evaluating the advantageous effects of islet transplantation on early dn . before transplantation , islet activity was evaluated by fda - pi staining with an aliquot of islets , and the results revealed a high level of islet activity ( > 99% , figure 2(b ) ) . the transplanted islets were distributed and were visible under the capsule ( figure 2(a ) ) . at four weeks after transplantation , immunohistochemical and immunofluorescence staining for insulin demonstrated high activity of the transplanted islets under the kidney capsule and indicated that they were still capable of normal insulin secretion ( figures 2(c ) and 2(d ) ) . throughout this experiment , the surviving islets retained normal insulin secretion function and maintained the blood glucose level within the normal range ; immunofluorescence staining verified these results . at three days after transplantation , the blood glucose level in the it group was significantly decreased and was maintained at a normal level ( 5.96 1.81 mmol / l , figure 1(a ) ) . the body weights of the rats in the it and in groups gradually increased , whereas the growth curve for the it group was significantly steeper than that for the in group ( figure 1(b ) ) . the glucose level in the rats in the dn group continued to remain high , along with a continuous decrease in body weight . as shown in figure 1(c ) , after 4 weeks , the urinary acr decreased significantly in the it group ( 0.43 0.11 mg / mmol ) after islet transplantation . in contrast , it remained high in the in group ( 1.64 0.49 mg / mmol ) . compared to the model group ( 2.73 0.58 mg / mmol ) , the urinary acr was higher ( 5.18 1.36 mg / mmol ) in the dn group . the urine protein - to - creatinine ratio also obviously differed between the it and in groups ( figure 1(d ) ) . compared with the in ( 43.76 6.30 mg / mmol ) and dn groups ( 93.43 8.51 mg / mmol ) , there was a significant decline in this ratio , which dropped close to the normal level ( 14.37 1.17 mg / mmol ) , in the it group ( 17.95 2.20 mg / mmol ) . as shown in figure 3(a ) , we found that the thickening of the gbm , disordered endothelial cell arrangement , and podocyte depletion with fusion of the foot processes were not obvious in the it group compared with the nc group . in contrast , in the in and dn groups , the range of podocyte depletion with fusion of the foot processes was more extensive than that in the it group ( figure 3(a ) ) . additionally , as shown in figure 3(c ) , the mean thickness of the gbm was measured , and the data demonstrated that the thickness in the it group ( 0.24 0.02 m ) was significantly less than that in the in group ( 0.33 0.05 m , p = 0.001 ) or dn group ( 0.46 0.04 m , p < 0.001 ) . synaptopodin stains podocytes specifically , and podocyte density can be measured by the intensity of synaptopodin staining . as shown in figure 3(b ) , the intensity of synaptopodin staining was significantly decreased in the model and dn groups compared with the nc group . the strength of synaptopodin staining was obviously greater in the it group than in the in group , indicating a greater density of podocytes . this was also confirmed by the greater iod / area value of synaptopodin staining in the it group compared with the in group ( p = 0.003 ) , as shown in figure 3(d ) . to confirm and compare the ameliorative effects of islet transplantation and insulin therapy on renal fibrosis in early dn , immunohistochemical staining was performed to determine the expression of fibrosis - related factors . as shown in figures 4 and 5 , decreased expression of tgf-1 , ctgf , mcp-1 , and -sma and increased expression of hgf were detected in the it group compared with the in group ( p < 0.05 for each ) . additionally , the brown staining of the granules was significantly decreased in the it group compared with the dn group , and the it group also had a lower mean iod / area value for the related renal fibrosis factors ( p < 0.001 ) . western blotting analysis further demonstrated that the protein expression of each factor was obviously lower in the it group compared with the in and dn groups ( figure 6 ) ; these results coincide with the immunohistochemical staining results . over the past few decades , islet transplantation has emerged as an alternative therapeutic method for the use of conventional antidiabetic drugs and insulin for the treatment of diabetic patients . several studies have demonstrated that pancreas or islet transplantation is an effective treatment for diabetic patients with complications . in recent years , remarkable results researchers have demonstrated that the kidney graft survival rates for uremic patients with type 1 diabetes mellitus can improve significantly after successful islet transplantation . our study was designed to investigate the efficacy of islet transplantation for reversing early dn and to establish whether this treatment is superior to insulin therapy for treating diabetic complications in model rats . the transplanted islets were able to maintain the blood glucose level within the normal range without the use of immunosuppressants in sd rats with diabetic nephropathy . the results of our study demonstrated that all of the measured clinical signs of diabetes mellitus were abolished after islet transplantation and that renal injury was significantly improved ; these signs included a decrease in the blood glucose level , attenuation of proteinuria and albuminuria , and normalization of the glomerular filtration barrier . in contrast , with insulin treatment , the blood glucose level , acr , and protein - to - creatinine ratio were not well controlled , and the expression of related renal fibrosis factors was obviously higher than that observed following islet transplantation in this study . all of these data demonstrated that early dn could be reversed after islet transplantation , and the ameliorative effects achieved with transplantation were significantly better than those obtained with insulin therapy for the treatment of diabetic complications . furthermore , tem was also used in this study to detect microscopic changes of the glomerular basement membrane ( gbm ) and podocytes in the glomerulus . wide application of this technology would allow for more accurate analysis of kidney diseases in the clinical setting . changes of microscopic structures ( podocytes and gbm ) are considered to play an important role during the progression of renal disease . podocytes are highly differentiated epithelial cells in the glomerulus that attach to the gbm to maintain efficient glomerular filtration . their interdigitating foot processes are bridged by a slit diaphragm to control the patency through the transcytotic clearance mechanisms . recent research has indicated that podocyte damage plays a crucial role in progressive dn and that it can directly result in progressive renal hypofunction [ 22 , 23 ] . in the early stage of diabetes , podocyte dysfunction is manifested histologically by the broadening of foot processes and functionally by various physiological alterations . injury to the gbm or podocytes could cause barrier damage and protein loss , which might explain why progressive proteinuria was the typical manifestation of early dn . in our study , islet transplantation significantly ameliorated the thickening of the gbm and the podocyte depletion with fusion of the foot processes . growing evidence indicates that the signaling abnormalities in podocytes in dn mainly involve the tgf- family . the blockage of inflammation - induced injury or intervention of inflammatory mechanisms in podocytes could ameliorate renal fibrosis in early dn . renal fibrosis is one of the typical features of early dn , and the degree of fibrosis is strongly associated with the progression of dn . progressive accumulation of myofibroblasts in the glomerulus and tubules is critical for the development of dn ; therefore , early prevention or intervention of renal fibrosis is extremely important . studies have also indicated that the actions of fibrotic factors and cytokines , such as tgf-1 , hgf , ctgf , -sma , and mcp-1 , play significant roles in the progression of renal fibrosis in early dn . compared with the untreated dn rats , the expression of hgf was obviously elevated , accompanied by a significant decrease in the expression of other factors , after islet transplantation . tgf-1 is regarded as the central fibrogenic factor in the pathogenesis of progressive renal fibrosis in dn . its overexpression stimulates renal fibrosis by promoting podocyte and tubular epithelial and glomerular endothelial cell apoptosis , activating interstitial fibroblasts , and inducing the activation of -sma expression and mesangial cells to produce large amounts of ecm components . hgf might inhibit tgf-1 expression and prevent the progression of renal fibrosis in various animal models . components of the diabetic milieu also could promote the recruitment of macrophages by stimulating the expression of mcp-1 in the kidneys in response to renal injury . the results of our study showed that renal fibrosis was improved significantly after islet transplantation compared with that observed after insulin therapy . because it is associated with a lower risk of surgery and fewer side effects than full - pancreas transplantation , pancreatic islet transplantation had been widely investigated as a promising strategy for treating diabetes mellitus and/or its complications . it is well known that sustained hyperglycemia is one of the major causes of dn . regular oral insulin administration or insulin injection can not always maintain plasma glucose within the normal range , resulting in fluctuations in the blood glucose level [ 31 , 32 ] . however , because islet transplantation is capable of physiologic regulation in vivo , it can control the secretion of insulin automatically to maintain the blood glucose level within the normal range . hypoglycemia is considered one of the most dangerous complications occurring in clinically diabetic patients , and intensive insulin therapy protocols have been widely used in the clinical intensive care of diabetes mellitus ; notably , the incidence of hypoglycemia is markedly increased if the blood glucose level is not constantly monitored . nevertheless , because of the self - regulation of physiological systems , excessive insulin secretion does not occur after islet transplantation . during this study , although a rat 's blood glucose level was not fully maintained within the normal range after transplantation , the renal damage and fibrosis of dn of this rat were still alleviated as observed in the late examination . these findings could indicate that transplantation of a small amount of transplanted islets also had ameliorative effects on dn , but further study is required for clarification . relevant signaling pathways about the ameliorative effects of islet transplantation have not been illuminated very clearly , and these topics also will be the main focuses of our next experiments . recent studies have demonstrated that abnormalities in signaling pathways could also contribute to the pathologic signs of dn . an increasing number of studies have attempted to elucidate the molecular mechanisms of dn to facilitate the development of preventative strategies and effective therapies . fiorina and his colleagues have found the induction of immune - related protein b7 - 1 is associated with the progression of proteinuria in human and animal glomerular diseases . the expression of b7 - 1 in podocytes is upregulated in high glucose conditions , which can induce podocyte morphologic abnormalities and promote cell death . targeting b7 - 1 has been suggested as a promising effective method on preventing cure diabetic nephropathy in their studies . these new pathogenic mechanisms have been clarified by manipulation of gene expression in animal models , and the use of such models might contribute to the understanding and clinical therapy of dn . due to the limitations of the present study , many important issues remain to be addressed , particularly the survival of the transplanted islets and the minimum equivalent required to treat diabetes mellitus . meanwhile , research has indicated that c - peptide might play an important role in the control of blood glucose in diabetic patients . johansson et al . have found that the combined administration of insulin , proinsulin , and c - peptide has greater efficacy for the treatment of diabetes than insulin injection alone . whether islet transplantation can regulate the secretion of c - peptide is also a vital issue . the encouraging studies and results of the present study could provide a basis for the clinical application of islet transplantation . in conclusion , our results demonstrated that islet transplantation could reverse various symptoms of early dn in a rat model , and the ameliorative effects on kidney injury and renal fibrosis were obviously better with islet transplantation than with insulin therapy . the recovery of impaired podocyte structure and function is the key to the treatment of diabetic nephropathy . in this study , islet transplantation under the kidney capsule significantly ameliorated the impaired renal function and microscopic damage in the early diabetic nephropathy rats . these findings may provide new possibilities to treat or prevent early dn and other complications in patients with diabetes mellitus in the future .

objective . diabetic nephropathy ( dn ) is a common microvascular complication of diabetes mellitus , and insulin therapy has many side effects in the treatment of dn . islet transplantation has emerged as a promising therapy for diabetic patients . this study was established to investigate its advantageous effects in a rat model of early dn . methods . streptozotocin was administered to the rats to induce diabetes . twelve weeks later , the diabetic rats were divided into 3 groups : the islet - transplanted group ( it group ) , the insulin - treated group ( in group ) , and the untreated group ( dn group ) . renal injury and kidney structure were assessed by urinalysis and transmission electron microscopy ( tem ) detection . immunohistochemical staining and western blotting were performed to assess renal fibrosis levels . results . the early dn features were reversed and the glomerular filtration barrier and basement membrane structures were improved at 4 weeks after islet transplantation . the urine microalbumin - to - creatinine ratio ( acr ) , protein - to - creatinine ratio , and mean thickness of the glomerular basement membrane ( gbm ) were significantly decreased in the it group . the expression of renal fibrotic factors was also significantly decreased . conclusions . these data suggest that early dn can be reversed after islet transplantation , and they may facilitate the development of a clinical therapeutic strategy for human diabetes mellitus .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

diabetic nephropathy ( dn ) is one of the common microvascular complications of diabetes , and it is characterized by expansion of the mesangial matrix , thickening of the glomerular basement membrane ( gbm ) , persistent proteinuria , and progressive renal dysfunction in diabetic patients with hyperglycemia ; it has been estimated that approximately 20%30% of type 1 and type 2 diabetic patients develop dn . the occurrence and development of dn seriously affect the quality of life , prognosis , and even survival of diabetic patients [ 4 , 5 ] . components of the high glucose milieu in diabetes are the main causes of renal injury in dn ; therefore , controlling the plasma glucose level is considered the most crucial goal during treatment . in addition to patients with type 1 diabetes mellitus , those with type 2 diabetes might also eventually require insulin therapy . an increasing number of studies have found that although diabetic patients start taking medication or insulin at the early disease stage , most of them still develop progressive dn , indicating that appropriate therapeutic targets and additional treatments are urgently needed . in the past few decades , with a general understanding of diabetes mellitus , various therapeutic protocols have been proposed to treat diabetes mellitus or its late complications , including vascularized , whole - pancreas transplantation and islet cell transplantation . recent studies have suggested that pancreas or islet transplantation can ameliorate the clinical signs of diabetes mellitus , stabilize further progression , and even reverse the related renal injury in early dn [ 911 ] , primarily because it is more suitable for use under physiological conditions in vivo . nevertheless , the complexity of the vascularized whole - pancreas transplantation procedure may , along with the preexisting diseases present in many diabetic patients , increase the risks of operative and postoperative complications . recently , with gradual advances in methods of islet isolation , islet cell transplantation has progressed from the laboratory to the clinical setting . compared with whole - pancreas transplantation , it is a simpler operation with lower morbidity , and it has become an attractive alternative therapeutic method to conventional insulin injection or pancreas transplantation for treating diabetes and its complications . the purpose of this study was to investigate the efficacy of islet transplantation for reversing early dn and to establish whether this treatment is superior to insulin therapy for the regression of dn . furthermore , commonly measured parameters were assessed , as described in previous studies , including the blood glucose level , body weight , and the urine protein level . podocyte abnormalities are crucial components of progressive renal injury and are known as good predictors of early dn . studies of the role of podocytes in renal injury have highlighted their importance in recent years . in this study , we have elaborated upon the significance of the recovery of podocyte structure and function during the treatment of diabetic nephropathy . additionally , the roles of profibrogenic factors and interstitial cytokines , including tgf-1 , hgf , ctgf , -sma , and mcp-1 , during the progression of renal fibrosis were assessed in early dn . the links between these fibrotic factors and the possible mechanisms of podocyte injury in early dn are also discussed in this study . a total of 50 male sprague - dawley ( sd ) rats weighing 200220 g were provided by the experimental animal center of wenzhou medical university , and some of the rats ( n = 12 ) were used as islet donors . all rats were housed with a 12 h light / dark cycle at 24c 1c and provided with food ad libitum for a week before initiation of the study . one week later , when the nonfasting blood glucose concentration was 16.67 mmol / l for 3 days , the experimental diabetic rat model was established . after 12 weeks , the urine acr and protein - to - creatinine ratio were determined , and transmission electron microscopy ( tem ) was performed to assess whether the early dn rat model had been successfully established . next , the rats were randomly divided into three different groups : in the untreated group ( dn group , n = 8) , the rats were left untreated and studied 4 weeks later ; in the islet - transplanted group ( it group , n = 8) , the rats underwent islet transplantation under the kidney capsule and were studied 4 weeks later ; and , in the insulin treatment group ( in group , n = 8) , the rats were given glargine insulin ( wanbang pharmaceuticals , jiangsu , china ) by subcutaneous injection at 9 a.m. and 9 p.m. every day for the following 4 weeks ( 3 u each ) . briefly , the rats were anesthetized by intraperitoneal injection of chloral hydrate , and laparotomy was performed to expose the pancreas . the entrance of the common bile duct into the intestine was located and ligated , and 8 ml collagenase v ( 0.8 mg / ml , dissolved with hanks solution ) was injected into the common bile duct by retrograde intubation . based on the methods of lembert and others , the cell clusters were counted , and the diameters were measured with a microscope eyepiece scale . , the final purified islets of approximately 8001000 ieq were aspirated into a 1 ml syringe connected to p50 polyethylene tubing , and the islet was transferred to the head end . the recipient rat was anesthetized by intraperitoneal injection of chloral hydrate , the left flank was shaved , and the kidney was exposed through a small lumbar incision . capsulotomy of the kidney was performed on the caudal outer surface , and the tip of the polyethylene tubing was inserted and advanced gently under the kidney capsule . the islet in the tubing was pushed out slowly and carefully , and the tube was removed after complete transfer of the islet into the capsule . the urine protein concentration was measured using the sulfosalicylic acid precipitation method , and the creatinine concentration was determined with a creatinine assay kit ( bioassay systems , hayward , ca ) . , the paraffin tissue sections were incubated overnight with primary antibodies to tgf-1 , hgf , ctgf , -sma , mcp-1 , and synaptopodin at 4c . more than 10 fields for each section in each group were evaluated , and the mean value was used . the secondary antibody used for this analysis was changed to goat anti - rabbit igg - fitc , and the nucleus was stained with hoechst staining solution . the thickness of the gbm was measured with image - pro plus 6.0 image analysis software . the proteins were extracted , and the total protein concentration was determined by protein bca assay ( beyotime , jiangsu , china ) . all statistical data were analyzed using spss version 19.0 statistical software ( spss , chicago , il usa ) , and the values are expressed as the mean standard deviation . multiple comparisons between groups were performed by one - way anova , and post hoc analyses were conducted using the least significant difference test . as shown in figure 1 , the urine microalbumin - to - creatinine ratio ( acr ) and protein - to - creatinine ratio were determined to assess renal injury , and transmission electron microscopy ( tem ) detection of the kidney tissue was performed to identify pathological changes . as local gbm thickening , podocyte depletion with fusion of foot processes , mesangial expansion , disordered endothelial cell arrangement , and glomerular filtration barrier structure abnormalities were evident , and the mean acr ( 2.73 0.58 mg / mmol ) and urine protein - to - creatinine ratio ( 66.14 7.25 mg / mmol ) were significantly higher compared to those in the nc group ( 0.28 0.06 mg / mmol and 14.37 1.17 mg / mmol , resp . ( figures 1(c ) and 1(d ) ) , p < 0.05 ) ; the early dn rat model was considered to have been established successfully , as described previously . thus , this rat model was considered suitable for evaluating the advantageous effects of islet transplantation on early dn . before transplantation , islet activity was evaluated by fda - pi staining with an aliquot of islets , and the results revealed a high level of islet activity ( > 99% , figure 2(b ) ) . at four weeks after transplantation , immunohistochemical and immunofluorescence staining for insulin demonstrated high activity of the transplanted islets under the kidney capsule and indicated that they were still capable of normal insulin secretion ( figures 2(c ) and 2(d ) ) . at three days after transplantation , the blood glucose level in the it group was significantly decreased and was maintained at a normal level ( 5.96 1.81 mmol / l , figure 1(a ) ) . the body weights of the rats in the it and in groups gradually increased , whereas the growth curve for the it group was significantly steeper than that for the in group ( figure 1(b ) ) . the glucose level in the rats in the dn group continued to remain high , along with a continuous decrease in body weight . as shown in figure 1(c ) , after 4 weeks , the urinary acr decreased significantly in the it group ( 0.43 0.11 mg / mmol ) after islet transplantation . in contrast , it remained high in the in group ( 1.64 0.49 mg / mmol ) . compared to the model group ( 2.73 0.58 mg / mmol ) , the urinary acr was higher ( 5.18 1.36 mg / mmol ) in the dn group . the urine protein - to - creatinine ratio also obviously differed between the it and in groups ( figure 1(d ) ) . compared with the in ( 43.76 6.30 mg / mmol ) and dn groups ( 93.43 8.51 mg / mmol ) , there was a significant decline in this ratio , which dropped close to the normal level ( 14.37 1.17 mg / mmol ) , in the it group ( 17.95 2.20 mg / mmol ) . as shown in figure 3(a ) , we found that the thickening of the gbm , disordered endothelial cell arrangement , and podocyte depletion with fusion of the foot processes were not obvious in the it group compared with the nc group . in contrast , in the in and dn groups , the range of podocyte depletion with fusion of the foot processes was more extensive than that in the it group ( figure 3(a ) ) . additionally , as shown in figure 3(c ) , the mean thickness of the gbm was measured , and the data demonstrated that the thickness in the it group ( 0.24 0.02 m ) was significantly less than that in the in group ( 0.33 0.05 m , p = 0.001 ) or dn group ( 0.46 0.04 m , p < 0.001 ) . synaptopodin stains podocytes specifically , and podocyte density can be measured by the intensity of synaptopodin staining . as shown in figure 3(b ) , the intensity of synaptopodin staining was significantly decreased in the model and dn groups compared with the nc group . the strength of synaptopodin staining was obviously greater in the it group than in the in group , indicating a greater density of podocytes . this was also confirmed by the greater iod / area value of synaptopodin staining in the it group compared with the in group ( p = 0.003 ) , as shown in figure 3(d ) . to confirm and compare the ameliorative effects of islet transplantation and insulin therapy on renal fibrosis in early dn , immunohistochemical staining was performed to determine the expression of fibrosis - related factors . as shown in figures 4 and 5 , decreased expression of tgf-1 , ctgf , mcp-1 , and -sma and increased expression of hgf were detected in the it group compared with the in group ( p < 0.05 for each ) . additionally , the brown staining of the granules was significantly decreased in the it group compared with the dn group , and the it group also had a lower mean iod / area value for the related renal fibrosis factors ( p < 0.001 ) . western blotting analysis further demonstrated that the protein expression of each factor was obviously lower in the it group compared with the in and dn groups ( figure 6 ) ; these results coincide with the immunohistochemical staining results . over the past few decades , islet transplantation has emerged as an alternative therapeutic method for the use of conventional antidiabetic drugs and insulin for the treatment of diabetic patients . several studies have demonstrated that pancreas or islet transplantation is an effective treatment for diabetic patients with complications . in recent years , remarkable results researchers have demonstrated that the kidney graft survival rates for uremic patients with type 1 diabetes mellitus can improve significantly after successful islet transplantation . our study was designed to investigate the efficacy of islet transplantation for reversing early dn and to establish whether this treatment is superior to insulin therapy for treating diabetic complications in model rats . the results of our study demonstrated that all of the measured clinical signs of diabetes mellitus were abolished after islet transplantation and that renal injury was significantly improved ; these signs included a decrease in the blood glucose level , attenuation of proteinuria and albuminuria , and normalization of the glomerular filtration barrier . in contrast , with insulin treatment , the blood glucose level , acr , and protein - to - creatinine ratio were not well controlled , and the expression of related renal fibrosis factors was obviously higher than that observed following islet transplantation in this study . all of these data demonstrated that early dn could be reversed after islet transplantation , and the ameliorative effects achieved with transplantation were significantly better than those obtained with insulin therapy for the treatment of diabetic complications . furthermore , tem was also used in this study to detect microscopic changes of the glomerular basement membrane ( gbm ) and podocytes in the glomerulus . changes of microscopic structures ( podocytes and gbm ) are considered to play an important role during the progression of renal disease . podocytes are highly differentiated epithelial cells in the glomerulus that attach to the gbm to maintain efficient glomerular filtration . in the early stage of diabetes , podocyte dysfunction is manifested histologically by the broadening of foot processes and functionally by various physiological alterations . injury to the gbm or podocytes could cause barrier damage and protein loss , which might explain why progressive proteinuria was the typical manifestation of early dn . in our study , islet transplantation significantly ameliorated the thickening of the gbm and the podocyte depletion with fusion of the foot processes . the blockage of inflammation - induced injury or intervention of inflammatory mechanisms in podocytes could ameliorate renal fibrosis in early dn . renal fibrosis is one of the typical features of early dn , and the degree of fibrosis is strongly associated with the progression of dn . progressive accumulation of myofibroblasts in the glomerulus and tubules is critical for the development of dn ; therefore , early prevention or intervention of renal fibrosis is extremely important . studies have also indicated that the actions of fibrotic factors and cytokines , such as tgf-1 , hgf , ctgf , -sma , and mcp-1 , play significant roles in the progression of renal fibrosis in early dn . compared with the untreated dn rats , the expression of hgf was obviously elevated , accompanied by a significant decrease in the expression of other factors , after islet transplantation . tgf-1 is regarded as the central fibrogenic factor in the pathogenesis of progressive renal fibrosis in dn . components of the diabetic milieu also could promote the recruitment of macrophages by stimulating the expression of mcp-1 in the kidneys in response to renal injury . the results of our study showed that renal fibrosis was improved significantly after islet transplantation compared with that observed after insulin therapy . because it is associated with a lower risk of surgery and fewer side effects than full - pancreas transplantation , pancreatic islet transplantation had been widely investigated as a promising strategy for treating diabetes mellitus and/or its complications . it is well known that sustained hyperglycemia is one of the major causes of dn . hypoglycemia is considered one of the most dangerous complications occurring in clinically diabetic patients , and intensive insulin therapy protocols have been widely used in the clinical intensive care of diabetes mellitus ; notably , the incidence of hypoglycemia is markedly increased if the blood glucose level is not constantly monitored . nevertheless , because of the self - regulation of physiological systems , excessive insulin secretion does not occur after islet transplantation . during this study , although a rat 's blood glucose level was not fully maintained within the normal range after transplantation , the renal damage and fibrosis of dn of this rat were still alleviated as observed in the late examination . relevant signaling pathways about the ameliorative effects of islet transplantation have not been illuminated very clearly , and these topics also will be the main focuses of our next experiments . recent studies have demonstrated that abnormalities in signaling pathways could also contribute to the pathologic signs of dn . an increasing number of studies have attempted to elucidate the molecular mechanisms of dn to facilitate the development of preventative strategies and effective therapies . the expression of b7 - 1 in podocytes is upregulated in high glucose conditions , which can induce podocyte morphologic abnormalities and promote cell death . targeting b7 - 1 has been suggested as a promising effective method on preventing cure diabetic nephropathy in their studies . these new pathogenic mechanisms have been clarified by manipulation of gene expression in animal models , and the use of such models might contribute to the understanding and clinical therapy of dn . due to the limitations of the present study , many important issues remain to be addressed , particularly the survival of the transplanted islets and the minimum equivalent required to treat diabetes mellitus . meanwhile , research has indicated that c - peptide might play an important role in the control of blood glucose in diabetic patients . have found that the combined administration of insulin , proinsulin , and c - peptide has greater efficacy for the treatment of diabetes than insulin injection alone . the encouraging studies and results of the present study could provide a basis for the clinical application of islet transplantation . in conclusion , our results demonstrated that islet transplantation could reverse various symptoms of early dn in a rat model , and the ameliorative effects on kidney injury and renal fibrosis were obviously better with islet transplantation than with insulin therapy . the recovery of impaired podocyte structure and function is the key to the treatment of diabetic nephropathy . in this study , islet transplantation under the kidney capsule significantly ameliorated the impaired renal function and microscopic damage in the early diabetic nephropathy rats . these findings may provide new possibilities to treat or prevent early dn and other complications in patients with diabetes mellitus in the future .

while autografts are currently the best available option for bone grafting , the pain and morbidity associated with second surgeries combined with the limited availability of autograft sources highlight the need for alternative grafting materials [ 13 ] . three - dimensional bone tissue engineering strategies provide scaffolding for new bone tissue to grow into as well as a cellular source for new tissue growth ( osteogenic ) [ 36 ] . however , one of the biggest limitations to overcome is the maintenance of cellular viability within the center of three - dimensional constructs upon in vivo implantation [ 711 ] . vascularization is critical for successful bone growth and development and is one of the first phases seen during fracture healing [ 1116 ] . without proper vascularization , bone tissue would face limited growth and hypoxia . as the vasculature provides bone tissue with oxygen , nutrients , growth factors , and the removal of waste , it is not surprising that vascularization is also essential for successful implant integration . currently , implanted bone grafts rely on the ingrowth of existing vessels from host tissue [ 7 , 9 ] , which can take significant amounts of time , leading to poor vascularization and implant rejection . one solution to this clinical problem is to introduce tissue - engineered bone that is prevascularized by a rudimentary vascular system [ 7 , 9 , 10 , 1720 ] . a common approach to this solution is the co - culture of bone cells and endothelial cells on three - dimensional constructs . there has been some success with co - culturing bone cells and endothelial cells to overcome the lack of vasculature in tissue - engineered bone [ 710 , 17 , 18 , 21 ] . these studies have shown that endothelial cells can form a pre - vascular network during co - culture [ 9 , 10 , 17 ] , and endothelial cells enhance osteoblastic activity when both cell types are in direct contact [ 8 , 9 , 17 , 22 , 23 ] . additionally , recent work has shown that three - dimensional cocultures will integrate with host tissue in vivo , with the formation of an improved vascular network upon implantation [ 2426 ] . although static cocultures have had some success , it is not known how cocultures respond to a dynamic environment . perfusion bioreactors deliver culture medium to cell - seeded porous scaffolds through pump - driven fluid flow . as oxygen 's poor diffusion capacity largely governs the production of three - dimensional tissue cultures in vitro [ 27 , 28 ] , perfusion bioreactors mitigate hypoxic ( oxygen - depleted ) conditions by providing oxygen delivery to the center of three - dimensional cell - seeded scaffolds , which increases cellular infiltration and viability within three - dimensional scaffolds [ 27 , 2933 ] . perfusion also leads to upregulation in gene expression [ 29 , 31 , 34 ] , increases in protein production [ 30 , 33 , 3538 ] , and enhanced calcium deposition [ 30 , 36 , 3941 ] in osteoblast - seeded scaffolds . furthermore , fluid flow activates endothelial cells , increasing angiogenic gene expression and the formation of tube - like structures [ 12 , 22 , 4245 ] . taken together , these results suggest that the use of a perfusion bioreactor with osteoblast / endothelial cell cocultures may improve osteoblast activity and accelerate the vascularization of tissue - engineered bone by alleviating the hypoxic state of three - dimensional culture . the goal of this study was to help to elucidate the effects that a dynamic environment has on osteoblast / endothelial cell three - dimensional cocultures . we hypothesized that co - culturing osteoblasts and endothelial cells increases osteogenic and angiogenic gene expressions and decreases hypoxic gene expression in three - dimensional calcium phosphate scaffolds . furthermore , we hypothesized that perfusion flow increases cell and matrix coverage area throughout three - dimensional scaffolds . mouse osteoblast - like cells ( mc3t3-e1 subclone 4 , atcc , manassas , va , usa ) were cultured in alpha - mem ( invitrogen , carlsbad , ca , usa ) with 10% fbs ( hyclone , logan , ut , usa ) and 1% pen / strep ( cellgro , herndon , va , usa ) . mouse microvascular endothelial cells ( ec ) ( eoma , atcc , manassas , va , usa ) were cultured in dulbecco 's mem ( invitrogen , carlsbad , ca , usa ) with 10% fbs ( hyclone , logan , ut , usa ) and 1% pen / strep ( cellgro , herndon , va , usa ) . once cells reached ~70% confluency , they were trypsinized , resuspended in culture medium and statically seeded onto three - dimensional calcium phosphate scaffolds ( bd biosciences , san jose , ca , usa ) in 25 l medium ( 1,000,000 cells / scaffold : 100% osteoblasts or 98% osteoblasts and 2% endothelial cells ) . the scaffolds have an interconnected porosity of ~60% with an average pore size of 200400 microns . this is comparable to trabecular bone ( 5090% porosity , 5001500 micron pore size ) and within the range suggested to be optimal for bone regeneration ( 150650 microns ) in porous scaffolds . cell - seeded scaffolds were incubated for 1 hour to allow cell adhesion , then covered with media ( alpha - mem , 10% fbs , and 1% pen / strep ) , and incubated for 24 hours before experimentation . during 7- and 14-day experimentation , osteoblast differentiation media was used by adding 10 mm beta - glycerol phosphate and 50 mg / ml ascorbic acid . during 1- and 2-day experimentation osteoblast - seeded scaffolds were evaluated after 1 or 2 days of static or perfusion ( n = 5 per group ) culture . four treatment groups ( n = 3 per group ) were tested at 7- and 14-day time points : two cell populations ( osteoblasts alone and osteoblast / endothelial cell co - culture ) with two culture methods ( static culture and perfusion flow ) . static samples were cultured in 24-well plates , while perfusion samples were cultured in bioreactor chambers . all samples remained incubated at 37c with 5% co2 for the duration of the experiment . flow was initiated immediately after samples were placed into the bioreactor chambers and continued for the duration of the experiment . after 1 , 2 , 7 , or 14 days of culture ( static or perfusion ) , rna was isolated from the cell - seeded scaffolds , or the scaffolds were fixed in 10% buffered formalin for histological processing . gene expression was assessed at all - time points , and histological analysis was performed at the 14 day timepoint . a custom perfusion bioreactor was used to deliver culture media to the cell - seeded calcium phosphate scaffolds . each bioreactor chamber consists of a top and bottom aluminum block , between which a polycarbonate block is secured ( figure 1 ) . a 6 mm hole was drilled in the polycarbonate block to fit the scaffolds ( 5 mm diameter ; 3.5 mm height ) . grooves were machined around the holes on each side of the polycarbonate block to fit a # 11 viton o - ring ( allorings , hampton falls , nh , usa ) . a 3 mm barbed pipe connector was threaded into both the top and bottom aluminum blocks and connected to 3 mm silicone tubing ( harvard apparatus , holliston , ma , usa ) . tubing connected the top of the chamber to a 140 ml syringe fluid reservoir and the bottom of the chamber to syringe pump ( harvard apparatus ) . the pump delivered media at a rate of 0.075 ml / min , with flow being reversed every 24 hours . this flow rate produced an estimated shear stress of < 0.001 pa . media was replenished every third day . after 1 , 2 , 7 , and 14 days of culture , cell - seeded scaffolds were placed in 2 ml tubes and crushed in sv lysis buffer with a glass rod . after lysis , dilution buffer was added along with 200 mm phosphate buffer to elute the rna . rna was isolated from cells using the promega sv total rna isolation kit according to manufacturer 's instructions . rna concentration and quality were determined using spectrophotometer ( nanodrop nd-1000 spectrophotometer , nanodrop technologies , rockland , de , usa ) readings at 260 , 230 , and 280 nm . rna was reverse transcribed into cdna using a reaction mix consisting of superscript ii reverse transcriptase ( invitrogen , calrsbad , ca , usa ) , 1x first strand buffer ( invitrogen ) , 800 m dntps ( promega ) , rnase out recombinant ribonuclease inhibitor ( invitrogen ) , 48 mm dithiothreitol ( invitrogen ) and 0.5 g oligo(dt)12 - 18 pimer at 42c for 20 minutes , 50c for 10 minutes , and 42c for 1 hour in the mastercycler gradient thermocycler ( eppendorf , westbury , ny , usa ) . cdna was then used for real - time pcr for the genes of interest and the housekeeping genes ( table 1 ) . all reactions were performed in the steponeplus real - time pcr system ( applied biosystems ) under the following cycle parameters : hot start at 95c for 15 minutes followed by 40 cycles of 95c for 15 seconds , 60c for 30 seconds , and 72c for 15 seconds . threshold fluorescence was set to 1500 dr and the ct value was determined for all reactions . ct values were used to determine the relative up - or downregulation for each gene using the relative standard curve method and normalizing to housekeeping genes that do not change between treatments . following fixation , 14-day samples were placed in cassettes , covered in oct freeze medium , and placed under vacuum for 48 hours to remove air bubbles . samples were then removed , flash frozen , and sectioned on a soft tissue microtome ( microm international , waldorf , germany ) at a thickness of 10 microns . sections were taken from mid - way through the scaffold running parallel to the direction of perfusion flow ( figure 2 ) . sections were decalcified in 0.5% edta for 20 minutes . after washing with pbs , biotin - conjugated antibody ( 1 : 250 antimouse cd31endothelial cell - specific ) was added and samples were incubated for 1 hour . sections were again washed and the secondary antibody ( 1 : 500 , alexa fluor 755 ) was added and samples were incubated for 45 minutes . following a brief rinse , sections were also stained with hematoxylin and eosin . prior to fluorescent microscopy , dapi was added to the sections to identify cell nuclei . images were captured for the entire section from each sample at 100x total magnification using both brightfield and fluorescent microscopy . nine images were taken in total , three from the upper , middle , and lower thirds of each section ( figure 2 ) . fluorescent images were used to distinguish between cell types , as dapi identifies all cell nuclei and cd31 stains membrane receptors on endothelial cells . brightfield images were used to determine total cell number , cell surface coverage , and cell / matrix area coverage . cell number , cell surface coverage , and cell / matrix area coverage were determined around the periphery of the scaffold as well as in the scaffold center , allowing for the assessment of whether perfusion flow is more effective in areas of poor diffusion . for the scaffold periphery , regions 18 were combined . for the scaffold center , only the center image ( region 9 ) was used for measurement ( figure 2 ) . cell number was determined by counting the total cell number in each region within a section . in the scaffold periphery , cell number was averaged for all 8 regions ( regions 18 , figure 2 ) for comparison with the scaffold center ( region 9 , figure 2 ) . cell surface coverage was calculated as the ratio of the length of cell - covered scaffold surface to the total length of scaffold surface using bioquant ( bioquant osteo , nashville , tn , usa ) software ( figure 2 ) . cell / matrix area coverage was calculated as the ratio of cell and matrix area within pore spaces to total pore space area ( figure 3 ) . to measure the length of endothelial cell aggregations , fluorescent microscopy was used to distinguish between cell types , and bioquant was used to determine endothelial cell aggregate length based on standard scale measurements ( figure 4 ) . the number of endothelial cell aggregates was small due to the low ratio of endothelial cells seeded initially . thus , additional sections were taken from 25% and 75% of the way through each sample for additional measurements of aggregation length . a one - factor anova was used to compare mrna levels in the 1- and 2-day osteoblast cultures between static and perfusion incubation . a two - factor anova ( cell population and incubation method ) was used for the 7- and 14-day cocultures . both cell population and incubation methods had model effects for some genes , so one - factor anovas were performed within both cultures alone to compare incubation methods , and within static incubation to compare cell populations ( osteoblasts alone versus co - culture ) . a two - factor anova ( cell population and culture method ) was used to assess the histological outcome variables . no significance was associated with cell population ( osteoblast versus co - culture ) for cell number ( p = 0.94 ) , surface coverage ( p = 0.64 ) , or cell / matrix area coverage ( p = 0.43 ) , and no interactions existed between factors . a one - factor anova was then used to compare cell number , surface coverage , and cell / matrix area coverage between static culture and perfusion flow in both the scaffold perimeter ( regions 18 combined ) and the scaffold center ( region 9 ) ( figure 2 ) . a one - way anova was used to compare endothelial cell aggregation lengths between static and perfusion cultures . jmp in 5.1 statistical software package ( sas , cary , nc , usa ) was used for all statistical analyses . after 24 hours of flow , opn ( a marker of both bone development and the inflammatory response ) was downregulated ( p = 0.03 ) by 10% as was vegf ( an angiogenic growth factor ) by 48% ( p = 0.02 ) . in contrast , cox-2 , another marker of both bone development and the inflammatory response , was upregulated ( p = 0.02 ) by perfusion flow by 67% ( table 2 ) . after 48 hours of perfusion flow , while the same trend existed for opn , vegf , and cox-2 at 24 hours , the responses were not statistically significant ( p > 0.20 ) . however , hif-1 , the major transcription factor driving the hypoxic response , was downregulated ( 0.02 ) by 55% ( table 3 ) . runx-2 , col1 , m - csf , bfgf , and gbe-1 were not significantly different between perfusion and static treatments at either time points ( p > 0.20 ) . after 7 days of culture , mrna levels of both alp and ocn were upregulated ( over static culture ) by 142% ( p = 0.001 ) and 819% ( p = 0.02 ) , respectively , when osteoblast cultures alone was exposed to perfusion flow ( table 3 ) . similarly , alp and ocn were upregulated ( over static culture ) by 143% ( p = 0.04 ) and 1072% ( p = 0.07 ) , respectively , when cocultures were exposed to perfusion flow ( table 4 ) , although ocn only approached significance . this was expected , as alp and ocn , both markers of osteoblast differentiation , have been shown to be increased in three - dimensional scaffolds exposed to perfusion culture [ 29 , 30 , 36 , 40 , 50 ] . runx-2 , opn , col1 , and vegf did not change after 7 days of culture ( p > 0.2 ) . after 14 days of culture , mrna levels of runx-2 and vegf were downregulated by 42% ( p = 0.01 ) and 85% ( p < 0.0001 ) , respectively , when osteoblast cultures were exposed to perfusion flow ( table 3 ) . similarly , perfusion flow downregulated mrna levels of runx-2 by 55% ( p = 0.01 ) when cocultures were exposed to perfusion flow ( table 4 ) . these results were unexpected as both genes have been shown to be upregulated as osteoblasts differentiate , and 7-day mrna levels indicated an increase in differentiation - related genes ( alp and ocn ) . co - culturing endothelial cells with osteoblasts ( static and perfusion combined ) caused changes in only one gene at both time points . after 7 and 14 days of static culture , runx-2 was downregulated in cocultures by 57% ( p = 0.01 ) and 26% ( p = 0.04 ) compared to osteoblast alone . cell number was 60% lower ( p = 0.003 ) in the scaffold center ( region 9 ) as compared to the scaffolds ' periphery ( regions 18 ) ( figure 5 ) . cell surface coverage and cell / matrix area coverage were not significantly different between the scaffold center and periphery ( p > 0.45 ) . around the scaffold periphery , perfusion flow had no effect on surface coverage ( p = 0.58 ) . however , perfusion - induced increases in cell number ( 35% ) and cell / matrix area coverage ( 140% ) approached significance ( p = 0.09 and p = 0.06 , resp . ) . furthermore , in the scaffold center , perfusion flow increased cell number ( p = 0.03 ) , surface coverage ( p = 0.02 ) , and cell / matrix area coverage ( p = 0.03 ) by 220% , 84% , and 280% , respectively , compared to static culture ( figures 6 , 7 , and 8) . these findings suggest the scaffolds ' centers are more responsive to perfusion flow than the periphery . endothelial cells were found along the surfaces of the scaffold rather than within the matrix formed by osteoblasts inside the scaffold pores . additionally , endothelial cell aggregations were found in all 9 sample regions of the scaffold and did not appear to be more highly concentrated in any particular region of the scaffolds ( periphery versus center ) . furthermore , endothelial cells were not found as individual cells , but rather as aggregations of many cells . aggregations seemed to be oriented in all directions on the scaffold surface and did not tend to be aligned in the direction of fluid flow . there was no difference ( p = 0.31 ) in the aggregate number between the scaffold periphery and the scaffold center . furthermore , there was no difference ( p = 0.20 ) in aggregate number between static culture and perfusion flow ( figure 9 ) . however , the average endothelial cell aggregate length increased by almost 50% ( p = 0.007 ) in perfusion flow samples compared to static samples ( figure 10 ) . there was no difference in total endothelial cell aggregate length in static culture versus perfusion flow ( figure 11 ) . the ultimate goal of tissue engineering is to produce high - quality bone tissue in vitro to be used as a clinical alternative to autografting . three - dimensional structures cultured in vitro encounter limited vascular invasion upon implantation ( 710 ) . here , we co - cultured endothelial cells and osteoblasts to develop a method which might improve vascularization following implantation of tissue - engineered bone constructs in vivo . specifically , we used perfusion flow to increase the length of endothelial cell aggregations within cocultures , osteoblast - specific gene expression , and the cell number , cell coverage of the scaffold perimeter , and matrix area in the scaffold pores in the center of the scaffold . although there is no existing literature on the effects of perfusion flow on cocultures , there have been studies which have achieved vascular - like network formation in static osteoblast / endothelial cell cocultures . while we showed that perfusion flow can increase the length of endothelial cell aggregations , we did not provide evidence of the formation of vascular - like networks among endothelial cell populations . to form a functioning vascular system , it will be advantageous for endothelial cell aggregates to eventually connect with one another . by increasing aggregate length , perfusion flow may make it more likely for neighboring endothelial cell aggregates to connect and communicate effectively . the low ratio ( 2% ) of endothelial cell seeding may not have allowed for communication between populations of endothelial cells , which is necessary for network formation . alternatively , while the scaffold pores are interconnected , the porosity of the 3d scaffolds used here is relatively low in comparison to scaffolds used in previous studies . in the future , it may be necessary to increase the ratio of endothelial cells and/or the porosity of the scaffolds to increase interaction between aggregations , enhancing network formation . in static culture , perfusion flow successfully increased cell number as well as cell coverage of the scaffold perimeter and matrix area in the scaffold pores in the center of the scaffold . this amplified response within the center of the scaffold may be explained by considering diffusional limitations associated with three - dimensional tissue engineering [ 4 , 27 , 28 , 51 ] . three - dimensional cell - seeded constructs have reduced cell numbers and cellular activity towards their centers due to poor oxygen and nutrient delivery . perfusion flow has been used to mitigate these limitations by increasing oxygen and nutrient delivery , consequently increasing cell number and matrix production in the center of three - dimensional constructs . these results may be attributed to the interconnected pore structure of 3d scaffolds as the medium allows for communication between cells via paracrine signaling . it is well known that successful tissue - engineered scaffolds require a high degree of pore interconnectedness . here , we provide evidence that perfusion flow affected the scaffolds ' center to a greater degree than it affected the scaffolds ' periphery , suggesting that perfusion bioreactors capitalize on a scaffold 's porous structure . previous research has shown that perfusion bioreactors reduce hypoxia in three - dimensional scaffolds [ 27 , 30 , 36 ] . hypoxia elicits a well - documented response from mesenchymal stem cells and osteoblasts , including the upregulation of vascular endothelial growth factor ( vegf ) , a major mediator of both angiogenesis and hypoxia [ 52 , 53 ] . we found that perfusion flow reduces the expression of vegf in three - dimensional osteoblast monocultures ( 1- and 2-day cultures ) and in three - dimensional osteoblast monocultures ( 14-day culture ) . perfusion also reduced hif-1 , a major regulator of hypoxia , in three - dimensional osteoblast monocultures ( 2-day culture ) . in addition , cell number , cell coverage of the scaffold perimeter , and matrix area in the scaffold pores are all increased in the scaffold center with perfusion flow ( 14-day culture ) . thus , hypoxia - specific gene regulation supports histological evidence of increased cellular activity , suggesting that perfusion flow mitigates a hypoxic state by providing adequate oxygen delivery to the center of three - dimensional constructs . in addition to histological and hypoxia - specific evidence , our results also indicate that perfusion flow increases bone specific gene expression of alp and ocn at seven days in both osteoblast monocultures and osteoblast / endothelial cell cocultures , which is consistent with previous reports [ 29 , 30 , 36 ] . however , in contrast to these results , runx-2 was reduced in both osteoblast and cocultures with perfusion flow after 14 days of culture . osteoblast differentiation genes tend to follow a well - defined pattern of expression throughout the process of maturation . alp , ocn , and opn are all upregulated transiently , tend to peak after 2 - 3 weeks , and subsequently drop in expression as osteoblasts differentiate into osteocytes [ 54 , 55 ] . as runx-2 is a transcription factor responsible for osteoblast differentiation , it is not surprising that its expression precedes osteoblast - specific genes . a possible explanation for the decrease in runx-2 expression with perfusion flow is that it has reached its peak expression earlier in perfused samples and has begun its decline in expression at 14 days . the increase in endothelial cell aggregation length highlights the potential for perfusion bioreactors in three - dimensional bone tissue engineering . by increasing the in vitro activity of ecs in bone tissue constructs , perfusion flow could serve to speed up the process of host acceptance in future clinical applications . a higher quality vascular - like network in vitro will have a greater likelihood of attaining successful anastomosis with host vasculature in vivo . even if functional anastomosis is not fully achieved , perfusion - induced angiogenic activity could potentiate paracrine signaling between cells within the scaffold and host vasculature , advancing vascular infiltration . these results suggest the potential for perfusion bioreactors to improve in vitro co - culturing to maximize the quality and effectiveness of vascularized bone tissue cultures .

maintaining cellular viability in vivo and in vitro is a critical issue in three - dimensional bone tissue engineering . while the use of osteoblast / endothelial cell cocultures on three - dimensional constructs has shown promise for increasing in vivo vascularization , in vitro maintenance of cellular viability remains problematic . this study used perfusion flow to increase osteogenic and angiogenic gene expression , decrease hypoxic gene expression , and increase cell and matrix coverage in osteoblast / endothelial cell co - cultures . mouse osteoblast - like cells ( mc3t3-e1 ) were cultured alone and in co - culture with mouse microvascular endothelial cells ( eoma ) on three - dimensional scaffolds for 1 , 2 , 7 , and 14 days with or without perfusion flow . mrna levels were determined for several osteogenic , angiogenic , and hypoxia - related genes , and histological analysis was performed . perfusion flow downregulated hypoxia - related genes ( hif-1 , vegf , and opn ) at early timepoints , upregulated osteogenic genes ( alp and ocn ) at 7 days , and downregulated runx-2 and vegf mrna at 14 days in osteoblast monocultures . perfusion flow increased cell number , coverage of the scaffold perimeter , and matrix area in the center of scaffolds at 14 days . additionally , perfusion flow increased the length of endothelial cell aggregations within co - cultures . these suggest perfusion stimulated co - cultures provide a means of increasing osteogenic and angiogenic activity .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

three - dimensional bone tissue engineering strategies provide scaffolding for new bone tissue to grow into as well as a cellular source for new tissue growth ( osteogenic ) [ 36 ] . however , one of the biggest limitations to overcome is the maintenance of cellular viability within the center of three - dimensional constructs upon in vivo implantation [ 711 ] . vascularization is critical for successful bone growth and development and is one of the first phases seen during fracture healing [ 1116 ] . without proper vascularization , bone tissue would face limited growth and hypoxia . as the vasculature provides bone tissue with oxygen , nutrients , growth factors , and the removal of waste , it is not surprising that vascularization is also essential for successful implant integration . a common approach to this solution is the co - culture of bone cells and endothelial cells on three - dimensional constructs . there has been some success with co - culturing bone cells and endothelial cells to overcome the lack of vasculature in tissue - engineered bone [ 710 , 17 , 18 , 21 ] . these studies have shown that endothelial cells can form a pre - vascular network during co - culture [ 9 , 10 , 17 ] , and endothelial cells enhance osteoblastic activity when both cell types are in direct contact [ 8 , 9 , 17 , 22 , 23 ] . additionally , recent work has shown that three - dimensional cocultures will integrate with host tissue in vivo , with the formation of an improved vascular network upon implantation [ 2426 ] . as oxygen 's poor diffusion capacity largely governs the production of three - dimensional tissue cultures in vitro [ 27 , 28 ] , perfusion bioreactors mitigate hypoxic ( oxygen - depleted ) conditions by providing oxygen delivery to the center of three - dimensional cell - seeded scaffolds , which increases cellular infiltration and viability within three - dimensional scaffolds [ 27 , 2933 ] . perfusion also leads to upregulation in gene expression [ 29 , 31 , 34 ] , increases in protein production [ 30 , 33 , 3538 ] , and enhanced calcium deposition [ 30 , 36 , 3941 ] in osteoblast - seeded scaffolds . furthermore , fluid flow activates endothelial cells , increasing angiogenic gene expression and the formation of tube - like structures [ 12 , 22 , 4245 ] . taken together , these results suggest that the use of a perfusion bioreactor with osteoblast / endothelial cell cocultures may improve osteoblast activity and accelerate the vascularization of tissue - engineered bone by alleviating the hypoxic state of three - dimensional culture . the goal of this study was to help to elucidate the effects that a dynamic environment has on osteoblast / endothelial cell three - dimensional cocultures . we hypothesized that co - culturing osteoblasts and endothelial cells increases osteogenic and angiogenic gene expressions and decreases hypoxic gene expression in three - dimensional calcium phosphate scaffolds . furthermore , we hypothesized that perfusion flow increases cell and matrix coverage area throughout three - dimensional scaffolds . mouse osteoblast - like cells ( mc3t3-e1 subclone 4 , atcc , manassas , va , usa ) were cultured in alpha - mem ( invitrogen , carlsbad , ca , usa ) with 10% fbs ( hyclone , logan , ut , usa ) and 1% pen / strep ( cellgro , herndon , va , usa ) . mouse microvascular endothelial cells ( ec ) ( eoma , atcc , manassas , va , usa ) were cultured in dulbecco 's mem ( invitrogen , carlsbad , ca , usa ) with 10% fbs ( hyclone , logan , ut , usa ) and 1% pen / strep ( cellgro , herndon , va , usa ) . once cells reached ~70% confluency , they were trypsinized , resuspended in culture medium and statically seeded onto three - dimensional calcium phosphate scaffolds ( bd biosciences , san jose , ca , usa ) in 25 l medium ( 1,000,000 cells / scaffold : 100% osteoblasts or 98% osteoblasts and 2% endothelial cells ) . cell - seeded scaffolds were incubated for 1 hour to allow cell adhesion , then covered with media ( alpha - mem , 10% fbs , and 1% pen / strep ) , and incubated for 24 hours before experimentation . during 1- and 2-day experimentation osteoblast - seeded scaffolds were evaluated after 1 or 2 days of static or perfusion ( n = 5 per group ) culture . four treatment groups ( n = 3 per group ) were tested at 7- and 14-day time points : two cell populations ( osteoblasts alone and osteoblast / endothelial cell co - culture ) with two culture methods ( static culture and perfusion flow ) . after 1 , 2 , 7 , or 14 days of culture ( static or perfusion ) , rna was isolated from the cell - seeded scaffolds , or the scaffolds were fixed in 10% buffered formalin for histological processing . gene expression was assessed at all - time points , and histological analysis was performed at the 14 day timepoint . a 6 mm hole was drilled in the polycarbonate block to fit the scaffolds ( 5 mm diameter ; 3.5 mm height ) . tubing connected the top of the chamber to a 140 ml syringe fluid reservoir and the bottom of the chamber to syringe pump ( harvard apparatus ) . after 1 , 2 , 7 , and 14 days of culture , cell - seeded scaffolds were placed in 2 ml tubes and crushed in sv lysis buffer with a glass rod . rna concentration and quality were determined using spectrophotometer ( nanodrop nd-1000 spectrophotometer , nanodrop technologies , rockland , de , usa ) readings at 260 , 230 , and 280 nm . rna was reverse transcribed into cdna using a reaction mix consisting of superscript ii reverse transcriptase ( invitrogen , calrsbad , ca , usa ) , 1x first strand buffer ( invitrogen ) , 800 m dntps ( promega ) , rnase out recombinant ribonuclease inhibitor ( invitrogen ) , 48 mm dithiothreitol ( invitrogen ) and 0.5 g oligo(dt)12 - 18 pimer at 42c for 20 minutes , 50c for 10 minutes , and 42c for 1 hour in the mastercycler gradient thermocycler ( eppendorf , westbury , ny , usa ) . all reactions were performed in the steponeplus real - time pcr system ( applied biosystems ) under the following cycle parameters : hot start at 95c for 15 minutes followed by 40 cycles of 95c for 15 seconds , 60c for 30 seconds , and 72c for 15 seconds . samples were then removed , flash frozen , and sectioned on a soft tissue microtome ( microm international , waldorf , germany ) at a thickness of 10 microns . sections were taken from mid - way through the scaffold running parallel to the direction of perfusion flow ( figure 2 ) . nine images were taken in total , three from the upper , middle , and lower thirds of each section ( figure 2 ) . fluorescent images were used to distinguish between cell types , as dapi identifies all cell nuclei and cd31 stains membrane receptors on endothelial cells . brightfield images were used to determine total cell number , cell surface coverage , and cell / matrix area coverage . cell number , cell surface coverage , and cell / matrix area coverage were determined around the periphery of the scaffold as well as in the scaffold center , allowing for the assessment of whether perfusion flow is more effective in areas of poor diffusion . for the scaffold center , only the center image ( region 9 ) was used for measurement ( figure 2 ) . in the scaffold periphery , cell number was averaged for all 8 regions ( regions 18 , figure 2 ) for comparison with the scaffold center ( region 9 , figure 2 ) . cell surface coverage was calculated as the ratio of the length of cell - covered scaffold surface to the total length of scaffold surface using bioquant ( bioquant osteo , nashville , tn , usa ) software ( figure 2 ) . cell / matrix area coverage was calculated as the ratio of cell and matrix area within pore spaces to total pore space area ( figure 3 ) . to measure the length of endothelial cell aggregations , fluorescent microscopy was used to distinguish between cell types , and bioquant was used to determine endothelial cell aggregate length based on standard scale measurements ( figure 4 ) . the number of endothelial cell aggregates was small due to the low ratio of endothelial cells seeded initially . a one - factor anova was used to compare mrna levels in the 1- and 2-day osteoblast cultures between static and perfusion incubation . both cell population and incubation methods had model effects for some genes , so one - factor anovas were performed within both cultures alone to compare incubation methods , and within static incubation to compare cell populations ( osteoblasts alone versus co - culture ) . no significance was associated with cell population ( osteoblast versus co - culture ) for cell number ( p = 0.94 ) , surface coverage ( p = 0.64 ) , or cell / matrix area coverage ( p = 0.43 ) , and no interactions existed between factors . a one - factor anova was then used to compare cell number , surface coverage , and cell / matrix area coverage between static culture and perfusion flow in both the scaffold perimeter ( regions 18 combined ) and the scaffold center ( region 9 ) ( figure 2 ) . after 48 hours of perfusion flow , while the same trend existed for opn , vegf , and cox-2 at 24 hours , the responses were not statistically significant ( p > 0.20 ) . runx-2 , col1 , m - csf , bfgf , and gbe-1 were not significantly different between perfusion and static treatments at either time points ( p > 0.20 ) . after 7 days of culture , mrna levels of both alp and ocn were upregulated ( over static culture ) by 142% ( p = 0.001 ) and 819% ( p = 0.02 ) , respectively , when osteoblast cultures alone was exposed to perfusion flow ( table 3 ) . similarly , alp and ocn were upregulated ( over static culture ) by 143% ( p = 0.04 ) and 1072% ( p = 0.07 ) , respectively , when cocultures were exposed to perfusion flow ( table 4 ) , although ocn only approached significance . this was expected , as alp and ocn , both markers of osteoblast differentiation , have been shown to be increased in three - dimensional scaffolds exposed to perfusion culture [ 29 , 30 , 36 , 40 , 50 ] . runx-2 , opn , col1 , and vegf did not change after 7 days of culture ( p > 0.2 ) . after 14 days of culture , mrna levels of runx-2 and vegf were downregulated by 42% ( p = 0.01 ) and 85% ( p < 0.0001 ) , respectively , when osteoblast cultures were exposed to perfusion flow ( table 3 ) . similarly , perfusion flow downregulated mrna levels of runx-2 by 55% ( p = 0.01 ) when cocultures were exposed to perfusion flow ( table 4 ) . these results were unexpected as both genes have been shown to be upregulated as osteoblasts differentiate , and 7-day mrna levels indicated an increase in differentiation - related genes ( alp and ocn ) . co - culturing endothelial cells with osteoblasts ( static and perfusion combined ) caused changes in only one gene at both time points . after 7 and 14 days of static culture , runx-2 was downregulated in cocultures by 57% ( p = 0.01 ) and 26% ( p = 0.04 ) compared to osteoblast alone . cell number was 60% lower ( p = 0.003 ) in the scaffold center ( region 9 ) as compared to the scaffolds ' periphery ( regions 18 ) ( figure 5 ) . cell surface coverage and cell / matrix area coverage were not significantly different between the scaffold center and periphery ( p > 0.45 ) . around the scaffold periphery , perfusion flow had no effect on surface coverage ( p = 0.58 ) . however , perfusion - induced increases in cell number ( 35% ) and cell / matrix area coverage ( 140% ) approached significance ( p = 0.09 and p = 0.06 , resp . ) furthermore , in the scaffold center , perfusion flow increased cell number ( p = 0.03 ) , surface coverage ( p = 0.02 ) , and cell / matrix area coverage ( p = 0.03 ) by 220% , 84% , and 280% , respectively , compared to static culture ( figures 6 , 7 , and 8) . endothelial cells were found along the surfaces of the scaffold rather than within the matrix formed by osteoblasts inside the scaffold pores . additionally , endothelial cell aggregations were found in all 9 sample regions of the scaffold and did not appear to be more highly concentrated in any particular region of the scaffolds ( periphery versus center ) . furthermore , endothelial cells were not found as individual cells , but rather as aggregations of many cells . aggregations seemed to be oriented in all directions on the scaffold surface and did not tend to be aligned in the direction of fluid flow . there was no difference ( p = 0.31 ) in the aggregate number between the scaffold periphery and the scaffold center . however , the average endothelial cell aggregate length increased by almost 50% ( p = 0.007 ) in perfusion flow samples compared to static samples ( figure 10 ) . there was no difference in total endothelial cell aggregate length in static culture versus perfusion flow ( figure 11 ) . the ultimate goal of tissue engineering is to produce high - quality bone tissue in vitro to be used as a clinical alternative to autografting . three - dimensional structures cultured in vitro encounter limited vascular invasion upon implantation ( 710 ) . here , we co - cultured endothelial cells and osteoblasts to develop a method which might improve vascularization following implantation of tissue - engineered bone constructs in vivo . specifically , we used perfusion flow to increase the length of endothelial cell aggregations within cocultures , osteoblast - specific gene expression , and the cell number , cell coverage of the scaffold perimeter , and matrix area in the scaffold pores in the center of the scaffold . although there is no existing literature on the effects of perfusion flow on cocultures , there have been studies which have achieved vascular - like network formation in static osteoblast / endothelial cell cocultures . while we showed that perfusion flow can increase the length of endothelial cell aggregations , we did not provide evidence of the formation of vascular - like networks among endothelial cell populations . to form a functioning vascular system , it will be advantageous for endothelial cell aggregates to eventually connect with one another . by increasing aggregate length , perfusion flow may make it more likely for neighboring endothelial cell aggregates to connect and communicate effectively . the low ratio ( 2% ) of endothelial cell seeding may not have allowed for communication between populations of endothelial cells , which is necessary for network formation . alternatively , while the scaffold pores are interconnected , the porosity of the 3d scaffolds used here is relatively low in comparison to scaffolds used in previous studies . in the future , it may be necessary to increase the ratio of endothelial cells and/or the porosity of the scaffolds to increase interaction between aggregations , enhancing network formation . in static culture , perfusion flow successfully increased cell number as well as cell coverage of the scaffold perimeter and matrix area in the scaffold pores in the center of the scaffold . this amplified response within the center of the scaffold may be explained by considering diffusional limitations associated with three - dimensional tissue engineering [ 4 , 27 , 28 , 51 ] . three - dimensional cell - seeded constructs have reduced cell numbers and cellular activity towards their centers due to poor oxygen and nutrient delivery . perfusion flow has been used to mitigate these limitations by increasing oxygen and nutrient delivery , consequently increasing cell number and matrix production in the center of three - dimensional constructs . previous research has shown that perfusion bioreactors reduce hypoxia in three - dimensional scaffolds [ 27 , 30 , 36 ] . we found that perfusion flow reduces the expression of vegf in three - dimensional osteoblast monocultures ( 1- and 2-day cultures ) and in three - dimensional osteoblast monocultures ( 14-day culture ) . perfusion also reduced hif-1 , a major regulator of hypoxia , in three - dimensional osteoblast monocultures ( 2-day culture ) . in addition , cell number , cell coverage of the scaffold perimeter , and matrix area in the scaffold pores are all increased in the scaffold center with perfusion flow ( 14-day culture ) . thus , hypoxia - specific gene regulation supports histological evidence of increased cellular activity , suggesting that perfusion flow mitigates a hypoxic state by providing adequate oxygen delivery to the center of three - dimensional constructs . in addition to histological and hypoxia - specific evidence , our results also indicate that perfusion flow increases bone specific gene expression of alp and ocn at seven days in both osteoblast monocultures and osteoblast / endothelial cell cocultures , which is consistent with previous reports [ 29 , 30 , 36 ] . however , in contrast to these results , runx-2 was reduced in both osteoblast and cocultures with perfusion flow after 14 days of culture . alp , ocn , and opn are all upregulated transiently , tend to peak after 2 - 3 weeks , and subsequently drop in expression as osteoblasts differentiate into osteocytes [ 54 , 55 ] . as runx-2 is a transcription factor responsible for osteoblast differentiation , it is not surprising that its expression precedes osteoblast - specific genes . a possible explanation for the decrease in runx-2 expression with perfusion flow is that it has reached its peak expression earlier in perfused samples and has begun its decline in expression at 14 days . the increase in endothelial cell aggregation length highlights the potential for perfusion bioreactors in three - dimensional bone tissue engineering . by increasing the in vitro activity of ecs in bone tissue constructs , perfusion flow could serve to speed up the process of host acceptance in future clinical applications . a higher quality vascular - like network in vitro will have a greater likelihood of attaining successful anastomosis with host vasculature in vivo . even if functional anastomosis is not fully achieved , perfusion - induced angiogenic activity could potentiate paracrine signaling between cells within the scaffold and host vasculature , advancing vascular infiltration . these results suggest the potential for perfusion bioreactors to improve in vitro co - culturing to maximize the quality and effectiveness of vascularized bone tissue cultures .

the ideal fluid type ( crystalloids or colloids ) and the optimum dosage of fluids are much debated issues ( 1 - 4 ) . hydroxyethyl starches ( 4 ) are widely used as fluid expanders in the perioperative period and are the preferred colloids in many intensive care units ( icu ) ( 5,6 ) . hydroxyethyl starch ( he s ) may impair blood coagulation by reducing the levels of von willebrand factor and factor viii and by decreasing the platelet count and function ( 7 - 10 ) . it has been suggested that the new generation he s ( third generation , low molecular weight , low degree of substitution ) is less prone to cause blood coagulation disorders than the old compounds ( 11 ) , but this issue remains controversial ( 7,12,13 ) primarily because few studies evaluated the issue ( 4 ) . in the perioperative period , even the new generation starches inspire concerns about their safety , especially in terms of coagulation disorders ( 1,7 ) , . patients submitted to oncologic surgery are subjected to long surgery procedures , with a great need of fluid resuscitation and blood products transfusion . it has been suggested that surgical cancer patients are more prone to being transfused with blood products than non - cancer patients ( 16 ) . no study has evaluated the effects of the intraoperative use of a new generation he s on the intraoperative and postoperative red blood cells and other blood products transfusion in patients submitted to elective major oncologic surgery . we hypothesized that patients submitted to oncologic surgery that received a new generation he s during surgery would require more red blood cells transfusions during and after the surgery than patients that did not receive he s . to test our hypothesis , we evaluated whether a new generation he s led to more red blood cell transfusions during the first 48 hours after major elective oncologic surgery than patients that received only crystalloids using a propensity - matched approach . secondary objectives included clotting profile , use of blood products other than red cells , icu length of stay and mortality between the two groups . patients : the ethics committee of the hospital ac camargo approved this study and waived patient consent due to its retrospective observational nature . from september 2007 to september 2009 , all patients admitted to the hospital icu ( three units with a total of 33 beds ) of our teaching hospital after an elective major oncologic surgery ( head and neck , neurological , thoracic , abdominal and other sites ) were included in the study . data collection : in the hospital , all data are recorded in a computerized physician order entry and an electronic medical record system . we collected the data recorded during the surgery and the following 48 hours . during the intraoperative period , we collected the number of packs of blood products transfused ( red blood cells , fresh frozen plasma , platelets and cryoprecipitate ) , the surgery length and the volume of crystalloids and colloids infused . during icu admission , we collected the simplified acute physiology score 3 ( saps 3 ) and performed laboratory exams that included creatinine levels and clotting profile ( composed of the platelets count and prothrombin time ( pt ) . during the 48-hour period after the surgery , we collected the number of packages of blood products transfused and performed laboratory exams , including serum creatinine . we divided the 48-hour period after the surgery into two periods of 24 hours because the hypothetical he s side effect could be evanescent . the data were analyzed for three consecutive periods : the intraoperative period , the period from the end of the surgery to the first 24 hours after post - surgery ( 24-hour period ) and the period between from 24 to 48 hours after the surgery ( 24- to 48-hour period ) . the patients were categorized in two groups according to the use of he s during the intraoperative period ( he s and no - hes group ) . the patients in the he s group received at least 500 ml of new generation he s ( voluven - 6% 130/0.4 , fresenius , germany ) . perioperative transfusion policy : the hospital transfusion policy recommends that patients should be admitted to the operating room with a hemoglobin level greater than 10 mg / dl and platelet count above 50 10 units / ml . coagulation disorders , as assessed by aptt and pt , were treated with preoperative fresh frozen plasma transfusion until both values were within the normal range ( international normalized ratio and aptt ratio less or equal to 1.5 ) . other causes of coagulopathy , such as vitamin k deficiency , were assessed as appropriate . all anesthesiologists in the hospital are part of a small team that follows protocols for surgery resuscitation and blood transfusion . there are five major surgical teams in the hospital ( thoracic , abdominal , pelvic , neurological and soft tissue ) and each has a closed staff , i.e. , surgery at each specific site is usually performed by a very restricted number of surgeons . the icu policy for transfusion suggests that red blood cell packs should be transfused if the hemoglobin level is below 7 g / dl or below 8 g / dl with signs of active bleeding and/or impaired tissue perfusion ( delayed capillary refill time , elevated lactate levels and nonchloremic metabolic acidosis ) ( 17 ) . transfusion with hemoglobin levels above 9 g / dl was not encouraged and was performed only at the discretion of the intensivist if massive blood lost was detected ( high output in the drain with hemodynamic instability ) . platelets were transfused if the total count was less than 50 10 units / ml with active bleeding or after neurosurgery . cryoprecipitate transfusion was indicated in patients with fibrinogen levels less than 100 mg / dl . fresh frozen plasma was administered if the coagulation times were two - fold higher the normal value range in patients with active bleeding or after neurosurgery . colloid use policy : at the time of the study , the only synthetic colloid available at our institution was a new generation 6% he s 130/0.4 ( voluven ) . he s use on the icu postoperative period is not part of our postoperative resuscitation protocol and is discouraged . albumin was not used during the postoperative period of elective oncologic surgery . statistical analysis : categorical and continuous data are presented as percentages and the mean sd ( or median and 25%-75% interquartile range [ iqr ] ) , respectively . categorical variables were compared using the chi - square or fisher exact test , as appropriate . quantitative continuous variables were compared using an unpaired t test or mann - whitney test for parametric or nonparametric variables , respectively . patients were clustered according to the administration of he s during surgery . because the use of colloids was not randomized , there were two unbalanced groups ( table 1 ) . because this was a retrospective analysis ( patients were not randomized ) , the creation of a propensity score is an effective method to reduce bias . propensity - matched analyses are increasingly being used in research due to its ability to improve the power of retrospective and prospective non - randomized analysis ( 18,19 ) . propensity score has been defined as the condition probability of being treated given the covariates ( 20 ) . propensity - matched analyses are able to take into account as many variables related to the outcome evaluated as needed , which reduces bias ( 20 ) . to construct the propensity score , a logistic regression was fitted , and variables were chosen for inclusion according to the methods of brookhart et al . ( 18 ) . we included variables that could be related to the study outcomes based on the propensity score . to identify the variables potentially associated with the outcome , a univariate analysis was performed to evaluate potential variables related to blood transfusion during the entire perioperative period ( from the surgical procedure to up to 48 hours after surgery ) . the variables included on the propensity score were as follows : age , gender , body - mass index , metastatic disease , surgery length , volume of crystalloid infused and surgical sites . although age and body mass index were not associated with the outcome , we believed that they were important variables that could alter the fluid dynamics during the surgical procedures and included them in the model ( 21 ) . the best caliper was 0.05 , which was obtained by the austin method ( 22 ) . after the propensity score was created , the patients were matched according to their respective propensity scores . as reported in the current literature , matching on the propensity score has now been clearly demonstrated to be the best method to attempt to provide an unbiased estimation of the treatment effect ( 19 ) . first , based on the crude analysis , we pre - defined a match considering one patient without he s use to a maximum of three patients with he s use . the match procedure was performed with an optimal matching method , and to avoid overinflation , we did not permit replacement of matched patients . the number of patients analyzed using the propensity score was smaller than the total number of patients studied because matching was not possible for all patients . our analysis was performed with fewer patients than the original study population . even with fewer patients , an analysis using the propensity score is more reliable than traditional statistical methods because imbalance between groups is reduced ( 18,23 ) . the correct construction of the propensity score was performed with the box - plot method . after the propensity score matches were performed , we performed a diagnostic to ensure good balance in the matched population through the bias reduction method and the stabilized standardized difference ( because we were using 1:n matching instead of 1:1 matching ) ( 19,22 ) . after matching , ( ctv ) instead of crystalloid volume as a variable in the propensity score ( see appendix ) . ctv was defined as total crystalloid volume 0.3 plus 1.4 infused colloid volume ( 21,24,25 ) . this alternative analysis was conducted to balance the effective volume used for expansion in both groups ( see appendix for details ) . all the statistical analysis were performed in spss 19.0 , and a p - value of 0.05 for was considered significant for all comparisons . we included 894 patients ; 614 received he s , and 280 received only crystalloids during surgery . before the use of the propensity score , the patients in the he s group had longer surgical times , had received more crystalloids , were younger and were less likely to have any comorbidity compared with the no - hes group ( table 1 ) . after the creation of the matched groups , 385 propensity - matched patients ( 97 in the crystalloids group and 288 in the he s group ) were analyzed . the median volume of infused he s was 1 l [ 0.5 - 1.0 ] . no patient received less than 0.5 l. creatinine values were similar for both groups in all the observation periods . the incidence of acute kidney injury ( aki ) ( defined according to the rifle criteria of risk , i.e. , an increase of serum creatinine of 50% or more over the baseline ) ( 26,27 ) was similar between the groups ( 7% versus 7% , p>0.99 ) . more detailed results regarding the other propensity matched analysis ( using ctv ) , standard logistic regression and non - propensity matched results analysis can be found in the appendix . red blood cells and other blood products transfusions : the variables related to transfusion of red blood cell pack transfusion for all the perioperative transfusions are shown in table 2 . most patients in both groups did not require blood products transfusion . in the he s group , a higher percentage of patients received red blood cell transfusion in the intraoperative and 24-hour postoperative period , but the percentage was similar for the 24- to 48-hour postoperative period ( figure 1 ) . for the patients in both groups that received red blood cell transfusions , the number of packs per patient was higher in the he s group during the intraoperative period ( 0.55 versus 0.39 ; p = 0.028 ) . there was no difference regarding the use of fresh frozen plasma , cryoprecipitate and platelets concentrate in any period . the patients that received he s had a small but statistically significant lower hemoglobin level at icu admission ( 11.21.9 versus 12.01.7 ; p<0.001 ) but not at the 24-hour ( 11.11.8 versus 11.31.6 ; p = 0.11 ) and 48-hour ( 10.21.7 versus 10.21.5 ; p = 0.20 ) postoperative periods . the transfusion rate was similar between the groups when we employed the propensity analysis that included ctv ( additional table 2 ) . the results of the non - propensity analysis ( see appendix , additional table 3 and 4 ) showed that red blood cell transfusion was more frequent in the he s patients . the multivariate analysis showed that the factors associated with red blood cell transfusion in the perioperative period included age , metastatic disease , total operative time , crystalloid volume used and colloid use ( any dose ) . thoracic and head and neck surgery were protective factors against transfusion ( see additional table 5 ) . secondary objectives : patients in the he s group showed lower levels of platelets at icu admission compared to the no - hes group ( table 3 ) . the international normalized ratio ( inr ) was also higher in the he s patients at icu admission and in the first 24 hours after surgery ( table 3 ) . table 4 shows the coagulation values for transfused and non - transfused patients in both groups ( no - hes and he s ) . the inr was higher in transfused no - hes patients than in non - transfused no - hes patients . the inr was also higher in the transfused he s patients than the non - transfused he s patients at icu admission . there were no differences between transfused patients in the he s and no - hes groups regarding platelet count or inr at any period studied . non - transfused he s patients also had a higher inr than non - transfused no - hes patients . icu mortality and length of stay were similar between he s and no - hes groups ( table 3 ) . in this retrospective analysis using a propensity - matched cohort of surgical patients , he s use was associated with more frequent red blood cells transfusions during major oncologic surgery and in the first 24 hours after the procedure . he s are considered more efficient volume expanders than crystalloids ; i.e. , less volume is needed to obtain the same hemodynamic effect ( 28,29 ) . a recent randomized controlled trial suggested that patients with penetrating trauma required less total volume when resuscitated by he s compared with saline ; there was no difference , however , in patients with blunt trauma ( 30 ) . a recently published trial in septic patients suggested that less fluid was needed to reach hemodynamic stability in patients resuscitated with he s ( 31 ) . a larger multicenter randomized trial has shown no difference regarding the total fluid need in septic patients ( 24 ) . the need for less fluid to obtain the same hemodynamic effect would be particularly interesting during the intraoperative period , when hemodynamic optimization associated with less total fluid volume may be beneficial ( 32 ) . he s and other colloids are not related to any robust positive clinical outcome , such as mortality or reduced icu length of stay ( 33 ) . in the recently published 6s trial , septic patients that were randomized to fluid resuscitation with he s had higher mortality , suggesting that at least in this specific population , he s use should be strongly discouraged ( 24 ) . the doubtful greater efficiency of he s may be counteracted by significant side effects , including coagulation abnormalities and aki ( 13 ) . the impact of these side effects on outcome , especially for he s 6% 130/0.4 , is largely unknown ( 4 ) . oncologic patients may have coagulation abnormalities related to their illness and chemotherapy side effects ( 34 ) . we aimed to evaluate the effects of he s on the perioperative need for blood transfusion in this specific population . this outcome is important because blood transfusion may be associated with worse outcome after oncologic surgery , including long - term survival ( 35 - 38 ) . blood transfusion is not harmless and has been associated with several clinical side effects , including transfusion related acute lung injury and fluid overload ( 39 ) . in critically ill patients , blood transfusion is associated with poorer outcomes ( 39 ) . our propensity - matched data showed that he s use is independently related to a greater need for red blood cell transfusion in the perioperative period . as previously reported , even low doses of he s could result in coagulation abnormalities ( 14 ) . in addition to requiring more red blood cell transfusions , he s patients also had lower hemoglobin levels at icu admission , suggesting that the difference was not only due to over transfusion of the he s group . corroborating with our results , other studies have shown a higher need for blood products in critically ill patients who received he s ( 24,40 ) . patients with blunt trauma resuscitated with he s required more blood transfusions than patients who received saline , although this conclusion is likely confounded by a higher injury severity in the hes - resuscitated patients ( 30 ) . our standard multivariate analysis showed that colloid use was a risk factor for the perioperative need for red blood cell transfusion . nevertheless , because this analysis included fewer patients ( with only 41 patients in the no - hes group see appendix ) , it is possible that the smaller sample reduced the power of the study to detect any difference . it can be argued that because the use of he s is associated with a greater degree of plasma volume expansion , and considering that the two groups were paired to the crystalloid volume infused , our findings may be related only to the increased hemodilution caused by he s use ( 41 ) . the increase in the frequency of red blood cell pack transfusions in the he s group in the main propensity analysis might to some degree be related to the expansion effect of he s and not to its impact on blood coagulation . it should be stated that the effectiveness of he s as a dilutional agent appears to be reduced after major injury ( 42 ) and that our patients also used a small volume of starch that was below the suggested maximum daily dose of 50 ml / kg . there were small differences in the coagulation profile between the he s and no - hes groups . those differences are likely without clinical significance because the values were very similar between the groups ( table 3 ) . there were no differences in the coagulation profile ( including the inr and platelet count ) between the transfused patients in both groups ( table 4 ) . the higher need for transfusions can not be explained by the differences in coagulation features after surgery . in our multivariate analysis , it may be speculated that because restrictive fluid therapy is frequently used in thoracic surgery ( 43 ) , patients received less fluid , with less hemodilution and bleeding related to dilutional coagulopathy , and therefore required less red blood cell transfusion . the icu length of stay and mortality were similar between groups , but our analysis was likely underpowered to detect a significant difference in a population with low mortality ( below 2% ) and short icu length of stay ( one day of median icu stay ) . in the univariate analysis , icu length of stay was higher in the he s group than in the no - hes group ( additional table 6 ) . third , due to its retrospective nature , we were unable to control for personal preferences variables regarding red blood cell transfusion and specific tumor staging ( which could be related to technical difficulties during the surgical procedure ) , although we included metastatic disease in the propensity score . fourth , although our hospital has strict policies for transfusion and perioperative fluid therapy , we can not guarantee that these protocols were followed in all of the patients analyzed . fifth , our study was underpowered to evaluate the icu length of stay and mortality . our finding that he s use is associated with a greater need for blood transfusion is biologically plausible and has been reported in other clinical scenarios ( 24,40 ) . if these findings are confirmed in large , prospective studies , he s use should be questioned in the subjects undergoing elective oncologic surgery due to its unproven clinically efficacy over crystalloids ( 4,13 ) . the intraoperative use of he s 6% 130/0.4 during major elective oncologic surgery is associated with an increase in red blood cell transfusions in the perioperative period of major oncologic surgery . there were no differences in icu length of stay and mortality . impact of intraoperative he s 6% 130/0.4 on the need for blood transfusion after major oncologic surgery : a propensity - matched analysis our propensity score analysis using ctv instead of infused crystalloid volume resulted in 97 patients in the no - hes group and 162 patients in the he s group . the major difference between this analysis and the previous analysis was the use of the corrected total volume ( ctv ) as a variable instead of the volume of infused crystalloid . we sought to minimize the role of hemodilution as a trigger to transfusion ; i.e. , the propensity - matched patients in this analysis would have received the same amount of plasma expansion . because the expansion effects of colloids are different from crystalloids , we created a variable that would account for the corrected amount of fluid expansion received ( ctv ) . we defined the ctv in patients in the no - hes group as equivalent to the crystalloid volume infused multiplied by 0.3 1 . the ctv in the he s group was defined as the sum of the crystalloid volume multiplied by 0.3 plus the volume of he s multiplied by 1.4 ( ctv = [ crystalloid volume 0.3 ] + [ 1.4 colloid volume ] ) . the use of the 1.4 ratio aimed at accounting for the theoretical greater expansion effects of the colloids 2,3 . this alternative propensity analysis included 41 patients in the no - hes group and 118 patients in the he s group ( additional table 1 ) . there was no difference in the need for red blood cell transfusion between the groups when this analysis was performed ( additional table 2 ) . the international normalized ration ( inr ) was higher at icu admission in the he s group , but the other coagulation variables were similar ( additional table 3 ) . the stepwise backward method was used to determine the factors associated with red blood cell transfusion in the perioperative time . the initial model consisted of all of the independent variables that had a p value of less than 0.25 in the bivariate analysis associated with red cell blood transfusion or between the he s and non - hes groups in the 894 patients ( the hosmer - lemeshow logistic regression ) . the variables were removed one at a time if they did not contribute to the model assessed according to a likelihood ratio test ( p < 0.050 ) . single colinearity was evaluated with pearson 's correlation between the independent variables , and multi - colinearity was evaluated with the variance inflation factor . the discriminative ability of the model to predict the outcome of patients was assessed by the area under the receiver operating characteristic ( auc ) curve . the calibration ability for the model was evaluated with hosmer - lemeshow goodness - of - fit statistics . the major outcomes regarding the need for blood transfusion on crude analysis are displayed in additional table 4 . the major difference between propensity - matched versus standard analysis was that before matching , the need for blood products other than red blood cell packs was more common in the he s patients . the he s patients in the unmatched analysis received fresh frozen plasma during surgery more frequently than the no - hes patients . the need for cryoprecipitate was also more frequent in the he s patients in the first 24 hours after the procedure . a multivariate analysis was performed to evaluate the risk factors for red blood cell transfusion from the intraoperative period up to 48 hours after the procedure ( additional table 5 ) . the factors associated with the red blood cell transfusions included age , metastatic disease , volume of crystalloid used , total operative time and use of any dose of he s . these factors may only highlight that patients that received blood transfusion were subject to more aggressive and/or technically difficult surgeries . the icu length of stay was higher in the he s group ( additional table 6 ) . our propensity score analysis using ctv instead of infused crystalloid volume resulted in 97 patients in the no - hes group and 162 patients in the he s group . the major difference between this analysis and the previous analysis was the use of the corrected total volume ( ctv ) as a variable instead of the volume of infused crystalloid . we sought to minimize the role of hemodilution as a trigger to transfusion ; i.e. , the propensity - matched patients in this analysis would have received the same amount of plasma expansion . because the expansion effects of colloids are different from crystalloids , we created a variable that would account for the corrected amount of fluid expansion received ( ctv ) . we defined the ctv in patients in the no - hes group as equivalent to the crystalloid volume infused multiplied by 0.3 1 . the ctv in the he s group was defined as the sum of the crystalloid volume multiplied by 0.3 plus the volume of he s multiplied by 1.4 ( ctv = [ crystalloid volume 0.3 ] + [ 1.4 colloid volume ] ) . the use of the 1.4 ratio aimed at accounting for the theoretical greater expansion effects of the colloids 2,3 . this alternative propensity analysis included 41 patients in the no - hes group and 118 patients in the he s group ( additional table 1 ) . there was no difference in the need for red blood cell transfusion between the groups when this analysis was performed ( additional table 2 ) . the international normalized ration ( inr ) was higher at icu admission in the he s group , but the other coagulation variables were similar ( additional table 3 ) . the stepwise backward method was used to determine the factors associated with red blood cell transfusion in the perioperative time . the initial model consisted of all of the independent variables that had a p value of less than 0.25 in the bivariate analysis associated with red cell blood transfusion or between the he s and non - hes groups in the 894 patients ( the hosmer - lemeshow logistic regression ) . the variables were removed one at a time if they did not contribute to the model assessed according to a likelihood ratio test ( p < 0.050 ) . single colinearity was evaluated with pearson 's correlation between the independent variables , and multi - colinearity was evaluated with the variance inflation factor . the discriminative ability of the model to predict the outcome of patients was assessed by the area under the receiver operating characteristic ( auc ) curve . the calibration ability for the model was evaluated with hosmer - lemeshow goodness - of - fit statistics . the major outcomes regarding the need for blood transfusion on crude analysis are displayed in additional table 4 . the major difference between propensity - matched versus standard analysis was that before matching , the need for blood products other than red blood cell packs was more common in the he s patients . the he s patients in the unmatched analysis received fresh frozen plasma during surgery more frequently than the no - hes patients . the need for cryoprecipitate was also more frequent in the he s patients in the first 24 hours after the procedure . a multivariate analysis was performed to evaluate the risk factors for red blood cell transfusion from the intraoperative period up to 48 hours after the procedure ( additional table 5 ) . the factors associated with the red blood cell transfusions included age , metastatic disease , volume of crystalloid used , total operative time and use of any dose of he s . these factors may only highlight that patients that received blood transfusion were subject to more aggressive and/or technically difficult surgeries . the icu length of stay was higher in the he s group ( additional table 6 ) .

objectives : to evaluate the effect of the intraoperative use of hydroxyethyl starch on the need for blood products in the perioperative period of oncologic surgery . the secondary end - points included the need for other blood products , the clotting profile , the intensive care unit mortality and length of stay.methods:retrospective observational analysis in a tertiary oncologic icu in brazil including 894 patients submitted to oncologic surgery for a two - year period from september 2007 . patients were grouped according to whether hydroxyethyl starch was used during surgery ( hydroxyethyl starch and no - hydroxyethyl starch groups ) and compared using a propensity score analysis . a total of 385 propensity - matched patients remained in the analysis ( 97 in the no - hydroxyethyl starch group and 288 in the hydroxyethyl starch group).results : a higher percentage of patients in the hydroxyethyl starch group required red blood cell transfusion during surgery ( 26% vs. 14% ; p = 0.016 ) and in the first 24 hours after surgery ( 5% vs. 0% ; p = 0.015 ) but not in the 24- to 48-hour period after the procedure . there was no difference regarding the transfusion of other blood products , intensive care unit mortality or length of stay.conclusion:hydroxyethyl starch use in the intraoperative period of major oncologic surgery is associated with an increase in red blood cell transfusions . there are no differences in the need for other blood products , intensive care unit length of stay or mortality .

INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Results of propensity score using corrected total volume and the results of standard multivariate analysis Corrected total volume (CTV) analysis Study results of standard analysis

hydroxyethyl starches ( 4 ) are widely used as fluid expanders in the perioperative period and are the preferred colloids in many intensive care units ( icu ) ( 5,6 ) . in the perioperative period , even the new generation starches inspire concerns about their safety , especially in terms of coagulation disorders ( 1,7 ) , . patients submitted to oncologic surgery are subjected to long surgery procedures , with a great need of fluid resuscitation and blood products transfusion . no study has evaluated the effects of the intraoperative use of a new generation he s on the intraoperative and postoperative red blood cells and other blood products transfusion in patients submitted to elective major oncologic surgery . we hypothesized that patients submitted to oncologic surgery that received a new generation he s during surgery would require more red blood cells transfusions during and after the surgery than patients that did not receive he s . to test our hypothesis , we evaluated whether a new generation he s led to more red blood cell transfusions during the first 48 hours after major elective oncologic surgery than patients that received only crystalloids using a propensity - matched approach . secondary objectives included clotting profile , use of blood products other than red cells , icu length of stay and mortality between the two groups . from september 2007 to september 2009 , all patients admitted to the hospital icu ( three units with a total of 33 beds ) of our teaching hospital after an elective major oncologic surgery ( head and neck , neurological , thoracic , abdominal and other sites ) were included in the study . during the intraoperative period , we collected the number of packs of blood products transfused ( red blood cells , fresh frozen plasma , platelets and cryoprecipitate ) , the surgery length and the volume of crystalloids and colloids infused . during icu admission , we collected the simplified acute physiology score 3 ( saps 3 ) and performed laboratory exams that included creatinine levels and clotting profile ( composed of the platelets count and prothrombin time ( pt ) . during the 48-hour period after the surgery , we collected the number of packages of blood products transfused and performed laboratory exams , including serum creatinine . we divided the 48-hour period after the surgery into two periods of 24 hours because the hypothetical he s side effect could be evanescent . the data were analyzed for three consecutive periods : the intraoperative period , the period from the end of the surgery to the first 24 hours after post - surgery ( 24-hour period ) and the period between from 24 to 48 hours after the surgery ( 24- to 48-hour period ) . the patients were categorized in two groups according to the use of he s during the intraoperative period ( he s and no - hes group ) . the patients in the he s group received at least 500 ml of new generation he s ( voluven - 6% 130/0.4 , fresenius , germany ) . there are five major surgical teams in the hospital ( thoracic , abdominal , pelvic , neurological and soft tissue ) and each has a closed staff , i.e. the icu policy for transfusion suggests that red blood cell packs should be transfused if the hemoglobin level is below 7 g / dl or below 8 g / dl with signs of active bleeding and/or impaired tissue perfusion ( delayed capillary refill time , elevated lactate levels and nonchloremic metabolic acidosis ) ( 17 ) . transfusion with hemoglobin levels above 9 g / dl was not encouraged and was performed only at the discretion of the intensivist if massive blood lost was detected ( high output in the drain with hemodynamic instability ) . colloid use policy : at the time of the study , the only synthetic colloid available at our institution was a new generation 6% he s 130/0.4 ( voluven ) . albumin was not used during the postoperative period of elective oncologic surgery . patients were clustered according to the administration of he s during surgery . because this was a retrospective analysis ( patients were not randomized ) , the creation of a propensity score is an effective method to reduce bias . propensity - matched analyses are increasingly being used in research due to its ability to improve the power of retrospective and prospective non - randomized analysis ( 18,19 ) . propensity - matched analyses are able to take into account as many variables related to the outcome evaluated as needed , which reduces bias ( 20 ) . to construct the propensity score , a logistic regression was fitted , and variables were chosen for inclusion according to the methods of brookhart et al . we included variables that could be related to the study outcomes based on the propensity score . to identify the variables potentially associated with the outcome , a univariate analysis was performed to evaluate potential variables related to blood transfusion during the entire perioperative period ( from the surgical procedure to up to 48 hours after surgery ) . the variables included on the propensity score were as follows : age , gender , body - mass index , metastatic disease , surgery length , volume of crystalloid infused and surgical sites . although age and body mass index were not associated with the outcome , we believed that they were important variables that could alter the fluid dynamics during the surgical procedures and included them in the model ( 21 ) . after the propensity score was created , the patients were matched according to their respective propensity scores . as reported in the current literature , matching on the propensity score has now been clearly demonstrated to be the best method to attempt to provide an unbiased estimation of the treatment effect ( 19 ) . the match procedure was performed with an optimal matching method , and to avoid overinflation , we did not permit replacement of matched patients . the number of patients analyzed using the propensity score was smaller than the total number of patients studied because matching was not possible for all patients . the correct construction of the propensity score was performed with the box - plot method . after the propensity score matches were performed , we performed a diagnostic to ensure good balance in the matched population through the bias reduction method and the stabilized standardized difference ( because we were using 1:n matching instead of 1:1 matching ) ( 19,22 ) . after matching , ( ctv ) instead of crystalloid volume as a variable in the propensity score ( see appendix ) . we included 894 patients ; 614 received he s , and 280 received only crystalloids during surgery . before the use of the propensity score , the patients in the he s group had longer surgical times , had received more crystalloids , were younger and were less likely to have any comorbidity compared with the no - hes group ( table 1 ) . after the creation of the matched groups , 385 propensity - matched patients ( 97 in the crystalloids group and 288 in the he s group ) were analyzed . more detailed results regarding the other propensity matched analysis ( using ctv ) , standard logistic regression and non - propensity matched results analysis can be found in the appendix . red blood cells and other blood products transfusions : the variables related to transfusion of red blood cell pack transfusion for all the perioperative transfusions are shown in table 2 . most patients in both groups did not require blood products transfusion . in the he s group , a higher percentage of patients received red blood cell transfusion in the intraoperative and 24-hour postoperative period , but the percentage was similar for the 24- to 48-hour postoperative period ( figure 1 ) . for the patients in both groups that received red blood cell transfusions , the number of packs per patient was higher in the he s group during the intraoperative period ( 0.55 versus 0.39 ; p = 0.028 ) . there was no difference regarding the use of fresh frozen plasma , cryoprecipitate and platelets concentrate in any period . the patients that received he s had a small but statistically significant lower hemoglobin level at icu admission ( 11.21.9 versus 12.01.7 ; p<0.001 ) but not at the 24-hour ( 11.11.8 versus 11.31.6 ; p = 0.11 ) and 48-hour ( 10.21.7 versus 10.21.5 ; p = 0.20 ) postoperative periods . the results of the non - propensity analysis ( see appendix , additional table 3 and 4 ) showed that red blood cell transfusion was more frequent in the he s patients . the multivariate analysis showed that the factors associated with red blood cell transfusion in the perioperative period included age , metastatic disease , total operative time , crystalloid volume used and colloid use ( any dose ) . secondary objectives : patients in the he s group showed lower levels of platelets at icu admission compared to the no - hes group ( table 3 ) . the international normalized ratio ( inr ) was also higher in the he s patients at icu admission and in the first 24 hours after surgery ( table 3 ) . table 4 shows the coagulation values for transfused and non - transfused patients in both groups ( no - hes and he s ) . there were no differences between transfused patients in the he s and no - hes groups regarding platelet count or inr at any period studied . non - transfused he s patients also had a higher inr than non - transfused no - hes patients . icu mortality and length of stay were similar between he s and no - hes groups ( table 3 ) . in this retrospective analysis using a propensity - matched cohort of surgical patients , he s use was associated with more frequent red blood cells transfusions during major oncologic surgery and in the first 24 hours after the procedure . a recent randomized controlled trial suggested that patients with penetrating trauma required less total volume when resuscitated by he s compared with saline ; there was no difference , however , in patients with blunt trauma ( 30 ) . a larger multicenter randomized trial has shown no difference regarding the total fluid need in septic patients ( 24 ) . the need for less fluid to obtain the same hemodynamic effect would be particularly interesting during the intraoperative period , when hemodynamic optimization associated with less total fluid volume may be beneficial ( 32 ) . he s and other colloids are not related to any robust positive clinical outcome , such as mortality or reduced icu length of stay ( 33 ) . we aimed to evaluate the effects of he s on the perioperative need for blood transfusion in this specific population . this outcome is important because blood transfusion may be associated with worse outcome after oncologic surgery , including long - term survival ( 35 - 38 ) . in critically ill patients , blood transfusion is associated with poorer outcomes ( 39 ) . our propensity - matched data showed that he s use is independently related to a greater need for red blood cell transfusion in the perioperative period . in addition to requiring more red blood cell transfusions , he s patients also had lower hemoglobin levels at icu admission , suggesting that the difference was not only due to over transfusion of the he s group . corroborating with our results , other studies have shown a higher need for blood products in critically ill patients who received he s ( 24,40 ) . patients with blunt trauma resuscitated with he s required more blood transfusions than patients who received saline , although this conclusion is likely confounded by a higher injury severity in the hes - resuscitated patients ( 30 ) . our standard multivariate analysis showed that colloid use was a risk factor for the perioperative need for red blood cell transfusion . nevertheless , because this analysis included fewer patients ( with only 41 patients in the no - hes group see appendix ) , it is possible that the smaller sample reduced the power of the study to detect any difference . it can be argued that because the use of he s is associated with a greater degree of plasma volume expansion , and considering that the two groups were paired to the crystalloid volume infused , our findings may be related only to the increased hemodilution caused by he s use ( 41 ) . the increase in the frequency of red blood cell pack transfusions in the he s group in the main propensity analysis might to some degree be related to the expansion effect of he s and not to its impact on blood coagulation . there were small differences in the coagulation profile between the he s and no - hes groups . there were no differences in the coagulation profile ( including the inr and platelet count ) between the transfused patients in both groups ( table 4 ) . the higher need for transfusions can not be explained by the differences in coagulation features after surgery . in our multivariate analysis , it may be speculated that because restrictive fluid therapy is frequently used in thoracic surgery ( 43 ) , patients received less fluid , with less hemodilution and bleeding related to dilutional coagulopathy , and therefore required less red blood cell transfusion . the icu length of stay and mortality were similar between groups , but our analysis was likely underpowered to detect a significant difference in a population with low mortality ( below 2% ) and short icu length of stay ( one day of median icu stay ) . in the univariate analysis , icu length of stay was higher in the he s group than in the no - hes group ( additional table 6 ) . third , due to its retrospective nature , we were unable to control for personal preferences variables regarding red blood cell transfusion and specific tumor staging ( which could be related to technical difficulties during the surgical procedure ) , although we included metastatic disease in the propensity score . fifth , our study was underpowered to evaluate the icu length of stay and mortality . our finding that he s use is associated with a greater need for blood transfusion is biologically plausible and has been reported in other clinical scenarios ( 24,40 ) . if these findings are confirmed in large , prospective studies , he s use should be questioned in the subjects undergoing elective oncologic surgery due to its unproven clinically efficacy over crystalloids ( 4,13 ) . the intraoperative use of he s 6% 130/0.4 during major elective oncologic surgery is associated with an increase in red blood cell transfusions in the perioperative period of major oncologic surgery . there were no differences in icu length of stay and mortality . impact of intraoperative he s 6% 130/0.4 on the need for blood transfusion after major oncologic surgery : a propensity - matched analysis our propensity score analysis using ctv instead of infused crystalloid volume resulted in 97 patients in the no - hes group and 162 patients in the he s group . the major difference between this analysis and the previous analysis was the use of the corrected total volume ( ctv ) as a variable instead of the volume of infused crystalloid . , the propensity - matched patients in this analysis would have received the same amount of plasma expansion . we defined the ctv in patients in the no - hes group as equivalent to the crystalloid volume infused multiplied by 0.3 1 . the ctv in the he s group was defined as the sum of the crystalloid volume multiplied by 0.3 plus the volume of he s multiplied by 1.4 ( ctv = [ crystalloid volume 0.3 ] + [ 1.4 colloid volume ] ) . the use of the 1.4 ratio aimed at accounting for the theoretical greater expansion effects of the colloids 2,3 . this alternative propensity analysis included 41 patients in the no - hes group and 118 patients in the he s group ( additional table 1 ) . there was no difference in the need for red blood cell transfusion between the groups when this analysis was performed ( additional table 2 ) . the stepwise backward method was used to determine the factors associated with red blood cell transfusion in the perioperative time . the initial model consisted of all of the independent variables that had a p value of less than 0.25 in the bivariate analysis associated with red cell blood transfusion or between the he s and non - hes groups in the 894 patients ( the hosmer - lemeshow logistic regression ) . the discriminative ability of the model to predict the outcome of patients was assessed by the area under the receiver operating characteristic ( auc ) curve . the major outcomes regarding the need for blood transfusion on crude analysis are displayed in additional table 4 . the major difference between propensity - matched versus standard analysis was that before matching , the need for blood products other than red blood cell packs was more common in the he s patients . the he s patients in the unmatched analysis received fresh frozen plasma during surgery more frequently than the no - hes patients . the need for cryoprecipitate was also more frequent in the he s patients in the first 24 hours after the procedure . a multivariate analysis was performed to evaluate the risk factors for red blood cell transfusion from the intraoperative period up to 48 hours after the procedure ( additional table 5 ) . the factors associated with the red blood cell transfusions included age , metastatic disease , volume of crystalloid used , total operative time and use of any dose of he s . the icu length of stay was higher in the he s group ( additional table 6 ) . our propensity score analysis using ctv instead of infused crystalloid volume resulted in 97 patients in the no - hes group and 162 patients in the he s group . the major difference between this analysis and the previous analysis was the use of the corrected total volume ( ctv ) as a variable instead of the volume of infused crystalloid . , the propensity - matched patients in this analysis would have received the same amount of plasma expansion . we defined the ctv in patients in the no - hes group as equivalent to the crystalloid volume infused multiplied by 0.3 1 . the ctv in the he s group was defined as the sum of the crystalloid volume multiplied by 0.3 plus the volume of he s multiplied by 1.4 ( ctv = [ crystalloid volume 0.3 ] + [ 1.4 colloid volume ] ) . the use of the 1.4 ratio aimed at accounting for the theoretical greater expansion effects of the colloids 2,3 . this alternative propensity analysis included 41 patients in the no - hes group and 118 patients in the he s group ( additional table 1 ) . there was no difference in the need for red blood cell transfusion between the groups when this analysis was performed ( additional table 2 ) . the stepwise backward method was used to determine the factors associated with red blood cell transfusion in the perioperative time . the initial model consisted of all of the independent variables that had a p value of less than 0.25 in the bivariate analysis associated with red cell blood transfusion or between the he s and non - hes groups in the 894 patients ( the hosmer - lemeshow logistic regression ) . the discriminative ability of the model to predict the outcome of patients was assessed by the area under the receiver operating characteristic ( auc ) curve . the major outcomes regarding the need for blood transfusion on crude analysis are displayed in additional table 4 . the major difference between propensity - matched versus standard analysis was that before matching , the need for blood products other than red blood cell packs was more common in the he s patients . the he s patients in the unmatched analysis received fresh frozen plasma during surgery more frequently than the no - hes patients . the need for cryoprecipitate was also more frequent in the he s patients in the first 24 hours after the procedure . a multivariate analysis was performed to evaluate the risk factors for red blood cell transfusion from the intraoperative period up to 48 hours after the procedure ( additional table 5 ) . the factors associated with the red blood cell transfusions included age , metastatic disease , volume of crystalloid used , total operative time and use of any dose of he s . the icu length of stay was higher in the he s group ( additional table 6 ) .

layered structures of semiconducting and magnetic materials have attracted a great deal of recent attention due to their huge potential in hybrid spintronic applications . in order to fabricate devices , magnetic materials compatible with the important group iii v and group iv semiconductors are required . the list of ideal characteristics for such materials is long : controllable magnetic states , high curie temperature , long spin diffusion and coherence lengths , well - behaved interfaces to the host semiconductor , suitable resistance - area ( ra ) product for spin injection , engineering compatibility for processing and annealing , and so forth . hence , many magnetic materials are under intensive investigation , including heusler alloys such as co2fesi , diluted magnetic semiconductors , transition metal pnictides ( tmps ) such as mnas , and simple magnetic metals and alloys such as nife . while these material classes have mutual advantages and disadvantages , the tmps are very well - suited to all - epitaxial spintronic structures due to their established structural and chemical compatibility with mainstream iii v semiconductors . both tmp epilayers on iii vs and more complex tmp / iii v multilayer heterostructures can be grown using molecular beam epitaxy ( mbe ) within conventional iii v chambers . this enables the creation and tailoring of a range of structures including spin light emitting diodes , ferromagnetic optical isolators , and spin valves . mnsb is an attractive tmp for spintronic applications being both ferromagnetic with a high curie temperature ( tc = 314 c ) and displaying a very large magneto - optical kerr rotation . the material is also a weak p - type metal ( p 10 cm ) which mitigates the well - known conductivity mismatch problem to semiconductors . mnsb adopts the double hexagonal close - packed niccolite ( n- ) crystal structure ( b81 ) and , unlike mnas , undergoes no structural or magnetic phase transitions in the pressure in addition , a major advantage of mnsb in device design and optimization is the ability to process the material using conventional iii v lithographic and etching techniques . this enables scalable processing of , for example , lateral nonlocal spintronic devices in which pure spin currents are manipulated . the successful growth of n - mnsb epilayers has been reported by several groups using gaas substrates , mainly aimed at magneto - optical applications for which iii v materials are a natural choice . combining tmps with si and ge would enable a wider range of hybrid spintronic devices to be developed , directly integrated with the dominant semiconductor materials systems used in conventional nanoelectronics . however , there have been relatively few studies of tmp epitaxial growth on si , and we are aware of none on ge . room temperature spin transport in si with spin diffusion lengths more than 100 nm has been established , and similarly efficient spin transport at 300 k in ge has also been demonstrated . one advantage of ge over si ( and gaas ) is its higher hole mobility : matching to a p - type ferromagnetic spin injection contact such as mnsb is therefore a natural choice for investigating and exploiting spin - polarized hole transport in semiconductors . the mbe growth conditions typically reported for stoichiometric n - mnsb are a substrate temperature in the range 200400 c and an sb / mn flux ratio greater than 1 . on gaas(001 ) , the principal epitaxial orientation is mnsb(1101)gaas(100 ) , which results in an oblique match with complicated interface and surface structures . a simpler epitaxy is found on gaas(111 ) , namely , mnsb(0001)gaas(111 ) and with mnsb[2110]gaas[110 ] . the bulk lattice parameters of n - mnsb are a = 4.128 and c = 5.789 , which results in an epitaxial mismatch of 3.2% when grown on gaas ( 111 ) surfaces ( the in - plane spacing on the gaas ( 111 ) surface being agaas/2 = 3.995 ) . the use of si(111 ) as a substrate for epitaxial mnsb(0001 ) growth is severely hampered by the large mismatch of 7.0% . furthermore , the growth of mn compounds such as mnbi or mnsb on si substrates appears to be difficult due to the formation of undesirable interfacial mn silicides . the use of ge(111 ) substrates reduces the epitaxial mismatch to a level that is similar to that found for gaas ( 111 ) ( i.e. , 3.2% ) . while the interfacial chemistry of ge with tmp growth is unknown , studies of the growth of mnxge1x thin - films suggest that mn5ge3 has the greatest thermodynamic stability , and layers of mn5ge3(0001 ) have been successfully grown on ge(111 ) substrates by solid phase epitaxy and mbe . a further issue in mnsb growth is the presence of multiple polymorphs in films grown on gaas(111 ) . we have recently demonstrated the growth by mbe of cubic c - mnsb polymorphs ( b3 structure ) on n - mnsb to thicknesses of tens of nm . because of its wide minority spin gap of around 1 ev , c - mnsb is predicted to be a robust half - metallic ferromagnet at room temperature . however , the growth mechanism is not known and pure n - mnsb polymorph is valuable for some device structures . nowadays , sige / si and ge / si virtual substrates are used for epitaxial growth of a huge variety of group - iv semiconductor heterostructures . they are ideal alternatives to expensive ge wafers , which are not always easily or cheaply available , while commercial sige substrates do not exist at all . a virtual substrate consists of a fully relaxed sige or ge buffer layer grown directly on a si substrate . because the epilayers are relaxed at their top surfaces , the in - plane lattice parameters relevant to subsequent epitaxial growth match those of equivalent bulk sige or ge crystals . here , we report the mbe growth of mnsb epitaxial films on high quality ge(111 ) virtual substrates , themselves grown on si wafers using reduced pressure chemical vapor deposition ( rpcvd ) . reflection high - energy electron diffraction ( rheed ) , high resolution synchrotron x - ray diffraction ( xrd ) with reciprocal space maps ( rsm ) , x - ray photoelectron spectroscopy ( xps ) , and superconducting quantum interference device ( squid ) magnetometry measurements were all used to characterize the properties of the resulting epilayers . we find that the quality of the mnsb layers is comparable to the best material grown on gaas , and unlike on gaas segregation of substrate material through the mnsb layer is not observed . there is no evidence for the formation of any interfacial alloy layers , and the mnsb grows in a single n - mnsb phase without any of the polymorphs seen on gaas substrates . an outline of the sample layer structure is shown in figure 1 together with the n - mnsb b81 crystal structure . for virtual substrate production , ge epilayers were grown on 100 mm diameter si(111 ) substrates using a germane ( geh4 ) gaseous precursor diluted in h2 carrier gas within a rpcvd reactor . a low temperature ( lt - 400 c ) ge seed layer 10 nm thick was grown first . following an anneal , a further ge layer around 500 nm thick was deposited at high temperature ( ht - 670 c ) . as shown schematically in figure 1 , the lt seed layer produces islands which serve to reduce the threading dislocation density and are efficiently planarized by the subsequent ht growth . the density of threading dislocations in the ge / si(111 ) virtual substrates is around 10 cm , and further details on this rpcvd intermediate islanding growth method can be found elsewhere . the top surface of the ge(111 ) virtual substrate is smooth , with a typical root - mean - square surface roughness of 2 nm . schematic layer structure of mnsb(0001 ) on ge / si(111 ) virtual substrates . the function of the ht and lt ge growth is described in the text , while the highlights a surface oxide removal stage after air transfer . the mbe chamber for mnsb growth contains shuttered mn , cr , ni , and sb effusion cells ( the latter having no separate thermal cracking stage ) and is equipped with an electron gun and phosphor screen for monitoring rheed throughout growth . a retractable beam flux gauge was used to monitor the elemental fluxes before and after growth . an adjoining chamber is equipped with an ion gun and annealing stage for surface preparation . small pieces of ge(111 ) virtual substrates , measuring approximately 8 mm 8 mm , were attached to stainless steel sample plates using spot - welded ta wires . the mounted samples were then cleaned to remove debris and dust by a cycle of acetone , isopropanol , and water rinsing prior to blow drying with dry nitrogen . the samples were then loaded into the vacuum via a fast - entry chamber and transferred into the preparation chamber . once under a vacuum , the substrates were heated for 1 h at 400 c for degassing . this was followed by a 10 min 500 ev ar ion bombardment and finally a 480 c anneal for 40 min . epilayers of mnsb were then grown using a substrate temperature of ( 410 10)c with the ratio of the sb to mn fluxes set at approximately 6.5:1 . growth was initiated by simultaneously opening the mn and sb shutters , with the directly measured mn beam equivalent pressure fixed at 8.0 10 mbar . these growth conditions are similar to those used in the epitaxy of mnsb films on gaas substrates . following growth , the samples were exposed to either sb or mn fluxes in order to examine the reconstruction behavior of the samples . once the mbe growth experiments were complete , samples were removed from the vacuum and transported in air with no special precautions to the national synchrotron light source at brookhaven national laboratory for xrd measurements . the structural characterization of the films was performed using high resolution triple - axis xrd on the x22c beamline . triple axis diffraction scans parallel to the [ 000l ] direction as well as rocking curves were collected at room temperature using a 10 kev x - ray beam and a ge(111 ) analyzer crystal . the surface stoichiometry of the films was investigated using xps using a monochromatized al k source and seven - channel hemispherical analyzer ( omicron gmbh ) . in order to check for the presence of surface - segregated ge and to investigate the native oxides of the mnsb films , magnetic hysteresis loops were obtained using a squid magnetometer ( quantum design inc . ) from a few - millimeter - sized pieces of the mnsb samples . the pieces were manually aligned so that the applied field was either in the plane of the layers or perpendicular to the plane . electrical properties of the mnsb films were measured at room temperature using sprung gold contacts in van der pauw geometry , after a 10 s dilute hcl etch ( 10% by volume ) to minimize contacting problems due to mn oxides . the rheed patterns of the films were monitored throughout growth , and the resulting patterns were recorded for the principal symmetry directions . after ar sputtering and annealing , the ge(111 ) surfaces exhibited a sharp c(2 8) reconstruction ( figure 2a , b ) . on the initiation of mnsb growth , this gave way to a weaker ( 1 1 ) pattern with a streak spacing different from that of the ge virtual substrate . the early stages of growth were characterized by the presence of faint transmission diffraction features which faded within 30 s. after this , a sharp and streaky ( 1 1 ) pattern formed , and this pattern was subsequently observed throughout growth . the measured streak spacing was 4.13(9 ) , in agreement with the bulk mnsb a lattice parameter of 4.128 . the observed symmetry of the rheed patterns is consistent with the formation of an orientated mnsb(0001 ) layer . the in - plane epitaxial orientations are then mnsb2110ge110 and mnsb1100ge211. following growth , and upon allowing the samples to cool to room temperature , a faint td(1 4 ) pattern developed indicating the formation of an sb - rich surface , and typical rheed patterns for this surface reconstruction are shown in figure 2d , e . after further exposure to a mn flux , a ( 2 2 ) periodicity developed , although upon closing the mn effusion cell shutter this developed into a mixed ( 2 2)/(3 3)r30 pattern after a few minutes as shown in panels ( g ) and ( h ) of figure 2 . the sharpness of the streaks and the presence of multiple laue zones and kikuchi features in these patterns indicate that the near - surface region is highly crystalline with well - ordered domains of both reconstructions present . exposure to an sb flux resulted in the appearance of the ( 1 1 ) and td(1 4 ) periodicities as described above . these reconstructions , and their appearance under more mn - rich or sb - rich conditions , are consistent with those observed in the mnsb(0001)/gaas(111 ) system . rheed patterns from ge(111 ) prior to growth and mnsb(0001 ) following growth . panels ( a ) and ( b ) demonstrate the c(2 8) reconstruction found on clean ge(111 ) substrates . panels ( d ) and ( e ) show the postgrowth td(1 4 ) observed on mnsb(0001 ) . panels ( g ) and ( h ) show the mixed ( 2 2)/(3 3)r30 pattern observed following mn deposition . panels ( c ) , ( f ) , and ( i ) show the expected reciprocal meshes for the three surfaces . in the case of panel ( i ) the dark gray circles indicate the ( 3 3)r30 mesh , while the light gray circles correspond to the ( 2 2 ) mesh . high - resolution triple axis xrd data for a sample of nominal thickness 70 nm are shown in figure 3 . panel ( a ) shows strong diffraction peaks which can be readily to assigned to the ( 111 ) , ( 222 ) , and ( 333 ) reflections from the ge and si components of the virtual substrate ; these occur at qz values precisely corresponding to the bulk lattice parameters . the sharpness and symmetry of the ge peaks highlights the full strain relaxation achieved in the two - stage rpcvd growth . a full reciprocal space map in the vicinity of the 111 reflections is shown in panel ( b ) with qx the in - plane component of the scattering vector . the ge(111 ) and si(111 ) features ( the lowest and central diamond shapes ) are intense and symmetrical in both qz and qx , confirming the high quality of the virtual substrate . triple axis out - of - plane xrd data from a 70 nm mnsb(0001 ) layer on ge(111 ) virtual substrate . in ( a ) , only three families of reflections are present : peaks are labeled on the figure , and the inset shows a expanded view of the lowest order set of reflections . panel ( b ) shows an rsm from the same sample covering the qz region as shown in the panel ( a ) inset , with the color scale ( black - blue to yellow - red ) representing intensity . strong peaks arising from reflections indexed to the mnsb(000l ) planes are present in figure 3a . the c lattice parameter derived from these is 5.790(1 ) , in good agreement with the reported value of bulk mnsb crystals . the widths of the rocking curves taken at the n - mnsb 000l reflections were independent of the diffraction order giving a value of 0.4 for the mosaic , which is comparable to the values observed in nisb / gaas epitaxy and in mnsb - gaas films . the inset in figure 3 shows a close - up of the mnsb(0002 ) region covering the 1.75 qz 2.35 range . the mnsb(0002 ) reflection shows a clear asymmetry toward higher qz values , with a distinct tail toward higher qz indicating a strain profile with part of the film having smaller c lattice parameters . the measured strain range corresponds to a lattice parameter change of c / cbulk 0.15% through the mnsb layers , again in agreement with mnsb - gaas layers . this asymmetry is also captured in the rsm where the mnsb(0002 ) peak appears as a feature around qz = 2.2 clearly elongated in the higher qz direction compared to the si and ge peaks . it is interesting to note that nisb - gaas films also exhibit high qz shoulders despite the much - reduced lattice mismatch , but more detailed experiments are needed to determine whether this is a surface feature or occurs at the substrate . a williamson - hall analysis was performed for the mnsb reflections which yields a grain size of ( 89 2 ) nm , comparable to the nominal film thickness and indicating high crystalline order throughout the mnsb film . we also note that there are no additional peaks in the xrd spectra obtained from mnsb films grown on ge(111 ) virtual substrates . on gaas(111 ) , both nisb and mnsb often show weak xrd peaks associated with alternative epitaxial orientations such as ( 1101 ) . such orientations have not been observed here , and evidence of polymorphic growth is also absent . when mnsb is grown on gaas ( and , in recent unpublished work , on ingaas ) under similar mnsb mbe growth conditions , diffraction features due to strained c - mnsb(111 ) epitaxial inclusions within the n - mnsb film can be observed . figure 4 shows typical xrd data for a 75 nm thick mnsb film grown on gaas(111 ) in direct comparison with the 70 nm film on ge(111 ) . a broad peak at qz 3.25 is present for the film on gaas , which can be assigned to the ( 222 ) reflection of c - mnsb , with corresponding lattice parameter 6.8(1 ) ( the evolution of the polymorph inclusion strain and grain size as a function of mnsb film thickness on gaas will be detailed in a future paper ) . such polymorph peaks are absent in all the xrd spectra and rsms from our samples grown on the ge virtual substrates , but a more detailed search of the mbe parameter space would be required to definitively rule out polymorphism in the mnsb - ge(111 ) epitaxial system . importantly , there is also no evidence for any interfacial mngex phases in the xrd spectra or rsm images , suggesting that even in the thin film limit n - mnsb can be grown . triple axis out - of - plane xrd data comparing 70 nm mnsb(0001 ) film on ge(111 ) virtual substrate with 75 nm mnsb(0001 ) film on gaas(111 ) . the broad peak at qz 3.25 is due to the presence of cubic c - mnsb inclusions in the film grown on gaas which are absent for the ge substrate . to explore the surface chemistry and to probe any substrate diffusion , panel ( a ) shows the shallow core region ( 2555 ev binding energy ) with the mn 3p , sb 4d , and ga 3d core levels highlighted ( the latter for a comparative growth on gaas ) . of note is the absence of a ge 3d peak at around 29 ev from the shallow core region , suggesting that no ge segregation occurs in these layers . this contrasts with mnsb / gaas(111 ) epilayers , where approximately a monolayer of segregated ga is observed at the surface of mnsb but is similar to the case of nisb / gaas(111 ) where no ga segregation occurs . the absence of surface ge can be checked using the ge 2p core level , whose larger atomic sensitivity and decreased probing depth relative to the 3d core level provide increased sensitivity in the detection of surface segregated ge . sometimes , a very weak peak can be observed : upper red curve , 90 takeoff angle ( toa ) . at 30 toa , where the surface sensitivity is enhanced , we ascribe the small signal in the 90 toa data to a piece of exposed ge substrate , probably due to postgrowth scratches or pregrowth defects caused during sample cutting , handling , or ta wire bonding . xps spectra from an as - loaded mnsb film on ge / si(111 ) virtual substrate . the solid line of panel ( a ) shows the shallow core region with the mn 3p and sb 4d core levels present , while the ge 3d core level is absent . for comparison , the dashed line in ( a ) shows a 75 nm film on gaas(111 ) , where a ga 3d peak is present . panel ( b ) is the ge 2p3/2 core level showing 90 and 30 toa data . panel ( c ) shows the sb 4d region , including fits , and indicates the presence of sb mn , sb comparison to mnsb growth on gaas should account for different growth conditions , which are likely to affect the kinetics of surface segregation . ono et al . grew mnsb on gaas(111 ) at lower substrate temperature ( 300 c ) and flux ratio ( 2:1 ) but similar growth rate ( 0.03 nm s compared to our 0.045 nm s ) . they found that a full monolayer of segregated ga was present at the surface , independent of mnsb film thickness above 2 nm up to their maximum thickness of around 25 nm ( figure 5 of ref ( 28 ) ) ; that is , the segregation was very efficient under these growth conditions . using xps we have observed significant surface ga signals for several much thicker mnsb films on gaas(111 ) with growth conditions similar to those used in the present work ( 420 c , flux ratio 6.35:1 , growth rate 0.04 nm s ) . example data are included in panel ( a ) of figure 5 ( dashed line ) for a 75 nm mnsb film on gaas(111 ) . a clear ga 3d peak is observed due to surface segregation . in order to determine the stoichiometry of the surface region , total elemental peak areas were obtained for the mn 3p and sb 4d core levels following subtraction of a linear background . the mn to sb ratio was found to be 1.27:1 with the increased mn content arising due to the preferential formation of mn oxides relative to sb oxides which is characteristic of oxidized mnsb thin films . shown in figure 5c is a close - up of the sb 4d region alongside a fit to the experimental data . three doublets are required to fit the core level with the doublets corresponding to sb sb , sb mn , and sb ox bonding environments , the corresponding binding energies of the sb 4d5/2 components are given in table 1 . the chemical shift between the sb sb and sb mn component is 0.34 ev , less than previously reported for mnsb films . the ferromagnetic properties of the films were studied by squid magnetometry . in figure 6 , we show hysteresis loops measured from a nominally 70 nm thick film recorded at t = 5 k. the film displays the expected ferromagnetic behavior , which is retained up to t = 300 k consistent with the reported bulk ordering temperature of mnsb tc of 314 c . the hysteresis loops in figure 6 show the magnetic response with the applied field perpendicular and parallel to the mnsb c - axis . the data are fitted piecewise to extract the coercive field using a langevin function to fit the hard axis and a pair of arctan functions to fit the ferromagnetic components . in the inset of figure 6 , the fits highlight that the shape of the top left / bottom right sections of the loop differs from that of the top right / bottom left sections . this implies a different switching behavior as the films reverse , the switch being more rounded as the films come from either positive or negative saturation . this behavior can be explained by a small misalignment of the film in the squid magnetometer : the applied field is not exactly in - plane , causing a field - asymmetric component from the hard axis to alter the switching . the overall magnetic behavior is , however , consistent with uniaxial magnetic anisotropy with the hard axis perpendicular to the ( 0001 ) plane of the films and the in - plane axis lying in the plane of the film . this agrees with the behavior of both thin ( 0001 ) films and bulk crystals of mnsb . an estimate of the effective moment per mn can be made using the nominal film thickness of 70 nm and the saturation magnetization which gives a value of ( 3.3 0.3)b . this is in broad agreement with previously determined values for mnsb of between 3.3 and 3.5 b . the coercive field , hc , in the easy plane is ( 111 2 ) oe , while along the hard axis it is more noisy but is approximately ( 1.2 0.6 ) koe . these values are consistent with those derived for mnsb(0001 ) films grown on gaas(111 ) , where hc is normally in the range 20200 oe ( easy ) and 300500 oe ( hard ) , depending on film thickness and exact growth conditions . squid magnetometry of a 70 nm mnsb layer on ge(111 ) virtual substrate . h hysteresis loops for the field applied parallel ( out - of - plane , blue squares ) and perpendicular ( in - plane , black circles ) to the [ 000l ] c - axis of mnsb . the inset shows a close - in loop for the field applied in - plane . room temperature hall measurements on the mnsb films on ge virtual substrates gave typical hole densities of p = ( 1.3 0.2 ) 10 cm with mobility around 90 cmv s , in agreement with thin film mnsb grown on gaas(111 ) . the ra product of the mnsb / ge interface , important for optimzing spin injection , can not be determined accurately from the present sample set due to the high series resistance of the undoped ge virtual substrate . however , a preliminary upper limit estimate of 10 to 10 cm is very promising ; measurements were based on making contacts with indium to exposed ge ( covered by ta foil during mnsb growth ) . further work on electrical characterization of the mnsb / ge interface will focus on p - doped ge virtual substrates to match the p - type mnsb contact . the structural , surface - chemical , and magnetic properties of mnsb thin films grown on ge / si(111 ) virtual substrates have been studied . the observed surface reconstructions are broadly consistent with those seen on mnsb(0001 ) films grown on gaas substrates , with rheed indicating that the surfaces are highly ordered and crystalline . xrd shows that the mnsb films exhibit a pure niccolite structure without other polymorphs or orientations . the n - mnsb is orientated ( 0001 ) on the ( 111 ) virtual substrates , with the lattice parameter relaxing to 5.790(1 ) after some tens of nanometers film thickness . magnetometry reveals the expected ferromagnetic behavior with properties similar to those observed on mnsb / gaas films . under the growth conditions employed here ( substrate temperature 410 c , sb / mn flux ratio 6.5:1 , growth rate 0.045 nm s , layer thickness 70 nm ) , surface segregation of ge substrate atoms through the mnsb film is suppressed , and no interfacial alloy layers form . the ability to grow mn pnictides on ge virtual substrates offers great promise for the integration of the tmp class of magnetic materials with si / sige in spintronic applications , especially exploiting spin - polarized hole transport . the growth of single - orientation , single - phase n - mnsb on ge(111 ) thus represents an important step in the realization of hybrid magnetoelectronic applications requiring carefully controlled magnetic properties in the epilayer .

molecular beam epitaxial growth of ferromagnetic mnsb(0001 ) has been achieved on high quality , fully relaxed ge(111)/si(111 ) virtual substrates grown by reduced pressure chemical vapor deposition . the epilayers were characterized using reflection high energy electron diffraction , synchrotron hard x - ray diffraction , x - ray photoemission spectroscopy , and magnetometry . the surface reconstructions , magnetic properties , crystalline quality , and strain relaxation behavior of the mnsb films are similar to those of mnsb grown on gaas(111 ) . in contrast to gaas substrates , segregation of substrate atoms through the mnsb film does not occur , and alternative polymorphs of mnsb are absent .

Introduction Experimental Details Results and Discussion Conclusions

the list of ideal characteristics for such materials is long : controllable magnetic states , high curie temperature , long spin diffusion and coherence lengths , well - behaved interfaces to the host semiconductor , suitable resistance - area ( ra ) product for spin injection , engineering compatibility for processing and annealing , and so forth . hence , many magnetic materials are under intensive investigation , including heusler alloys such as co2fesi , diluted magnetic semiconductors , transition metal pnictides ( tmps ) such as mnas , and simple magnetic metals and alloys such as nife . this enables the creation and tailoring of a range of structures including spin light emitting diodes , ferromagnetic optical isolators , and spin valves . the successful growth of n - mnsb epilayers has been reported by several groups using gaas substrates , mainly aimed at magneto - optical applications for which iii v materials are a natural choice . however , there have been relatively few studies of tmp epitaxial growth on si , and we are aware of none on ge . room temperature spin transport in si with spin diffusion lengths more than 100 nm has been established , and similarly efficient spin transport at 300 k in ge has also been demonstrated . a simpler epitaxy is found on gaas(111 ) , namely , mnsb(0001)gaas(111 ) and with mnsb[2110]gaas[110 ] . the bulk lattice parameters of n - mnsb are a = 4.128 and c = 5.789 , which results in an epitaxial mismatch of 3.2% when grown on gaas ( 111 ) surfaces ( the in - plane spacing on the gaas ( 111 ) surface being agaas/2 = 3.995 ) . the use of si(111 ) as a substrate for epitaxial mnsb(0001 ) growth is severely hampered by the large mismatch of 7.0% . the use of ge(111 ) substrates reduces the epitaxial mismatch to a level that is similar to that found for gaas ( 111 ) ( i.e. while the interfacial chemistry of ge with tmp growth is unknown , studies of the growth of mnxge1x thin - films suggest that mn5ge3 has the greatest thermodynamic stability , and layers of mn5ge3(0001 ) have been successfully grown on ge(111 ) substrates by solid phase epitaxy and mbe . a further issue in mnsb growth is the presence of multiple polymorphs in films grown on gaas(111 ) . nowadays , sige / si and ge / si virtual substrates are used for epitaxial growth of a huge variety of group - iv semiconductor heterostructures . because the epilayers are relaxed at their top surfaces , the in - plane lattice parameters relevant to subsequent epitaxial growth match those of equivalent bulk sige or ge crystals . here , we report the mbe growth of mnsb epitaxial films on high quality ge(111 ) virtual substrates , themselves grown on si wafers using reduced pressure chemical vapor deposition ( rpcvd ) . reflection high - energy electron diffraction ( rheed ) , high resolution synchrotron x - ray diffraction ( xrd ) with reciprocal space maps ( rsm ) , x - ray photoelectron spectroscopy ( xps ) , and superconducting quantum interference device ( squid ) magnetometry measurements were all used to characterize the properties of the resulting epilayers . we find that the quality of the mnsb layers is comparable to the best material grown on gaas , and unlike on gaas segregation of substrate material through the mnsb layer is not observed . there is no evidence for the formation of any interfacial alloy layers , and the mnsb grows in a single n - mnsb phase without any of the polymorphs seen on gaas substrates . for virtual substrate production , ge epilayers were grown on 100 mm diameter si(111 ) substrates using a germane ( geh4 ) gaseous precursor diluted in h2 carrier gas within a rpcvd reactor . the density of threading dislocations in the ge / si(111 ) virtual substrates is around 10 cm , and further details on this rpcvd intermediate islanding growth method can be found elsewhere . the top surface of the ge(111 ) virtual substrate is smooth , with a typical root - mean - square surface roughness of 2 nm . schematic layer structure of mnsb(0001 ) on ge / si(111 ) virtual substrates . the function of the ht and lt ge growth is described in the text , while the highlights a surface oxide removal stage after air transfer . the mbe chamber for mnsb growth contains shuttered mn , cr , ni , and sb effusion cells ( the latter having no separate thermal cracking stage ) and is equipped with an electron gun and phosphor screen for monitoring rheed throughout growth . small pieces of ge(111 ) virtual substrates , measuring approximately 8 mm 8 mm , were attached to stainless steel sample plates using spot - welded ta wires . the mounted samples were then cleaned to remove debris and dust by a cycle of acetone , isopropanol , and water rinsing prior to blow drying with dry nitrogen . epilayers of mnsb were then grown using a substrate temperature of ( 410 10)c with the ratio of the sb to mn fluxes set at approximately 6.5:1 . these growth conditions are similar to those used in the epitaxy of mnsb films on gaas substrates . following growth , the samples were exposed to either sb or mn fluxes in order to examine the reconstruction behavior of the samples . the structural characterization of the films was performed using high resolution triple - axis xrd on the x22c beamline . triple axis diffraction scans parallel to the [ 000l ] direction as well as rocking curves were collected at room temperature using a 10 kev x - ray beam and a ge(111 ) analyzer crystal . the surface stoichiometry of the films was investigated using xps using a monochromatized al k source and seven - channel hemispherical analyzer ( omicron gmbh ) . in order to check for the presence of surface - segregated ge and to investigate the native oxides of the mnsb films , magnetic hysteresis loops were obtained using a squid magnetometer ( quantum design inc . ) from a few - millimeter - sized pieces of the mnsb samples . the pieces were manually aligned so that the applied field was either in the plane of the layers or perpendicular to the plane . electrical properties of the mnsb films were measured at room temperature using sprung gold contacts in van der pauw geometry , after a 10 s dilute hcl etch ( 10% by volume ) to minimize contacting problems due to mn oxides . the rheed patterns of the films were monitored throughout growth , and the resulting patterns were recorded for the principal symmetry directions . on the initiation of mnsb growth , this gave way to a weaker ( 1 1 ) pattern with a streak spacing different from that of the ge virtual substrate . the early stages of growth were characterized by the presence of faint transmission diffraction features which faded within 30 s. after this , a sharp and streaky ( 1 1 ) pattern formed , and this pattern was subsequently observed throughout growth . the observed symmetry of the rheed patterns is consistent with the formation of an orientated mnsb(0001 ) layer . following growth , and upon allowing the samples to cool to room temperature , a faint td(1 4 ) pattern developed indicating the formation of an sb - rich surface , and typical rheed patterns for this surface reconstruction are shown in figure 2d , e . the sharpness of the streaks and the presence of multiple laue zones and kikuchi features in these patterns indicate that the near - surface region is highly crystalline with well - ordered domains of both reconstructions present . exposure to an sb flux resulted in the appearance of the ( 1 1 ) and td(1 4 ) periodicities as described above . these reconstructions , and their appearance under more mn - rich or sb - rich conditions , are consistent with those observed in the mnsb(0001)/gaas(111 ) system . panels ( d ) and ( e ) show the postgrowth td(1 4 ) observed on mnsb(0001 ) . panels ( c ) , ( f ) , and ( i ) show the expected reciprocal meshes for the three surfaces . panel ( a ) shows strong diffraction peaks which can be readily to assigned to the ( 111 ) , ( 222 ) , and ( 333 ) reflections from the ge and si components of the virtual substrate ; these occur at qz values precisely corresponding to the bulk lattice parameters . the sharpness and symmetry of the ge peaks highlights the full strain relaxation achieved in the two - stage rpcvd growth . the ge(111 ) and si(111 ) features ( the lowest and central diamond shapes ) are intense and symmetrical in both qz and qx , confirming the high quality of the virtual substrate . triple axis out - of - plane xrd data from a 70 nm mnsb(0001 ) layer on ge(111 ) virtual substrate . in ( a ) , only three families of reflections are present : peaks are labeled on the figure , and the inset shows a expanded view of the lowest order set of reflections . the widths of the rocking curves taken at the n - mnsb 000l reflections were independent of the diffraction order giving a value of 0.4 for the mosaic , which is comparable to the values observed in nisb / gaas epitaxy and in mnsb - gaas films . the inset in figure 3 shows a close - up of the mnsb(0002 ) region covering the 1.75 qz 2.35 range . the measured strain range corresponds to a lattice parameter change of c / cbulk 0.15% through the mnsb layers , again in agreement with mnsb - gaas layers . a williamson - hall analysis was performed for the mnsb reflections which yields a grain size of ( 89 2 ) nm , comparable to the nominal film thickness and indicating high crystalline order throughout the mnsb film . we also note that there are no additional peaks in the xrd spectra obtained from mnsb films grown on ge(111 ) virtual substrates . on gaas(111 ) , both nisb and mnsb often show weak xrd peaks associated with alternative epitaxial orientations such as ( 1101 ) . such orientations have not been observed here , and evidence of polymorphic growth is also absent . when mnsb is grown on gaas ( and , in recent unpublished work , on ingaas ) under similar mnsb mbe growth conditions , diffraction features due to strained c - mnsb(111 ) epitaxial inclusions within the n - mnsb film can be observed . figure 4 shows typical xrd data for a 75 nm thick mnsb film grown on gaas(111 ) in direct comparison with the 70 nm film on ge(111 ) . a broad peak at qz 3.25 is present for the film on gaas , which can be assigned to the ( 222 ) reflection of c - mnsb , with corresponding lattice parameter 6.8(1 ) ( the evolution of the polymorph inclusion strain and grain size as a function of mnsb film thickness on gaas will be detailed in a future paper ) . such polymorph peaks are absent in all the xrd spectra and rsms from our samples grown on the ge virtual substrates , but a more detailed search of the mbe parameter space would be required to definitively rule out polymorphism in the mnsb - ge(111 ) epitaxial system . triple axis out - of - plane xrd data comparing 70 nm mnsb(0001 ) film on ge(111 ) virtual substrate with 75 nm mnsb(0001 ) film on gaas(111 ) . the broad peak at qz 3.25 is due to the presence of cubic c - mnsb inclusions in the film grown on gaas which are absent for the ge substrate . to explore the surface chemistry and to probe any substrate diffusion , panel ( a ) shows the shallow core region ( 2555 ev binding energy ) with the mn 3p , sb 4d , and ga 3d core levels highlighted ( the latter for a comparative growth on gaas ) . this contrasts with mnsb / gaas(111 ) epilayers , where approximately a monolayer of segregated ga is observed at the surface of mnsb but is similar to the case of nisb / gaas(111 ) where no ga segregation occurs . at 30 toa , where the surface sensitivity is enhanced , we ascribe the small signal in the 90 toa data to a piece of exposed ge substrate , probably due to postgrowth scratches or pregrowth defects caused during sample cutting , handling , or ta wire bonding . xps spectra from an as - loaded mnsb film on ge / si(111 ) virtual substrate . for comparison , the dashed line in ( a ) shows a 75 nm film on gaas(111 ) , where a ga 3d peak is present . grew mnsb on gaas(111 ) at lower substrate temperature ( 300 c ) and flux ratio ( 2:1 ) but similar growth rate ( 0.03 nm s compared to our 0.045 nm s ) . they found that a full monolayer of segregated ga was present at the surface , independent of mnsb film thickness above 2 nm up to their maximum thickness of around 25 nm ( figure 5 of ref ( 28 ) ) ; that is , the segregation was very efficient under these growth conditions . using xps we have observed significant surface ga signals for several much thicker mnsb films on gaas(111 ) with growth conditions similar to those used in the present work ( 420 c , flux ratio 6.35:1 , growth rate 0.04 nm s ) . example data are included in panel ( a ) of figure 5 ( dashed line ) for a 75 nm mnsb film on gaas(111 ) . in order to determine the stoichiometry of the surface region , total elemental peak areas were obtained for the mn 3p and sb 4d core levels following subtraction of a linear background . three doublets are required to fit the core level with the doublets corresponding to sb sb , sb mn , and sb ox bonding environments , the corresponding binding energies of the sb 4d5/2 components are given in table 1 . the hysteresis loops in figure 6 show the magnetic response with the applied field perpendicular and parallel to the mnsb c - axis . this agrees with the behavior of both thin ( 0001 ) films and bulk crystals of mnsb . an estimate of the effective moment per mn can be made using the nominal film thickness of 70 nm and the saturation magnetization which gives a value of ( 3.3 0.3)b . these values are consistent with those derived for mnsb(0001 ) films grown on gaas(111 ) , where hc is normally in the range 20200 oe ( easy ) and 300500 oe ( hard ) , depending on film thickness and exact growth conditions . room temperature hall measurements on the mnsb films on ge virtual substrates gave typical hole densities of p = ( 1.3 0.2 ) 10 cm with mobility around 90 cmv s , in agreement with thin film mnsb grown on gaas(111 ) . the ra product of the mnsb / ge interface , important for optimzing spin injection , can not be determined accurately from the present sample set due to the high series resistance of the undoped ge virtual substrate . further work on electrical characterization of the mnsb / ge interface will focus on p - doped ge virtual substrates to match the p - type mnsb contact . the structural , surface - chemical , and magnetic properties of mnsb thin films grown on ge / si(111 ) virtual substrates have been studied . the observed surface reconstructions are broadly consistent with those seen on mnsb(0001 ) films grown on gaas substrates , with rheed indicating that the surfaces are highly ordered and crystalline . xrd shows that the mnsb films exhibit a pure niccolite structure without other polymorphs or orientations . the n - mnsb is orientated ( 0001 ) on the ( 111 ) virtual substrates , with the lattice parameter relaxing to 5.790(1 ) after some tens of nanometers film thickness . magnetometry reveals the expected ferromagnetic behavior with properties similar to those observed on mnsb / gaas films . under the growth conditions employed here ( substrate temperature 410 c , sb / mn flux ratio 6.5:1 , growth rate 0.045 nm s , layer thickness 70 nm ) , surface segregation of ge substrate atoms through the mnsb film is suppressed , and no interfacial alloy layers form . the ability to grow mn pnictides on ge virtual substrates offers great promise for the integration of the tmp class of magnetic materials with si / sige in spintronic applications , especially exploiting spin - polarized hole transport . the growth of single - orientation , single - phase n - mnsb on ge(111 ) thus represents an important step in the realization of hybrid magnetoelectronic applications requiring carefully controlled magnetic properties in the epilayer .

recent systematic studies have shown that mrna and protein abundances within an organism are less correlated than expected both in eukaryotes and prokaryotes . surprisingly , the abundances of orthologous proteins in caenorhabditis elegans and drosophila melanogaster were shown to be highly conserved and correlated better with each other ( rs = 0.79 ) than with the corresponding mrna concentrations within each organism ( rs = 0.44 in c. elegans and rs = 0.36 in d. melanogaster ) . a later analysis of seven different organisms ( two prokaryotes and five eukaryotes ) confirmed that orthologous protein abundances were generally more correlated between organisms than the abundances of mrna and protein within organisms . on the basis of these comparisons , we hypothesized that each protein may exhibit an evolutionarily conserved preference for certain steady - state abundances but that the precise mechanisms employed to set these levels ( i.e. , the relative contributions played by transcriptional , post - transcriptional , translational , and/or degradative processes ) may differ between organisms . thus , the degree to which mrna - level and post - mrna - level processes contribute to the setting of a given protein s steady - state abundance may differ between organisms provided that the final target levels of the protein are properly set . although it is technically difficult to measure the contributions of transcriptional and post - transcriptional processes to establishing mrna and protein abundances inside cells , by measuring steady - state levels under defined conditions it is possible to identify differences in protein and mrna abundances . ( note that we will generally use the term post - transcriptional to indicate the combined effect of all forms of protein abundance regulation acting after the setting of mrna levels , including translational and degradative mechanisms . ) in particular , protein - to - mrna ratios form a simple summary statistic that is useful both for detecting specific genes likely to be regulated post - transcriptionally and for measuring the evolutionary conservation of all post - transcriptional regulatory processes . several previous studies have measured protein and mrna abundances in bacteria . however , protein and mrna abundances in desulfovibrio vulgaris were analyzed under multiple growth conditions ; however , because of low mass spectrometry resolution , the number of proteins consistently observed under multiple conditions allowed only for the investigation of global trends in post - transcriptional regulation . similarly , protein abundances from mycoplasma pneumoniae were measured under different growth conditions and integrated with previously published transcriptome data . the protein and mrna abundances were not correlated within m. pneumoniae , and the authors concluded that post - transcriptional regulation plays a large role in this bacterium . however , it is difficult to compare these results to those of other species because of the small number of m. pneumoniae genes . more advanced techniques , such as single - cell imaging combined with in situ hybridization and transcriptome profiling with short - read sequencing ( rna seq ) , have been used to measure protein and mrna abundances in bacteria . such studies have confirmed that mrna abundances are insufficient to predict protein abundances and that some key regulatory genes , including virulence factors , are post - transcriptionally regulated . however , published studies have used different growth conditions for each bacterium , so it is difficult to determine whether the divergences between protein and mrna abundances are conserved for orthologous genes across organisms . to investigate the divergence of protein and mrna expression when controlling for sequence divergence , we measured the protein and mrna concentrations of 703 genes from two strains of the bacterium pseudomonas aeruginosa , strain pao1 and strain ucbpp - pa14 ( hereafter referred to as pao1 and pa14 ) . although these two strains have highly similar genomes ( 96.7% of total pao1 genes and 90.7% of total pa14 genes are orthologous as one - to - one relations ) , previous studies have reported that they are physiologically quite different , including virulence in various model organisms . both strains were grown under identical conditions , and mrna and protein abundances were measured using the identical microarray and lc ms / ms platforms . because the majority of genes are highly conserved at the nucleotide sequence level between these strains , any measurement bias derived from divergence between orthologous genes is therefore minimized . we further controlled for such bias by limiting our analyses to microarray probes with perfect matches to the corresponding genomes . we confirmed previous observations that protein and mrna abundances between the two strains are more correlated than the protein and mrna abundances within each strain . we further showed that the protein - to - mrna ratios between pao1 and pa14 are well - correlated , suggesting that mechanisms regulating protein abundances downstream of transcription are conserved . despite this high correlation , there were important differences between the two strains , and we showed that in these cases protein and mrna measurements can be used to identify post - transcriptional regulation . p. aeruginosa pao1 ( a subline from barbara iglewski ) and ucbpp - pa14 were grown in 25 ml of synthetic cystic fibrosis sputum medium ( scfm ) in 250 ml flasks , which mimics the nutritional environment of the cystic fibrosis lung , at 37 c with shaking at 250 rpm . cells for transcriptome and proteome analyses were not collected simultaneously , but they were grown under identical conditions . we also performed growth curve assays by diluting overnight cultures grown in scfm to od600 0.01 in 25 ml of scfm ( using 250 ml flasks ) . the od600 was measured every 30 min to generate a growth curve , from which the doubling time ( 3040 min for both strains ) was determined ( data not shown ) . briefly , cultures were mixed 1:1 with rnalater ( ambion ) , an rna - stabilizing agent . rna was isolated using the rneasy mini kit ( qiagen ) , and cdna was prepared for hybridization to affymetrix genechip microarrays ( array identifier pae_g1a ) . genechips were washed , stained , and scanned using an affymetrix fluidics station at the university of iowa dna core facility . these data are available at the ncbi geo database ( accession number gsm546278gsm546281 ) as part of a previous study . we preprocessed microarray cel files with the rma method using the affy package ( version 1.32.1 ) in r ( version 2.14.1 ) with default options . to assign microarray probesets to genes , we downloaded probe sequences from the affymetrix web site ( http://www.affymatrix.com ) and mapped them to both the pao1 genome ( genbank accession number nc_002516.2 ) and the pa14 genome ( genbank accession number nc_008463.1 ) using exonerate ( version 2.20 ) . probes that mapped uniquely were then remapped to p. aeruginosa pao1 and pa14 cdna sequences ( downloaded from pseudocap , version 2009-nov-23 ) . we assigned probe sets to genes if 1214 probes in a probeset were mapped to a single gene . differential expression analysis between the two strains was conducted using an empirical bayesian method implemented in the limma package ( version 3.10.3 ) . genes with greater than 2-fold changes and less than 0.05 false discovery rate ( fdr ) cutoffs were considered differentially expressed . briefly , cells were lysed three times with a french press , and cellular lysate was collected from the supernatant after centrifugation for 20 min at 10 000 rpm . lysis buffer consisted of 25 mm tris - hcl ( ph 7.5 ) , 5 mm dtt , 1.0 mm edta , and 1 cpiops ( calbiochem protease inhibitor cocktail ) . fifty microliters of diluted cell lysate ( 2 mg / ml ; diluted with 50 mm tris - hcl buffer ) was incubated at 55 c for 45 min with 50 l of trifluoroethanol ( tfe ) and 15 mm dithiothreitol ( dtt ) and was then incubated with 55 mm iodoacetamide ( iam ) in the dark for 30 min . after diluting the sample to 1 ml with buffer ( 50 mm trishcl , ph 8.0 ) , 1:50 w / w trypsin was added for a 4.5 h digestion , which was then halted by adding 20 l of formic acid , resulting in 2% v / v . the sample was lyophilized , resuspended with buffer c ( 95% h2o , 5% acetonitrile , and 0.01% formic acid ) , and contaminants removed with c18 tips ( thermo fisher ) . the eluted sample was again lyophilized , resuspended with 120 l of buffer c , and filtered through a microcon-10 filter ( for 45 min at 14 000 g at 4 c ) . each sample was injected five times into an ltq - orbitrap classic mass spectrometer ( thermo electron ; mass resolution 60 000 ; top12 ms2 selection strategy ) , and data were collected in a 090% acetonitrile gradient over 5 h with a agilent zorbax c18 column . the raw files from the ms / ms experiments are available at http://www.marcottelab.org/index.php/pseae_ref.2013 , and the data were also deposited to the proteomexchange under identifier pxd000684 . raw files were searched independently using the p. aeruginosa pao1 and pa14 protein sequence database ( downloaded from the pseudocap database , version 2009-nov-23 ) . the database for each strain contained the same number of randomly shuffled protein sequences as the decoy database . we used bioworks / sequest ( thermo electron ; version 3.3.1 sp1 ) , x!tandem with k - score ( version 2009.10.01.1 labkey and isb , included in tpp 4.3.1 package ) , inspect ( version 20100331 ) , and ms - gfdb ( version 06/16/2011 ) for the database search . we used the same search parameters as described previously except that ms - gfdb was newly added for the current study ( with the settings t 300 ppm c13 1 nnet 0 n 2 ) . then , we combined these results with msblender and considered peptidespectrum matches with an estimated fdr less than 0.01 . subsequently , we calculated apex scores with weighted spectral counts per protein ( using a fdr < 0.01 estimated by msblender ) . because msblender only provides peptide - level probabilities , we set each protein probability as 1.0 using the original apex formulato estimate oi values for each protein , we analyzed both sequest and x!tandem results of each biological replicate with peptideprophet and proteinprophet , and this output was then used with the apex gui program . apex oi values were trained on proteins with proteinprophet probabilities greater than 0.99 using a fdr < 0.01 . we used 25 000 ( arbitrary unit of protein concentration ) as the apex normalization constant . we confirmed that the oi values calculated with sequest results and those from the x!tandem results were well - correlated , and we used the mean of these values as the representative oi values for individual proteins . oi values for both strains and all proteomics analysis data are available at http://www.marcottelab.org/index.php/pseae_ref.2013 . differentially expressed proteins were identified using qspec ( version 2 ; 5000 burn - ins and 20 000 iterations ; a 2-fold change and fdr < 0.05 were required to determine differentially expressed proteins ) on normalized apex scores . genes were omitted from the differential expression analysis if the sum of two strains apex scores was less than 1.0 . for ms1-intensity - based quantification , we analyzed same data with maxquant ( version 1.4.1.2 ) using the default option . to define orthologous genes between pao1 and pa14 , we used inparanoid ( version 4.1 ) with protein sequences of each strain downloaded from pseudocap ( version 2009-nov-23 ) . to analyze the 5 sequences of cdnas containing the shine dalgarno motif , we extracted 50 bp dna fragments around the translational start site of each gene ( from 25 to + 25 bp ) and calculated the gibbs free energy of hybridization to the 3 end of 16s rrna ( 33 bp for pao1 and 31 bp for pa14 ; pao1 differs in having an extra aa at the end ) . this analysis used the modified melt.pl wrapper script in unafold ( version 3.8 ) and the associated hybrid - min program . translationally repressed genes ( those for which no protein was observed in the shotgun proteomics analysis in spite of reasonably high mrna abundance ) were identified on the basis of calculating two mrna abundance distributions for genes with or without accompanying protein observations . we identified the mrna abundance value for which a protein had a 80% chance of being observed by proteomics . to increase the stringency further , we sorted all genes with mrna signals greater than the 80% protein observation threshold , and we removed the genes with the lowest 20% of all mrna abundance ( pao1 cutoff is 691.0 and pa14 cutoff is 758.0 ) . to identify genes with high or low protein - to - mrna ratios ( supporting information table s4 ) , we calculated protein - to - mrna ratios of all 703 gene pairs identified in both strains using the mean of the two replicates and then selected the top 50 and bottom 50 genes of the list . for kegg pathway enrichment analyses , we considered proteins observed in at least one strain or mrnas observed in both pao1 and pa14 as a background for testing pathway enrichment . p values of enrichment were estimated by measuring random chances that equal to or greater than the number of reported genes in each pathway were selected among 10 000 trials . pao1 and pa14 were diluted to od600 0.01 in 25 ml of scfm in 250 ml flasks . cells were grown at 37 c with shaking at 250 rpm until they reached the exponential phase ( od600 0.40.6 ) , at which point they were spread on half of an scfm agar plate using a sterile cotton swab . sterile discs containing 0 , 1.5 , and 2.5 mg / ml chloramphenicol were placed on the plates , which were then incubated overnight at 37 c . to investigate the relationship between protein and mrna abundance in two strains of the same species , we first analyzed the correlation between protein and mrna abundances in p. aeruginosa strains pao1 and pa14 . we were able to detect 5345 mrnas and 1652 proteins in both strains when considering only one - to - one orthologues . although all detectable mrnas and proteins were used to determine differential expression , to focus on the highest accuracy measurements , we analyzed the 703 gene pairs with consistent protein abundances in both strains for all correlation analyses ( summarized in figure 6 ; all values before filtering are available in supporting information table s7 ) . as shown in figure 1 , both the protein and mrna abundances were highly correlated between pao1 and pa14 ( rs = 0.95 for mrna and rs = 0.89 for protein ) , which is better than the correlation observed between protein and mrna abundance within each strain ( rs = 0.64 for pao1 and rs = 0.65 for pa14 ) . in contrast to previous studies showing that the correlation in protein abundance is higher than that of mrna abundance , we observed a better correlation between mrna abundances than for protein abundances . alternatively , previous studies measured mrna abundance in heterogeneous cell types and in organisms grown under different conditions , so it is possible that mechanisms setting transcript abundance are more sensitive than mechanisms setting protein abundance . ( a ) correlation between mrna abundances from p. aeruginosa pao1 and pa14 strains . genes were considered to be differentially expressed ( de ) if they exhibited a greater than 2-fold expression change and an fdr < 0.05 for both protein and mrna . genes without protein observations in either pao1 or pa14 and genes with high variation between biological replicates are omitted ( see supporting information figures s1 and s2 for details ) . a total of 703 genes are presented ( 3 de mrna genes , 72 de protein genes , and 3 de both genes ; see the text for details ) . de mrna : differentially expressed genes at the mrna level but not at the protein level between two strains . de protein : differentially expressed genes at the protein level but not at the mrna level . next , we investigated the differentially expressed genes between pao1 and pa14 . among the 5377 gene pairs with probesets on the affymetrix microarray , 150 genes were significantly differentially expressed at the mrna level ( supporting information table s1 ) . similarly , among the 1730 gene pairs with associated protein abundances , 279 genes were significantly differentially expressed at the protein level ( supporting information table s2 ) . among the 703 genes that we analyzed for correlation between the two strains , 75 gene pairs with differential protein expression and 6 gene pairs with differential mrna expression ( 3 gene pairs with differential expression of both protein and mrna ) were identified . many of the gene pairs that were significantly differentially expressed between the two strains at the protein level ( red circles in figure 1b ) did not show differences at the mrna level ( red circles in figure 1a ) , suggesting that these genes are post - transcriptionally regulated . these differentially expressed proteins between the two strains did not exhibit systematic trends in the correlation between protein and mrna within each strain ( red circles in figure 1c , d ) , so neither the protein nor the mrna abundances themselves are the major factors of inconsistency between them . compared to the genes differentially expressed at the protein level , only a few genes that were differentially expressed at the mrna level were included in this analysis because most of those ( 96 out of 150 ) identified as differentially expressed at the mrna level between pao1 and pa14 were not observed in the shotgun proteomics analysis ( the sum of the apex scores for all four biological samples was less than 1.0 ) , likely because of their low abundance . the higher correlation in both protein and mrna abundances between the two strains , compared to the mrna and protein abundances within each strain , led us to speculate that the protein - to - mrna ratios should also be highly correlated between the strains . as shown in figure 2 , the protein - to - mrna ratios between the strains were indeed highly correlated ( rs = 0.84 ) and were considerably higher than the correlation of mrna to protein within each strain . we also observed that most of the differentially expressed genes at the protein level had different protein - to - mrna ratios ( red circles in figure 2a ) , supporting the notion that the differences in protein abundance between the two closely related strains were attributable to post - transcriptional regulation . protein - to - mrna ratios were calculated as the ratio of the log2-transformed apex score to the log2-transformed microarray signal ( ratio is not log transformed ) . although the two strains showed strong correlation in their protein - to - mrna ratios ( spearman rank correlation 0.84 , p value < 10 ) , most genes with statistically different expression at the protein level also showed a statistically significant difference between the protein - to - mrna ratios from the two strains ( red circles ) . only genes for which we detected proteins in both pao1 and pa14 are presented . de mrna : differentially expressed genes at the mrna level but not at the protein level between two strains . de protein : differentially expressed genes at the protein level but not at the mrna level . de both : differentially expressed genes at both the mrna and protein level . although many studies have used both pao1 and pa14 as reference strains , to our knowledge there has not been a systematic comparison of their molecular characteristics at the transcriptome and proteome levels . because the genetic differences between pao1 and pa14 are minor , we expected that phenotypic differences between the two strains might be predominantly explained by underlying differences in mrna and protein abundances . among the 114 conserved proteins that had significantly different protein abundances between pao1 and pa14 , 14 genes also showed significantly different mrna expression levels ( table 1 ) . a comprehensive list of differentially expressed genes is available in supporting information tables s1 and s2 . a > 2-fold - change and fdr<0.05 were applied to detect significant differences both in mrna and protein . the mrna abundance value represents the average normalized microarray signals of two biological replicates . differentially expressed genes at both the protein and mrna levels included well - known virulence and antibiotic resistance genes , such as algr and the pqs operon , and the mexef oprn operon . it has been shown that overexpression of mexef oprn increases resistance to chloramphenicol . we therefore hypothesized that pao1 , which shows higher expression of mexef oprn , may exhibit higher resistance to chloramphenicol compared to pa14 . to test this hypothesis , we used a disk diffusion assay to measure growth inhibition by chloramphenicol . as expected , the zones of inhibition were larger for pa14 with increasing chloramphenicol concentrations , whereas pao1 growth was minimally inhibited by chloramphenicol ( figure 3 ) . additionally , genes involved in the metabolism of several amino acids were differentially expressed between pao1 and pa14 ( table 2 ) , likely highlighting different metabolic characteristics of the two strains . . oprn operon explains differential chloramphanicol resistance in p. aeruginosa pao1 and pa14 strains . ( a ) protein expression levels of mexef oprn in pao1 and pa14 . two biological replicates are plotted for each strain , as shown by the same color bars . in pa14 , we could not detect mexf or oprn , and the protein abundance score for mexe was low ( 0.2 ) . ( b ) mrna levels of mexef oprn in pao1 and pa14 . two biological replicates are plotted for each strain , as shown by the same color bars . a hypergeometric test was used to evaluate significance of enrichment with 10 000-fold bootstrapping . two pathways marked in bold ( nucleotide excision repair and pyrimidine metabolism ) were enriched in genes showing different protein abundances but not differences in mrna abundances or shine dalgarno - binding free energies . we identified 114 differentially expressed proteins between pao1 and pa14 ( supporting information table s2 ) . fifty six of these proteins ( 49% ) also had concordant mrna levels to proteins , although only 14 were significant according to our statistical cutoffs . higher mrna levels most likely explain the differences in protein levels , suggesting that the remaining 58 genes have a different post - transcriptional regulation mechanism . in addition to differences in the gene repertoires between the two strains , these genes may prove to be a useful resource for choosing reference strains for p. aeruginosa experiments . to determine whether differential protein - to - mrna ratios for the remaining 58 genes can be explained by ribosomal binding energy , we analyzed the shine dalgarno motif under the assumption that high - affinity ribosome binding may correspond to increased translation efficiency ( supporting information table s5 ) . to normalize for the effect of differential mrna abundance , we compared protein - to - mrna ratios instead of protein abundance . differences in protein - to - mrna ratios for 14 gene pairs ( 12% ) could be accounted for by differences in ribosome binding energy . we could not identify the cause of the remaining 39% of gene pairs with differential protein abundances ( supporting information table s6 ) . in light of the high sequence similarity of orthologous genes between pao1 and pa14 , we suspect that intrinsic sequence features , such as sequence length , are unlikely , in general , to explain these differences . also , our group reported that various sequence signatures and mrna concentrations can only explain 66% of protein abundances in a human cell line ; thus , these sequences may be the target of extrinsic post - transcriptional regulation such as small noncoding rnas or rna - binding proteins . finally , the systematic measurement of protein and rna abundances allowed us to select specific candidate genes for translational regulation . in particular , if protein abundance is solely proportional to mrna abundance and the protein detection is mainly governed by abundance , then genes with high mrna signal but undetected protein should be candidates for translational repression . to search for such cases , we identified those genes that were not observed in the shotgun proteomics experiment but had reasonably high mrna expression levels . by comparing mrna levels of genes for which we detected protein to those of which we did not , we identified an mrna abundance threshold corresponding to an 80% chance of protein detection ( figure 4 ) . to enrich for true cases of translational repression further , we additionally filtered out the 20% of genes with the lowest mrna abundances ( supporting information table s4 ) . of the 181 genes identified as translationally repressed in this analysis , 97 genes showed significant repression at the protein level in both pao1 and pa14 . candidates for translation repression can be selected from histograms of mrna concentrations with and without corresponding protein observations : ( a ) pao1 and ( b ) pa14 . the gray line represents the frequencies of genes with a microarray signal ( log2-transformed ) for which proteins were not observed in shotgun proteomics . the blue line represents the frequencies of genes with a microarray signal for which we also measured the corresponding protein . some genes with high microarray signals are still not observed at the protein level ( gray line to the right of the red mark ) and may be translationally repressed or targeted for degradation . to identify candidates for such regulation , we considered mrnas for which protein was not observed with abundances above the red line ( corresponding to an 80% chance of detecting protein ) . to increase the stringency further , we sorted all genes with microarray signals greater than the cutoff and discarded the bottom 20% of the list . in total , 181 genes were identified as putatively translationally repressed , and 97 showed in both pao1 and pa14 ( supporting information table s4 ) . because it is possible that low detectability in shotgun proteomics can produce this bias , we corrected for mass spectrometry detectability using the apex method . using kegg pathway enrichment analysis , we identified ribosomal proteins ( pa3745 , pa4432 , and pa5049 ) with significantly high protein - to - mrna ratios . we also found that genes involved in terpenoid backbone biosynthesis ( pa3627 and pa4557 ) , nucleotide excision repair ( pa1529 and pa4234 ) , and one carbon ( folate ) metabolism ( pa0944 and pa1843 ) showed low protein - to - mrna ratios . genes involved in oxidative phosphorylation ( pa1582|sdhd , pa2643|nuoh , pa2645|nuoj , pa2646|nuok , pa2648|nuim , and pa4430 ) had reasonably high mrna levels , but we did not detect protein for them , suggesting that these genes may be translationally repressed . if most gene regulation occurred at the level of transcription and rna degradation , then we might expect protein abundance to be directly proportional to mrna abundance , as a constant level of protein is translated from mrna . however , recent studies show that variation in mrna concentrations can only explain a fraction ( one - third to one - half ) of the resulting variation in final protein concentrations . on the basis of the observed conservation of protein - to - mrna ratios across the two closely related p. aeruginosa strains , we argue that these ratios can predict post - transcriptional regulation . to model this , first we assumed a linear relationship between log - transformed protein and mrna concentrations within each organism ( figures 1 and 3 ) . with the additional assumption of steady state for both protein and mrna abundances ( degradation and synthesis are not considered separately , and they are assumed to be constant across the cell population over time ) , their linear relationship can be described aswhere p , m , and represent the protein abundance , the mrna abundance , and the random error term , respectively . here , speciesx is the global translational efficiency in species x , representing how many proteins can be produced from a given mrna amount . it should be noted that speciesx does not account for dynamic features in translation , such as protein and mrna degradation and time delay in translation . if gene - specific post - transcriptional regulation is negligible , then global translation efficiency should be dominant , resulting in a constant protein - to - mrna ratio for all genes in a given species ( speciesx ) . however , as shown in figure 2 , the protein - to - mrna ratios of pao1 and pa14 were not a constant , and those of orthologous genes in two strains were highly correlated to each other . on the basis of these observations , we revised the equation as followshere , speciesx , geney is the gene y - specific post - transcriptional regulation factor in species x , representing the adjustment of translation for individual genes to their mrna levels . we concluded that gene - specific post - transcriptional regulation factors , speciesx , geney , were conserved between pao1 and pa14 . indeed , as shown in figure 5 , we can predict protein abundance more accurately when we incorporate these gene - specific post - transcriptional regulation factors with mrna abundance . it should be noted that these post - transcriptional regulation factors will vary depending on environmental conditions and related post - transcriptional regulators such as noncoding rna and rna - binding proteins . although our model does not distinguish translation and degradation separately , the specific parameter speciesx and gene - specific factor speciesx , geney incorporate these processes implicitly . protein abundance is well - predicted from mrna abundance and conserved protein - to - mrna ratio . ( a ) pao1 predicted protein abundance was calculated with pao1 mrna abundance multiplied by pa14 protein - to - mrna ratio . ( b ) similarly , pa14 predicted protein abundance was calculated with pa14 mrna abundance multiplied by pao1 protein - to - mrna ratio . compared to figure 1 , it is clear that gene - specific protein - to - mrna ratios help to predict protein abundance from mrna abundance more accurately . in this study , we measured the correlation between mrna and protein abundances of 703 orthologous gene pairs in two p. aeruginosa reference strains , pao1 and pa14 ( summarized in figure 6 ) . ( fifteen such genes with significantly different protein abundance between pao1 and pa14 without accompanying differences in mrna abundance or 5 sequence are given in table 3 . ) similar to previous studies , we observed that mrna and protein abundances of orthologous genes are less well correlated within each strain ( rs = 0.640.65 ) than the protein and mrna abundances between the two strains ( rs = 0.89 and 0.95 , respectively ) . because we examined mrna and protein levels in two p. aeruginosa strains grown under identical conditions , we were able to focus the analysis on evolutionary differences ( e.g. , sequence divergence ) while controlling for the influence of different environments . ( a ) out of 5345 genes with one - to - one orthology between pao1 and pa14 , we measured proteome and transcriptome abundance of 703 genes with highly stringent criteria of reproducibility between biological replicates . ( b ) we observed that protein ( spearman rank correlation 0.89 ) and mrna ( spearman rank correlation 0.95 ) abundances are highly conserved , much more than those abundances within each strain . ( c ) out of 114 genes showing significantly different protein levels between pao1 and pa14 , about half of them ( 56 genes ) showed different mrna abundances and 43 genes showed different 5 sequences ( assuming that different 5 sequence may affect translation repression efficiency ) . however , fewer than half of 5 sequence differences were relevant to differences in the shine fifteen differentially expressed proteins with identical 5 sequences and similar mrna abundances may be regulated by strand - specific post - transcriptional mechanisms ( table 3 ) . the mrna abundance value represents the average normalized microarray signals of two biological replicates . in contrast to previous studies , we observed that for the two p. aeruginosa strains mrna abundances are more conserved than protein abundances ( 0.95 and 0.89 in comparison to roughly 0.01 and 0.57 from other studies ) . one possible explanation for this is that transcriptional regulation is more sensitive to environmental conditions than post - transcriptional regulation . a high correlation of mrna abundances would be observed only when the data are collected under very similar conditions , as was done in this study . alternatively , in previous studies , differences in microarray platforms and sequence hybridization may be larger than expected , accounting for the observed lower correlation of mrna concentrations . of course , the higher correlation in mrna abundances that we observed compared to protein abundances may also be explained by lower variation between biological replicates in the mrna measurements as compared to the protein measurements . however , after filtering out inconsistently observed genes between biological replicates ( see supporting information figures s1 and s2 for details ) , we found a very high correlation ( spearman rank correlation > 0.95 ) between replicates for both mrna and protein abundances , so it is unlikely that the experimental variance between mrna and protein abundances significantly affected our results . thus , the apparent post - transcriptional buffering of divergence of mrna concentrations across organisms does not seem to hold true when accounting for any differences in environmental conditions and focusing simply on two very closely related strains : both transcriptional and post - transcriptional regulation appears to diverge between the two p. aeruginosa strains and has additive effects on the final protein concentrations . it is also possible that a small group of highly abundant genes , such as housekeeping genes , may dominate the interspecies correlation patterns we observed in this study . unlike studies between more distant organisms , such as fly and worm , where the proportion of housekeeping genes may be higher among the conserved genes , here we analyzed two bacterial strains of the same species and do not expect housekeeping genes to dominate the set of orthologues . also , as shown in figure 1 , the correlation patterns are quite consistent over a wide range of abundance both in protein and mrna . thus , the correlation is unlikely to derive primarily from highly abundant housekeeping genes . we also identified genes with significantly high or low protein - to - mrna ratios ( supporting information table s3 ) and genes with high mrna abundances that we did not detect in our proteomics experiment ( supporting information table s4 ) . low protein - to - mrna ratios or translational repression may be alternatively explained by low detectability . shotgun proteomics can introduce certain biases because of inefficient peptide ionization and unavailability of informative tryptic peptides , which could potentially account for some of these proteins . one possible scenario is that post - translational protein modifications could be missed when searching the mass spectrometry data : proteins might appear to be at lower concentration when , in reality , they are post - translationally modified , which hinders their identification . however , most of the proteins we observed in this study were identified by two or more peptides ( 653 pao1 proteins and 664 pa14 proteins , respectively , out of 703 one - to - one protein pairs used in the correlation analysis ) , making it unlikely that low protein - to - mrna ratios were caused by systematic bias because of unidentified modified peptides . another possibility to explain translational repression is a systematic bias of mass spectrometry against certain types of proteins , such as membrane proteins . although we identified more proteins localized in the cytoplasm than those localized in other cellular compartments , we found no significant bias in protein - to - mrna ratios according to localization ( supporting information figure 3 ) , confirming that the translational repression we observed here was not due to technical bias . to validate our findings in the correlation analysis further , we reanalyzed all proteomics data using an alternative label - free quantification method based on ms1 intensities and confirmed the same trends for protein - to - mrna ratios to be more conserved across species than for protein abundances to be correlated with mrna abundances within species ( supporting information figure 5 and supporting information table 8) . recently developed techniques improving detectability and precision of both the proteome and transcriptome , such as selected reaction monitoring ( srm ) and rna seq , would be helpful to minimize the potential for systematic bias further , as has been already shown in several studies of single organisms . also , techniques discriminating newly synthesized mrnas and proteins by labeling should allow the contributions of different post - transcriptional regulatory mechanisms to the observed divergence of mrna and protein abundances to be determined . interestingly , several virulence genes in p. aeruginosa were differentially expressed at the protein level under our growth conditions . oprn operon was more highly expressed in pao1 at both the mrna and protein levels . we confirmed that pao1 is more resistant to chloramphenicol , a substrate of mexef oprn efflux pump , than pa14 . although mext is a known regulator of the mexef oprn operon , both its mrna and protein expression was low under our growth conditions . thus , the high expression of this operon in pao1 may be independent of mext expression . p. aeruginosa produces 4-quinolones , a structurally diverse group of small molecules that act as cell cell signals and antibiotics . the products required for 4-quinolone biosynthesis and regulation are encoded by the pqs operons ( pqsa e , pqsh , and pqsl ) . notably , pa14 expressed all of them at higher levels than pao1 ( supporting information figure s4 ) , confirming that they are differentially regulated between pao1 and pa14 in scfm . by testing for overrepresented kegg pathways among the differentially expressed proteins ( p value < 0.05 estimated by resampling ) , we also observed significant differences in several key metabolic pathways including amino acid metabolism , which can impact 4-quinolone levels because of the shared metabolic precursors between these pathways . given the low mrna abundances for genes in this pathway in pao1 , translational repression appears to be unlikely to explain these differences ; rather , pa14 appears to have upregulated this pathway relative to pao1 at the transcript level . our approach to measure protein and mrna abundances between closely related organisms complements previous studies on protein and mrna concentrations in a single organism under different conditions . although these studies report similarly discordant tendencies of protein and mrna abundances and the importance of post - transcriptional regulation , the detailed mechanisms of post - transcriptional regulation are still unclear . mechanisms of translational suppression can include altering the mrna structure of translation initiation sites and the presence of cis - encoded antisense rnas . recently , several studies have reported the genome - wide effect of trans - acting post - transcriptional regulation in bacteria , such as small rnas and rna - binding proteins , which can impact rna synthesis , stability , sequestration , and degradation . in the future , it will be interesting to investigate the contribution of these regulatory mechanisms to the observed discordance between protein and mrna levels in various organisms .

recent studies have shown that the concentrations of proteins expressed from orthologous genes are often conserved across organisms and to a greater extent than the abundances of the corresponding mrnas . however , such studies have not distinguished between evolutionary ( e.g. , sequence divergence ) and environmental ( e.g. , growth condition ) effects on the regulation of steady - state protein and mrna abundances . here , we systematically investigated the transcriptome and proteome of two closely related pseudomonas aeruginosa strains , pao1 and pa14 , under identical experimental conditions , thus controlling for environmental effects . for 703 genes observed by both shotgun proteomics and microarray experiments , we found that the protein - to - mrna ratios are highly correlated between orthologous genes in the two strains to an extent comparable to protein and mrna abundances . in spite of this high molecular similarity between pao1 and pa14 , we found that several metabolic , virulence , and antibiotic resistance genes are differentially expressed between the two strains , mostly at the protein but not at the mrna level . our data demonstrate that the magnitude and direction of the effect of protein abundance regulation occurring after the setting of mrna levels is conserved between bacterial strains and is important for explaining the discordance between mrna and protein abundances .

Introduction Materials and Methods Results Discussion

recent systematic studies have shown that mrna and protein abundances within an organism are less correlated than expected both in eukaryotes and prokaryotes . a later analysis of seven different organisms ( two prokaryotes and five eukaryotes ) confirmed that orthologous protein abundances were generally more correlated between organisms than the abundances of mrna and protein within organisms . on the basis of these comparisons , we hypothesized that each protein may exhibit an evolutionarily conserved preference for certain steady - state abundances but that the precise mechanisms employed to set these levels ( i.e. thus , the degree to which mrna - level and post - mrna - level processes contribute to the setting of a given protein s steady - state abundance may differ between organisms provided that the final target levels of the protein are properly set . although it is technically difficult to measure the contributions of transcriptional and post - transcriptional processes to establishing mrna and protein abundances inside cells , by measuring steady - state levels under defined conditions it is possible to identify differences in protein and mrna abundances . ( note that we will generally use the term post - transcriptional to indicate the combined effect of all forms of protein abundance regulation acting after the setting of mrna levels , including translational and degradative mechanisms . ) in particular , protein - to - mrna ratios form a simple summary statistic that is useful both for detecting specific genes likely to be regulated post - transcriptionally and for measuring the evolutionary conservation of all post - transcriptional regulatory processes . several previous studies have measured protein and mrna abundances in bacteria . however , protein and mrna abundances in desulfovibrio vulgaris were analyzed under multiple growth conditions ; however , because of low mass spectrometry resolution , the number of proteins consistently observed under multiple conditions allowed only for the investigation of global trends in post - transcriptional regulation . the protein and mrna abundances were not correlated within m. pneumoniae , and the authors concluded that post - transcriptional regulation plays a large role in this bacterium . more advanced techniques , such as single - cell imaging combined with in situ hybridization and transcriptome profiling with short - read sequencing ( rna seq ) , have been used to measure protein and mrna abundances in bacteria . however , published studies have used different growth conditions for each bacterium , so it is difficult to determine whether the divergences between protein and mrna abundances are conserved for orthologous genes across organisms . to investigate the divergence of protein and mrna expression when controlling for sequence divergence , we measured the protein and mrna concentrations of 703 genes from two strains of the bacterium pseudomonas aeruginosa , strain pao1 and strain ucbpp - pa14 ( hereafter referred to as pao1 and pa14 ) . although these two strains have highly similar genomes ( 96.7% of total pao1 genes and 90.7% of total pa14 genes are orthologous as one - to - one relations ) , previous studies have reported that they are physiologically quite different , including virulence in various model organisms . both strains were grown under identical conditions , and mrna and protein abundances were measured using the identical microarray and lc ms / ms platforms . because the majority of genes are highly conserved at the nucleotide sequence level between these strains , any measurement bias derived from divergence between orthologous genes is therefore minimized . we confirmed previous observations that protein and mrna abundances between the two strains are more correlated than the protein and mrna abundances within each strain . we further showed that the protein - to - mrna ratios between pao1 and pa14 are well - correlated , suggesting that mechanisms regulating protein abundances downstream of transcription are conserved . despite this high correlation , there were important differences between the two strains , and we showed that in these cases protein and mrna measurements can be used to identify post - transcriptional regulation . to define orthologous genes between pao1 and pa14 , we used inparanoid ( version 4.1 ) with protein sequences of each strain downloaded from pseudocap ( version 2009-nov-23 ) . translationally repressed genes ( those for which no protein was observed in the shotgun proteomics analysis in spite of reasonably high mrna abundance ) were identified on the basis of calculating two mrna abundance distributions for genes with or without accompanying protein observations . to identify genes with high or low protein - to - mrna ratios ( supporting information table s4 ) , we calculated protein - to - mrna ratios of all 703 gene pairs identified in both strains using the mean of the two replicates and then selected the top 50 and bottom 50 genes of the list . to investigate the relationship between protein and mrna abundance in two strains of the same species , we first analyzed the correlation between protein and mrna abundances in p. aeruginosa strains pao1 and pa14 . as shown in figure 1 , both the protein and mrna abundances were highly correlated between pao1 and pa14 ( rs = 0.95 for mrna and rs = 0.89 for protein ) , which is better than the correlation observed between protein and mrna abundance within each strain ( rs = 0.64 for pao1 and rs = 0.65 for pa14 ) . in contrast to previous studies showing that the correlation in protein abundance is higher than that of mrna abundance , we observed a better correlation between mrna abundances than for protein abundances . de mrna : differentially expressed genes at the mrna level but not at the protein level between two strains . de protein : differentially expressed genes at the protein level but not at the mrna level . next , we investigated the differentially expressed genes between pao1 and pa14 . among the 5377 gene pairs with probesets on the affymetrix microarray , 150 genes were significantly differentially expressed at the mrna level ( supporting information table s1 ) . similarly , among the 1730 gene pairs with associated protein abundances , 279 genes were significantly differentially expressed at the protein level ( supporting information table s2 ) . among the 703 genes that we analyzed for correlation between the two strains , 75 gene pairs with differential protein expression and 6 gene pairs with differential mrna expression ( 3 gene pairs with differential expression of both protein and mrna ) were identified . many of the gene pairs that were significantly differentially expressed between the two strains at the protein level ( red circles in figure 1b ) did not show differences at the mrna level ( red circles in figure 1a ) , suggesting that these genes are post - transcriptionally regulated . these differentially expressed proteins between the two strains did not exhibit systematic trends in the correlation between protein and mrna within each strain ( red circles in figure 1c , d ) , so neither the protein nor the mrna abundances themselves are the major factors of inconsistency between them . compared to the genes differentially expressed at the protein level , only a few genes that were differentially expressed at the mrna level were included in this analysis because most of those ( 96 out of 150 ) identified as differentially expressed at the mrna level between pao1 and pa14 were not observed in the shotgun proteomics analysis ( the sum of the apex scores for all four biological samples was less than 1.0 ) , likely because of their low abundance . the higher correlation in both protein and mrna abundances between the two strains , compared to the mrna and protein abundances within each strain , led us to speculate that the protein - to - mrna ratios should also be highly correlated between the strains . as shown in figure 2 , the protein - to - mrna ratios between the strains were indeed highly correlated ( rs = 0.84 ) and were considerably higher than the correlation of mrna to protein within each strain . we also observed that most of the differentially expressed genes at the protein level had different protein - to - mrna ratios ( red circles in figure 2a ) , supporting the notion that the differences in protein abundance between the two closely related strains were attributable to post - transcriptional regulation . protein - to - mrna ratios were calculated as the ratio of the log2-transformed apex score to the log2-transformed microarray signal ( ratio is not log transformed ) . although the two strains showed strong correlation in their protein - to - mrna ratios ( spearman rank correlation 0.84 , p value < 10 ) , most genes with statistically different expression at the protein level also showed a statistically significant difference between the protein - to - mrna ratios from the two strains ( red circles ) . de mrna : differentially expressed genes at the mrna level but not at the protein level between two strains . de protein : differentially expressed genes at the protein level but not at the mrna level . although many studies have used both pao1 and pa14 as reference strains , to our knowledge there has not been a systematic comparison of their molecular characteristics at the transcriptome and proteome levels . because the genetic differences between pao1 and pa14 are minor , we expected that phenotypic differences between the two strains might be predominantly explained by underlying differences in mrna and protein abundances . among the 114 conserved proteins that had significantly different protein abundances between pao1 and pa14 , 14 genes also showed significantly different mrna expression levels ( table 1 ) . differentially expressed genes at both the protein and mrna levels included well - known virulence and antibiotic resistance genes , such as algr and the pqs operon , and the mexef oprn operon . additionally , genes involved in the metabolism of several amino acids were differentially expressed between pao1 and pa14 ( table 2 ) , likely highlighting different metabolic characteristics of the two strains . in pa14 , we could not detect mexf or oprn , and the protein abundance score for mexe was low ( 0.2 ) . in addition to differences in the gene repertoires between the two strains , these genes may prove to be a useful resource for choosing reference strains for p. aeruginosa experiments . to determine whether differential protein - to - mrna ratios for the remaining 58 genes can be explained by ribosomal binding energy , we analyzed the shine dalgarno motif under the assumption that high - affinity ribosome binding may correspond to increased translation efficiency ( supporting information table s5 ) . to normalize for the effect of differential mrna abundance , we compared protein - to - mrna ratios instead of protein abundance . differences in protein - to - mrna ratios for 14 gene pairs ( 12% ) could be accounted for by differences in ribosome binding energy . in light of the high sequence similarity of orthologous genes between pao1 and pa14 , we suspect that intrinsic sequence features , such as sequence length , are unlikely , in general , to explain these differences . of the 181 genes identified as translationally repressed in this analysis , 97 genes showed significant repression at the protein level in both pao1 and pa14 . using kegg pathway enrichment analysis , we identified ribosomal proteins ( pa3745 , pa4432 , and pa5049 ) with significantly high protein - to - mrna ratios . we also found that genes involved in terpenoid backbone biosynthesis ( pa3627 and pa4557 ) , nucleotide excision repair ( pa1529 and pa4234 ) , and one carbon ( folate ) metabolism ( pa0944 and pa1843 ) showed low protein - to - mrna ratios . on the basis of the observed conservation of protein - to - mrna ratios across the two closely related p. aeruginosa strains , we argue that these ratios can predict post - transcriptional regulation . with the additional assumption of steady state for both protein and mrna abundances ( degradation and synthesis are not considered separately , and they are assumed to be constant across the cell population over time ) , their linear relationship can be described aswhere p , m , and represent the protein abundance , the mrna abundance , and the random error term , respectively . if gene - specific post - transcriptional regulation is negligible , then global translation efficiency should be dominant , resulting in a constant protein - to - mrna ratio for all genes in a given species ( speciesx ) . however , as shown in figure 2 , the protein - to - mrna ratios of pao1 and pa14 were not a constant , and those of orthologous genes in two strains were highly correlated to each other . protein abundance is well - predicted from mrna abundance and conserved protein - to - mrna ratio . ( a ) pao1 predicted protein abundance was calculated with pao1 mrna abundance multiplied by pa14 protein - to - mrna ratio . ( b ) similarly , pa14 predicted protein abundance was calculated with pa14 mrna abundance multiplied by pao1 protein - to - mrna ratio . compared to figure 1 , it is clear that gene - specific protein - to - mrna ratios help to predict protein abundance from mrna abundance more accurately . in this study , we measured the correlation between mrna and protein abundances of 703 orthologous gene pairs in two p. aeruginosa reference strains , pao1 and pa14 ( summarized in figure 6 ) . similar to previous studies , we observed that mrna and protein abundances of orthologous genes are less well correlated within each strain ( rs = 0.640.65 ) than the protein and mrna abundances between the two strains ( rs = 0.89 and 0.95 , respectively ) . because we examined mrna and protein levels in two p. aeruginosa strains grown under identical conditions , we were able to focus the analysis on evolutionary differences ( e.g. ( a ) out of 5345 genes with one - to - one orthology between pao1 and pa14 , we measured proteome and transcriptome abundance of 703 genes with highly stringent criteria of reproducibility between biological replicates . ( c ) out of 114 genes showing significantly different protein levels between pao1 and pa14 , about half of them ( 56 genes ) showed different mrna abundances and 43 genes showed different 5 sequences ( assuming that different 5 sequence may affect translation repression efficiency ) . in contrast to previous studies , we observed that for the two p. aeruginosa strains mrna abundances are more conserved than protein abundances ( 0.95 and 0.89 in comparison to roughly 0.01 and 0.57 from other studies ) . of course , the higher correlation in mrna abundances that we observed compared to protein abundances may also be explained by lower variation between biological replicates in the mrna measurements as compared to the protein measurements . however , after filtering out inconsistently observed genes between biological replicates ( see supporting information figures s1 and s2 for details ) , we found a very high correlation ( spearman rank correlation > 0.95 ) between replicates for both mrna and protein abundances , so it is unlikely that the experimental variance between mrna and protein abundances significantly affected our results . thus , the apparent post - transcriptional buffering of divergence of mrna concentrations across organisms does not seem to hold true when accounting for any differences in environmental conditions and focusing simply on two very closely related strains : both transcriptional and post - transcriptional regulation appears to diverge between the two p. aeruginosa strains and has additive effects on the final protein concentrations . we also identified genes with significantly high or low protein - to - mrna ratios ( supporting information table s3 ) and genes with high mrna abundances that we did not detect in our proteomics experiment ( supporting information table s4 ) . low protein - to - mrna ratios or translational repression may be alternatively explained by low detectability . however , most of the proteins we observed in this study were identified by two or more peptides ( 653 pao1 proteins and 664 pa14 proteins , respectively , out of 703 one - to - one protein pairs used in the correlation analysis ) , making it unlikely that low protein - to - mrna ratios were caused by systematic bias because of unidentified modified peptides . although we identified more proteins localized in the cytoplasm than those localized in other cellular compartments , we found no significant bias in protein - to - mrna ratios according to localization ( supporting information figure 3 ) , confirming that the translational repression we observed here was not due to technical bias . to validate our findings in the correlation analysis further , we reanalyzed all proteomics data using an alternative label - free quantification method based on ms1 intensities and confirmed the same trends for protein - to - mrna ratios to be more conserved across species than for protein abundances to be correlated with mrna abundances within species ( supporting information figure 5 and supporting information table 8) . in the future , it will be interesting to investigate the contribution of these regulatory mechanisms to the observed discordance between protein and mrna levels in various organisms .

conceptual knowledge for objects comprises a diverse set of information about their sensory qualities , motor plans and verbal associations . how are these disparate sources of information linked to form a concept ? according to one influential view , originally proposed by wernicke ( wernicke , 1900 ; as cited in eggert , 1977 ) , conceptual knowledge for objects arises from the co - activation of their sensory - motor properties within a network of modality - specific processing regions that are widely distributed throughout the cortex ( barsalou , 2008 ; martin , 2007 ; pulvermuller , 2001 ) . this approach makes two key predictions concerning the breakdown of conceptual knowledge under brain damage . first , damage to a single , modality - specific region should give rise to knowledge deficits that disproportionately affect properties in that modality and , by extension , categories of objects for which the affected modality is particularly central ( capitani , laiacona , mahon , & caramazza , 2003 ; mahon & caramazza , 2009 ; warrington & shallice , 1984 ) . so , for example , damage to regions of inferior parietal cortex involved in representing skilled actions should impair knowledge of how objects are manipulated and lead to a disproportionate deficit for tools ( buxbaum & saffran , 2002 ) . the second prediction concerns global , pan - modal conceptual impairments . according to wernicke and his modern counterparts , these should only occur as a result of global cortical damage , because only damage to all of the modality - specific regions would be sufficient to produce a global impairment . sd patients suffer from a global conceptual knowledge deficit that affects all categories of object and word ( hoffman & lambon ralph , 2011 ; lambon ralph , lowe , & rogers , 2007 ) and all sensory - motor modalities ( bozeat , lambon ralph , patterson , garrard , & hodges , 2000 ; bozeat , lambon ralph , patterson , & hodges , 2002 ; luzzi et al . , 2007 ; piwnica - worms , omar , hailstone , & warren , 2010 ) , yet the cerebral atrophy and hypometabolism that gives rise to this debilitating impairment is not global : it is focused bilaterally on the anterior ventrolateral and polar portions of the temporal lobes ( galton et al . evidence from functional neuroimaging ( binney , embleton , jefferies , parker , & lambon ralph , 2010 ; visser & lambon ralph , 2011 ) and transcranial magnetic stimulation ( pobric , jefferies , & lambon ralph , 2007 ; pobric , jefferies , & lambon ralph , 2010 ) in neurologically - intact participants confirms that ventrolateral anterior temporal lobe ( atl ) areas are involved in all forms of conceptual processing irrespective of the modality of the information or the category of object probed . the crucial role of this area in transmodal semantic representation also fits with recent in vivo tractography data demonstrating the convergence of multiple white - matter pathways into the atl . such results indicate that this region 's structural connectivity is ideal for blending different sources of verbal and nonverbal information into integrated , coherent concepts ( binney , parker , & lambon ralph , 2012 ) . to account for the global , pan - modal involvement of the ventrolateral atls in conceptual knowledge , we have developed an alternative framework for conceptual knowledge termed the hub - and - spoke model ( lambon ralph , sage , jones , & mayberry , 2010 ; patterson , nestor , & rogers , 2007 ; pobric et al . this model holds that in addition to modality - specific sources of information ( spokes ) and their inter - connections , representation of conceptual knowledge requires an integrative hub . the hub uses information from the modality - specific spoke regions to develop modality - invariant , conceptual representations that capture deeper patterns of conceptual similarity across all sensory - motor and verbal modalities . these integrated representations are necessary because similarity in any particular sensory - motor domain is , at best , only a partial guide to conceptual similarity ( dilkina & lambon ralph , 2013 ; lambon ralph et al . , 2010 ; smith & medin , 1981 ) . for example , though apples and bananas have different shapes , colours and tactile properties and are manipulated in different ways , the conceptual system must be able to recognise that they are similar types of object . in addition , true conceptual representation requires the integration of properties that are experienced in different times and situations , and representation of the complex , non - linear relationships between the concept 's verbal and nonverbal modality - specific properties and its conceptual significance ( see lambon ralph et al . the hub - and - spoke framework holds that the atl hub provides this critical aspect of conceptual representation through the formation of representations that integrate information from all sensory - motor - verbal domains . when this region is damaged , as in sd , the result is a breakdown in the complex boundaries that define different concepts , such that semantic decisions come to be made on the basis of superficial characteristics rather than their deeper conceptual properties . for example , sd patients may reject emu as an example of a bird but simultaneously over - extend the concept to accept butterfly previous work on the function of the ventrolateral atls has focused on their role in representing existing knowledge and its progressive deterioration as a result of atl atrophy in sd ( e.g. , binney et al . , 2010 ; rogers et al . , the hub - and - spoke framework also predicts that the atls play a key role in the acquisition of novel concepts ( rogers & mcclelland , 2004 ) . the response properties of anterior inferior temporal neurons change as monkeys learn novel associations between visual stimuli , suggesting a role for this region in the acquisition of concepts ( albright , 2012 ) . in the present study , we tested this hypothesis in humans by studying acquisition of new conceptual knowledge in patients with sd . the hub - and - spoke model predicts that the atls are critical for integrating the various sensory features of an object into a unified , coherent conceptual representation that can be generalised to new exemplars . we tested this prediction by training sd patients to recognise novel visual stimuli as members of two categories . previous research has shown that sd patients are able to apply well - defined rules to classify novel stimuli , when the classification rule is provided by the experimenter ( koenig , smith , & grossman , 2006 ) . here , we tested the patients ' ability to acquire more complex category structures that could not be captured by a simple rule and when no information about the nature of the categories was supplied by the experimenter . the structure of the two categories ( shown in fig . 1a ) was designed such that optimal performance could only be achieved by acquiring integrated representations of the various typical characteristics of each category . when presented en masse as in fig . members of category a usually contained squares while those in b contained circles , though there were exceptions in both categories . the same was true for the number of shapes ( members of a usually contain one shape ) and the colour of the background square ( usually blue for a ) . the colour of the internal shapes , though perceptually salient , was not diagnostic of category . this category structure , in which a number of features are associated with each category but no single feature is diagnostic , is termed a family resemblance structure and is characteristic of object categories in the real world ( rosch & mervis , 1975 ; smith & medin , 1981 ; wittgenstein , 1953 ) . within such a structure , it is impossible to classify with complete accuracy by learning only about a single feature dimension . optimum performance instead requires participants to form integrated representations that include second - order statistical information about the feature conjunctions that characterise each category , allowing them , for example , to correctly class an exemplar with two circles as a member of category b , even if it has a blue background . we predicted that forming such integrated representations is a key function of the atls and , therefore , that sd patients would be impaired in learning the categories . we deliberately selected an abstract , novel set of stimuli with little perceptual similarity to objects in the real word , to ensure that pre - existing conceptual knowledge would not influence the learning process . however , the novel stimuli 's underlying family resemblance structure meant that they shared several important attributes with real conceptual categories.1.items in a category shared a number of typical characteristics but no single feature was diagnostic of category membership ( e.g. , most creatures that fly are birds but there are also a number of flightless birds and some non - bird creatures that can fly).2.while there were no individual diagnostic features , the conjunction of a number of typical features was a good guide to category membership ( e.g. , a creature that lays eggs and has feathers and a beak is likely to be a bird , even if it can not fly).3.some features , though salient , were not useful in determining category membership ( e.g. , the colour of a creature is not helpful in deciding whether it is a bird or not ) . items in a category shared a number of typical characteristics but no single feature was diagnostic of category membership ( e.g. , most creatures that fly are birds but there are also a number of flightless birds and some non - bird creatures that can fly ) . while there were no individual diagnostic features , the conjunction of a number of typical features was a good guide to category membership ( e.g. , a creature that lays eggs and has feathers and a beak is likely to be a bird , even if it can not fly ) . some features , though salient , were not useful in determining category membership ( e.g. , the colour of a creature is not helpful in deciding whether it is a bird or not ) . our hypothesis was that the computational challenges posed by these complex , natural categories are met by the atls , which form integrated conceptual representations that allow us to categorise items based on the overall summation of their characteristics rather than relying on a single defining feature . we predicted that sd patients would be impaired in their ability to acquire these integrated representations , leading to an over - reliance on individual features to guide their category decisions . seven patients with sd were recruited from memory clinics in northwest and southwest england . all , 2011 ; hodges , patterson , oxbury , & funnell , 1992 ) , in that they presented with pan - modal conceptual knowledge deficit that affected receptive and expressive tasks . other aspects of cognition were preserved in all but the most severe cases : patients were well - oriented in time and space and presented with fluent , grammatically correct speech . however , the case - series was intended to span the full range of severity in semantic performance and one of the most severe cases ( n.h . ) , while initially presenting with a selective semantic impairment , had begun to show signs of decline on other cognitive tasks at the time of the study . structural neuroimaging indicated bilateral atrophy of the anterior temporal region in each case ( see fig . 2 ) . conceptual knowledge was assessed using elements of the cambridge semantic battery ( bozeat et al . , 2000 ) , consisting of tests of picture naming , spoken word picture matching , pictorial semantic association ( the camel and cactus test ) and verbal fluency for six semantic categories . all seven patients performed below the normal range on all tests . as expected , there was a broad range of impairment in conceptual knowledge from mild to very severe ( see table 1 ; patients are ordered from mild to severe based on word picture matching scores ) . general dementia severity was assessed with the addenbrooke 's cognitive examination - revised ( mioshi , dawson , mitchell , arnold , & hodges , 2006 ) and the mini mental state examination ( folstein , folstein , & mchugh , 1975 ) . visuospatial processing was tested using the rey figure copy and two subtests from the visual object and space perception battery ( warrington & james , 1991 ) . patients also completed tests of digit span ( wechsler , 1987 ) and raven 's coloured progressive matrices ( raven , 1962 ) . these tests revealed the expected pattern of relative preservation of other cognitive functions in most cases . at time of testing , n.h . had begun to show signs of more general cognitive decline . in contrast , e.t . performed strikingly well on the non - semantic tasks , despite severe semantic impairment . we included both patients in the case - series in order to assess the effects of severe conceptual knowledge impairment on learning ; however , it is possible that concomitant deficits may have affected n.h . importantly , the other six patients all demonstrated preservation of the basic perceptual and cognitive functions necessary to complete the category learning task . it also has a strong problem - solving element and requires understanding the notion of similarity relationships between stimuli . twenty - four abstract visual stimuli were created based on those used by waldron and ashby ( 2001 ) . stimuli varied on four dimensions : background colour , internal shape , number of shapes and shape colour . these dimensions each had two possible values ( e.g. , shape : circle or square ) and we refer to these as features . the shape colour dimension had three possible values ( red , black and green ) and was irrelevant for classification . a family resemblance structure was used to divide the stimuli into two categories , arbitrarily labelled a and b ( see fig . 1a ) . each of the three relevant dimensions had a feature reliably associated with each category , though no single dimension was fully diagnostic of category . three exemplars in each category possessed all of the three features associated with the category ( i.e. , the typical background , typical number and typical shape for their category , shown in the top row of fig . the remaining exemplars had two features that were typical of their category , while the remaining feature was more strongly associated with the opposing category . six exemplars were not presented at all during the learning task but were retained to later test the participants ' ability to generalise their learning to novel exemplars . , patients were told that they would see some abstract patterns and would attempt to learn which ones were as and which were bs . they were told that there was no simple rule for deciding but that it was something they would learn over time . on each trial , they saw one of the 18 exemplars , presented in the centre of a laptop computer screen on a white background . the letters a and b were presented in bottom left and right corners of the screen and the patient was asked whether the exemplar was an a or a b. they were then presented with a green tick if they decided correctly or a red cross if they chose the wrong category . verbal feedback was also given at first so that patients understood the significance of the ticks and crosses . at no point were participants told which aspects of the stimuli to attend to or how to make their decisions . the 144 trials were divided into eight blocks , with each exemplar presented once in each block . for the second session , the patients were told that they were continuing the task they started the previous day and that the identity of the a 's and b 's had not changed . to determine the degree to which participants were able to form integrated category representations , categorisation success during the second half of the second session was analysed in detail ( 72 trials ) . by this point , participants had completed 216 trials of the learning task , allowing them to form stable representations of the characteristics of each category . the generalisation test probed participants ' ability to apply their acquired knowledge of the categories to a new set of stimuli comprised the same features but in novel combinations . this allowed us to rule out an alternative basis for task performance : namely , that participants had used an episodic memory strategy and attempted to memorise the correct category for each individual stimulus , rather than learning the underlying properties that characterised the two categories . we reasoned that knowledge of the underlying category structure would generalise to a new set of stimuli that participants had not seen during learning . in contrast , if participants had only learned the categories for the specific stimuli presented during learning , they would not be able to classify new stimuli at an above - chance level . to test for generalisation , immediately after the second session participants were presented with six new exemplars , each of the six new exemplars was presented a total of four times . in a recent study , barense , rogers , bussey , saksida , and graham ( 2010 ) demonstrated that sd patients can have difficulty discriminating between visual objects when they have many overlapping features . specifically , patients were impaired when required to discriminate stimuli based on conjunctions of features , even in a purely perceptual task with no learning requirement . this raises the possibility that apparent deficits in learning could arise because sd patients have difficulty perceiving the stimuli correctly . to ensure that our patients were able to discriminate between the stimuli in our experiment , we tested them with a demanding odd - one - out task described by barense et al . ( 2010 ) . on each trial , patients were presented simultaneously with seven exemplars from the learning study . the seven stimuli consisted of three identical pairs and one odd - one - out and patients were asked to point to the odd - one - out . in the minimum ambiguity condition , the odd - one - out could be detected on the basis of a single stimulus dimension ( e.g. , in fig . in the medium ambiguity condition , it was necessary to perceive the conjunction of two dimensions to distinguish the odd - one - out ( e.g. , in fig . 1b , only the odd - one - out has squares on a yellow background ) . finally , in the maximum ambiguity condition , the odd - one - out could only be detected by integrating all three dimensions . they had a mean age of 69 years and educational level of 16.7 years , neither of which differed from the patients [ t(17 ) < 1.9 , p > .05 ] . their mean age was 68 and education was 16.0 years [ not significantly different from patients : t(11 ) < 1.0 , p > .05 ] . mean categorisation accuracy in the control group was 67% ( standard deviation = 9.7% ) , which indicates that learning the family resemblance category structure under experimental conditions was challenging even for healthy participants , as expected from previous studies ( medin , wattenmaker , & hampson , 1987 ) . sd patients also averaged 67% ( standard deviation = 4.7% ) and their accuracy was not significantly different to that of controls [ t(17 ) = .15 , p = .88 ] . importantly , binomial tests indicated that all seven patients were significantly above chance in their categorisation performance ( p < .0019 ) . this indicates that all of the patients understood the nature of the task ( i.e. , they were not guessing ) and were able to acquire some information about the novel stimuli . to determine the nature of the representations formed by our participants , we analysed performance on the final 72 trials of the learning task in more detail . these analyses revealed that learning in the sd group took a very different form to that seen in the control group , as we describe next . our key prediction was that sd patients would have difficulty forming integrated representations that included information about all three dimensions needed for optimal classification . to test this 3 shows the data from each patient and , for comparison purposes , from two representative controls . each participant 's responses have been split according to the exemplar 's features on each of the three critical dimensions . the y - axis shows how often the participant responded b to stimuli with each feature , so values close to one indicate items that were usually classed as b 's and values close to zero show items that were usually classed as a 's . control 1 showed an optimal pattern of responding : she successfully acquired knowledge about the typical features in all three dimensions ( this can be seen clearly by comparing her pattern of responses with the set of category members in fig . 1a ; for example , she correctly classified most of the circle exemplars as b 's and the squares as a 's ) . control 2 achieved much poorer learning overall ( 60% accuracy ) but showed a similar qualitative pattern . she also learned about all three dimensions equally , albeit to a much lesser extent . the pattern in the patients was rather different and indicates that they were unable to form coherent representations that combined all three dimensions . ) learned about only one of the three critical dimensions , as indicated by strong differentiation and one dimension and a lack of discrimination on the other two dimensions . for example , p.w . classified all stimuli based on their shape , ignoring their number and background colour.1 the remaining three patients showed a more ambiguous pattern of performance , with weak learning on two stimulus dimensions . to investigate these profiles in more detail , we calculated d scores for each participant . d is a signal detection measure that reflects a participant 's tendency to give a particular response when presented with a particular type of stimulus weighed against their propensity to make the same response to other stimuli . we computed d scores that expressed a participant 's sensitivity to the feature category associations in each of the three dimensions . according to our predictions , sd patients should show strong learning ( i.e. , high d values ) in one dimension but much weaker learning across the remaining dimensions . controls were expected to display a more even pattern of learning across the three dimensions . once d scores had been computed , an additional step was necessary to compare the results in the two groups . since different participants learned about different aspects of the stimuli ( e.g. , compare patient m.t . with p.w . ) , a simple averaging of the d scores in each dimension would mask the true effects . instead , we labelled the dimensions for each participant according to their d scores , with the dimension in which the greatest learning had occurred labelled as their strongest dimension ( so m.t . 's strongest dimension was number , her second dimension was shape and her weakest dimension was background colour ) . we were then able to average d scores within each group based on the strongest , second and weakest dimensions of each individual . it is important to note that interpretation of the d scores presented here is slightly different to most circumstances . in most studies , a particular stimulus feature is always associated with a particular response and optimum performance is signified by the maximum possible d value ( typically between 3 and 4 ) . because of the family resemblance structure employed here , each feature was only associated with its typical category on 78% of trials . as a consequence , the optimum d score was lower : a participant classifying with 100% accuracy would have d scores of 1.52 for each dimension ( indicated by the blue line in fig . scores higher than this indicate an over - extension of the learning in the strongest dimension , such that the information in this dimension was driving classification even for exemplars where the other two dimensions pointed towards a different category . this over - generalisation was present in four of the seven patients and is similar to the over - generalisation exhibited by sd patients when attempting to use their impaired conceptual knowledge of real objects ( see discussion ) . no patients demonstrated much learning in their second or weakest dimensions , in line with the prediction that they would be unable to form category representations that integrated all of the information required for optimum categorisation . as expected , there was a large disparity between the strongest dimension and the remaining two dimensions in sd , with a more balanced pattern of learning across the three dimensions in the control group . a 3 ( dimension ) 2 ( group ) anova was performed on these data . there was a main effect of dimension [ f(2,34 ) = 43 , p < .001 ] . there was no effect of group but there was a highly significant interaction between dimension and group [ f(2,34 ) = 6.83 , p = .003 ] . post - hoc t - tests indicated that sd patients showed significantly less learning on their weakest dimension than controls [ t(17 ) = 3.44 , p = .003 ] . there was also a trend towards poorer learning on the second dimension in sd patients , relative to controls [ t(17 ) = 1.95 , p = .07 ] . while the general pattern in the patient group was towards strong , single - dimension learning , we did observe some variation across patients , with j.w . displaying a less clear pattern than the other four patients . to investigate these differences , we tested whether these patients ' responses were influenced by the shape colour dimension , which was irrelevant for classification . we calculated a d measure of learning in this dimension in a similar manner to the other dimensions . since this dimension was irrelevant to classification , the optimum d was 0 . the four patients who achieved the most successful learning on their strongest dimension showed low d values , indicating that they were not influenced by the irrelevant dimension . however , patients n.h . and e.t . , and to a lesser extent j.w . , had higher d scores , indicating that their responses were incorrectly influenced by this dimension . this suggests a more severe impairment in these individuals , since their responses were guided by stimulus features that were not reliably associated with either category . in line with this hypothesis , the two patients with the most severe semantic deficit showed the largest effects ( n.h . and e.t . d scores in the sd group as a whole were also compared with those of the control group ( see fig . sd patients were more likely to be influenced by the irrelevant dimension than controls [ t(17 ) = 2.26 , p = .04 ] . the general picture emerging from the d analyses was that sd patients displayed relatively successful learning on their strongest dimension but were less successful in learning the category associations in the other two dimensions . this suggests that they failed to integrate the various stimulus features into a coherent conceptual representation . as a strong test of this interpretation , we re - analysed categorisation accuracy but now specifically considered trials on which an over - reliance on learning in one dimension would cause participants to choose the wrong category . trials from the final period of learning were divided into two conditions for each participant:1.consistent trials : on most trials ( 78% ) , the feature on the strongest dimension indicated the correct category for the exemplar . on these trials , participants could categorise correctly even if they had only acquired knowledge in a single dimension.2.inconsistent trials : due to the family resemblance structure , there were a minority of trials in which the feature in the participant 's strongest dimension did not indicate the correct category . participants could only give the correct response on these trials if they had also acquired some knowledge of the other two dimensions , which would direct them towards the correct response . consequently , we expected sd patients to have particular difficulty on these trials , because it was not possible for them to select the correct category unless they had achieved integrated learning across multiple dimensions . consistent trials : on most trials ( 78% ) , the feature on the strongest dimension indicated the correct category for the exemplar . on these trials , participants could categorise correctly even if they had only acquired knowledge in a single dimension . inconsistent trials : due to the family resemblance structure , there were a minority of trials in which the feature in the participant 's strongest dimension did not indicate the correct category . participants could only give the correct response on these trials if they had also acquired some knowledge of the other two dimensions , which would direct them towards the correct response . consequently , we expected sd patients to have particular difficulty on these trials , because it was not possible for them to select the correct category unless they had achieved integrated learning across multiple dimensions . the data were analysed with 2 2 mixed anova that included condition and group . this revealed main effects of both group [ f(1,17 ) = 10.7 , p = .005 ] and condition [ f(1,17 ) = 89 , p < .001 ] . critically , there was also a highly significant interaction [ f(1,17 ) = 10.8 , p = .004 ] . post - hoc tests indicated that patients performed as accurately as controls on consistent trials ( t < 1 ) but were substantially impaired on inconsistent trials [ t(19 ) = 4.15 , p = .001 ] . this supports the hypothesis that patients were less able to form representations that included information from multiple dimensions and instead responded solely on the basis of their strongest dimension . the generalisation test probed participants ' ability to apply their acquired knowledge of the categories to novel stimuli . performance on the new stimuli was above chance in both groups [ one - tailed one - sample t - tests : sd patients : t(6 ) = 1.94 , p = .05 ; controls : t(11 ) = 3.19 , p = .009 ] . we also compared performance on the generalisation stimuli with performance in the final block of the learning task , to assess how successfully learning transferred to new exemplars . for the purposes of this comparison , we excluded the six highly prototypical stimuli from the training set ( i.e. , the stimuli on the top row of fig . we excluded them because there were no equivalent stimuli in the generalisation set : all of the generalisation had at least one feature associated with the opposing category . a 2 2 anova revealed no difference between learning and generalisation [ f(1,17 ) = 1.79 , p = .2 ] , no effect of group [ f(1,17 ) = .91 , p = .4 ] and no interaction [ f(1,17 ) = .59 , p = .5 ] . based on these findings , it is unlikely that either patients or controls were memorising the correct category for individual stimuli . instead , they attempted to form more general representations of the characteristics of each category , which allowed them to generalise to new exemplars . the visual discrimination test measured participants ' ability to perceive the conjunctions of features present in the stimuli and to discriminate between them . patients and controls performed close to ceiling , even for the most demanding trials ( see fig . 2 ( group ) mixed anova comparing patients with controls revealed no main effect of either group [ f(1,11 ) = 1.65 , p = .2 ] or condition [ f(2,22 ) = .38 , p = .5 ] and no interaction [ f(2,22 ) = .60 , p = .6 ] . the performance of each individual patient was compared with the control group using the modified t - test ( crawford & howell , 1998 ) . no patient showed a significant impairment in any of the conditions ( all t < 1.4 , p > .1 ) , indicating that their abnormal performance on the learning task was not due to difficulty in discriminating visually between the exemplars . the atls are thought to play a central role in the representation of conceptual knowledge ( lambon ralph et al . , 2010 ; patterson et al . , 2007 ) . here , sd patients completed a category learning task , in which the category members conformed to a family resemblance structure designed to replicate the key computational challenges of acquiring real - world concepts . the patients were able to learn some information about the stimuli but did so in a sub - optimal fashion that differed from healthy controls in systematic and theoretically important ways . for optimal performance , it was necessary to integrate all three critical dimensions of the stimuli into a coherent representation . patients were unable to do this and instead based all of their category judgements on a single dimension . this deficit is consistent with the hub - and - spoke theory of conceptual knowledge and specifically with the theory that the atls act as a pan - modal representational hub , which integrates a concept 's disparate sensory - motor and verbal features into a single coherent representation ( lambon ralph et al . , 2010 ; with damage to the atls , sd patients largely retained the ability to associate individual stimulus features with novel categories but were unable to acquire the integrated feature structure necessary for optimal discrimination between the two categories . sd patients also demonstrated over - generalisation of the successful learning in their preferred dimension : information from one dimension dominated category decisions , even when the other features of the stimulus pointed towards an alternative response . this over - generalisation of remaining knowledge is also common when sd patients attempt to make use of their remaining conceptual knowledge in everyday life and in clinical assessment ( lambon ralph & patterson , 2008 ; lambon ralph et al . , 2010 ) . over the course of the disease , patients become increasingly likely to over - extend category boundaries on the basis of superficial characteristics ( e.g. , accepting a butterfly as a type of bird ; mayberry et al . , 2011 ) , to use a single , highly familiar concept label to refer to a whole class of items ( e.g. , all forms of fruit may be called apples ; hodges , graham , & patterson , 1995 ) , and to imbue items with over - generalised , stereotypical attributes in delayed - copy drawing ( e.g. , the case of the four - legged duck ; bozeat et al . , 2003 ; lambon ralph & howard , 2000 ) . in the present study , we were able to unmask one of the basic mechanisms underpinning this profound deterioration in conceptual representation : cerebral atrophy in sd affects integrated conceptual representations that bind together the various sources of information that characterise a particular set of items . without these coherent concepts , classification and identification of objects interestingly , another study indicates that sd patients can successfully make category judgements about novel items when they are not required to form integrated representations . ( 2006 ) investigated six sd patients ' ability to classify novel stimuli based on a category membership rule and on similarity to a prototype . koenig et al . 's study differs from ours in that koenig et al . explicitly provided patients with the appropriate rule to apply or prototype to compare during categorisation . in contrast , we required patients to learn the relevant category structure themselves through feedback . study performed similarly to controls and the authors attributed this good performance to intact attentional and executive processes . one possibility for the difference between the two studies is that the application of explicit rules to determine category membership depends heavily on executive and attentional processes , while the acquisition of multi - dimensional feature structure is a more automatic process involving implicit learning mechanisms in temporal regions . this assertion is supported by an investigation in healthy participants , on which the present learning task was based ( waldron & ashby , 2001 ) . as in our study , participants were trained to classify stimuli without being given any explicit instruction regarding the structure of the category . they were trained with category structures in which a single feature determined category membership as well ones that required integration of features . crucially , an executively - demanding concurrent task slowed learning of the single - feature categories but had little effect on the categories that required integration . the authors suggested that learning a single - feature category involved using executive resources to extract an explicit rule that governs category membership . in contrast , learning of the feature - integration categories was assumed to be an implicit stimulus - driven process ( see also ashby & ell , 2001 ) . relating these findings to our patient group , it appears that while integration of features was impaired , executively - mediated rule extraction was intact in most cases , hence their over - learning of a single feature dimension . however , the two most severe patients ( n.h . and e.t . ) were less successful in acquiring appropriate single - feature information , perhaps indicating a decline in executive processes as the disease progresses . which regions within the atls are critically involved in acquiring and storing coherent concepts ? in sd , atrophy affects the entire atl region , though it is concentrated in polar and ventrolateral regions ( gorno - tempini et al . , 2004 ; converging evidence from other methodologies has also implicated the ventral and lateral aspects of the atls in the representation of conceptual knowledge ( binney et al . , 2010 ; marinkovic et al . , 2003 ; pobric et al . , 2007 ; visser & lambon ralph , 2011 ) . a parallel line of work has implicated medial anterior temporal regions , particularly the perirhinal cortex , in the perception and learning of novel feature conjunctions , both in humans ( barense et al . , 2005 ; taylor , moss , stamatakis , & tyler , 2006 ) and non - human primates ( bussey , saksida , & murray , 2002 ; murray & richmond , 2001 ) . damage to this region is associated with deficits in discriminating between novel stimuli based on conjunctions of their features . medial and ventrolateral temporal regions also appear to interact in the acquisition and representation of concepts . for example , neurons in both the perirhinal and ventrolateral atls change their response characteristics as monkeys learn novel visual associations , suggesting that both areas are involved ( messinger , squire , zola , & albright , 2001 ) . it is likely that medial temporal regions play a critical role in the perception and initial encoding of new conceptual information , while ventrolateral temporal cortex is necessary for longer - term storage of concepts ( albright , 2012 ; squire , stark , & clark , 2004 ) . established theories of learning hold that this division of labour is necessary to avoid catastrophic interference between similar representations ( mcclelland , mcnaughton , & o'reilly , 1995 ) . sd patients were not generally amnesic for novel information , as would be expected in patients with medial temporal lobe dysfunction : they were able to learn single - feature information and maintain this between the two training sessions . nor were they impaired in perceptual discriminations based on conjunctions of features ( though another study has shown that sd patients can be impaired on such discriminations for meaningful items ; barense et al . , in contrast , their deficits stemmed from an inability to extract the underlying patterns of feature co - occurrence present over many trials to form representations of the two stimulus categories . however , a great deal more work is needed to determine precisely how different sub - regions within the atls work together to process complex feature conjunctions in a single experience and to integrate information acquired over many experiences into coherent concepts . the striatum and putamen are also involved in learning to classify stimuli when integration of two dimensions is required , particularly in the early stages of learning ( waldschmidt & ashby , 2011 ) . these subcortical structures are intact in sd ( mummery et al . , 2000 ) but their interaction with the damaged temporal cortex has not been investigated . in this study , we focused on the integration of stimulus features within the visual modality . however , it is important to note that the atls play an important role in integrating conceptual knowledge across modalities : they are equally activated during conceptual processing of visual and auditory stimuli , both verbally and non - verbally ( binney et al . , 2010 ; spitsyna , warren , scott , turkheimer , & wise , 2006 ; visser & lambon ralph , 2011 ) . in the primate literature , the atls have been associated with associative learning both within the visual modality ( albright , 2012 ; messinger et al . , 2001 ) and across different sensory modalities ( murray & richmond , 2001 ; parker & gaffan , 1998 ) . indeed , the atls are strongly connected to visual , auditory and other sensory cortices ( moran , mufson , & mesulam , 1987 ; pandya & seltzer , 1982 ) , making this region a key area of polysensory or transmodal cortex ( mesulam , 1998 ; patterson et al . , 2007 ; simmons & barsalou , 2003 ) . the hub - and - spoke model distinguishes between this transmodal cortex and spoke regions that are sensitive to structure in a single modality , though this distinction may be relative rather than absolute . recently , we have proposed that the anterior temporal region acts as a graded representational space ( plaut , 2002 ) , in which the type of information coded by each area of cortex is determined by the inputs it receives from sensory and unimodal association cortices ( binney et al . , 2012 ) . for example , the dorsolateral atl receives strong input from the posterior superior temporal gyrus , leading this area to exhibit relative specialisation for information in auditory and verbal modalities ( visser & lambon ralph , 2011 ) . ventromedial atl is strongly connected with ventral occipitotemporal cortex , leading to a prominent role in coding visual properties . critically , between these extremes lies equi - modal cortex in the inferior temporal and fusiform gyri that responds similarly across modalities and presumably codes transmodal structure . in summary , the process of extracting meaning from our experience with objects involves the fusion of complex sets of information from sensory inputs , motor programmes and verbal experience . we have demonstrated that one key aspect of this process , the integration of individual features into coherent concepts , depends critically on the atls .

recent evidence from multiple neuroscience techniques indicates that regions within the anterior temporal lobes ( atls ) are a critical node in the neural network for representing conceptual knowledge , yet their function remains elusive . the hub - and - spoke model holds that atl regions act as a transmodal conceptual hub , distilling the various sensory - motor features of objects and words into integrated , coherent conceptual representations . single - cell recordings in monkeys suggest that the atls are critically involved in visual associative learning ; however , investigations of this region in humans have focused on existing knowledge rather than learning . we studied acquisition of new concepts in semantic dementia patients , who have cortical damage centred on the ventrolateral aspects of the atls . patients learned to assign abstract visual stimuli to two categories . the categories conformed to a family resemblance structure in which no individual stimulus features were fully diagnostic ; thus the task required participants to form representations that integrate multiple features into a single concept . patients were unable to do this , instead responding only on the basis of individual features . the study reveals that integrating disparate sources of information into novel coherent concepts is a critical computational function of the atls . this explains the central role of this region in conceptual representation and the catastrophic breakdown of concepts in semantic dementia .

Introduction Method Results Discussion

conceptual knowledge for objects comprises a diverse set of information about their sensory qualities , motor plans and verbal associations . how are these disparate sources of information linked to form a concept ? according to one influential view , originally proposed by wernicke ( wernicke , 1900 ; as cited in eggert , 1977 ) , conceptual knowledge for objects arises from the co - activation of their sensory - motor properties within a network of modality - specific processing regions that are widely distributed throughout the cortex ( barsalou , 2008 ; martin , 2007 ; pulvermuller , 2001 ) . this approach makes two key predictions concerning the breakdown of conceptual knowledge under brain damage . first , damage to a single , modality - specific region should give rise to knowledge deficits that disproportionately affect properties in that modality and , by extension , categories of objects for which the affected modality is particularly central ( capitani , laiacona , mahon , & caramazza , 2003 ; mahon & caramazza , 2009 ; warrington & shallice , 1984 ) . so , for example , damage to regions of inferior parietal cortex involved in representing skilled actions should impair knowledge of how objects are manipulated and lead to a disproportionate deficit for tools ( buxbaum & saffran , 2002 ) . according to wernicke and his modern counterparts , these should only occur as a result of global cortical damage , because only damage to all of the modality - specific regions would be sufficient to produce a global impairment . sd patients suffer from a global conceptual knowledge deficit that affects all categories of object and word ( hoffman & lambon ralph , 2011 ; lambon ralph , lowe , & rogers , 2007 ) and all sensory - motor modalities ( bozeat , lambon ralph , patterson , garrard , & hodges , 2000 ; bozeat , lambon ralph , patterson , & hodges , 2002 ; luzzi et al . , 2007 ; piwnica - worms , omar , hailstone , & warren , 2010 ) , yet the cerebral atrophy and hypometabolism that gives rise to this debilitating impairment is not global : it is focused bilaterally on the anterior ventrolateral and polar portions of the temporal lobes ( galton et al . evidence from functional neuroimaging ( binney , embleton , jefferies , parker , & lambon ralph , 2010 ; visser & lambon ralph , 2011 ) and transcranial magnetic stimulation ( pobric , jefferies , & lambon ralph , 2007 ; pobric , jefferies , & lambon ralph , 2010 ) in neurologically - intact participants confirms that ventrolateral anterior temporal lobe ( atl ) areas are involved in all forms of conceptual processing irrespective of the modality of the information or the category of object probed . the crucial role of this area in transmodal semantic representation also fits with recent in vivo tractography data demonstrating the convergence of multiple white - matter pathways into the atl . such results indicate that this region 's structural connectivity is ideal for blending different sources of verbal and nonverbal information into integrated , coherent concepts ( binney , parker , & lambon ralph , 2012 ) . to account for the global , pan - modal involvement of the ventrolateral atls in conceptual knowledge , we have developed an alternative framework for conceptual knowledge termed the hub - and - spoke model ( lambon ralph , sage , jones , & mayberry , 2010 ; patterson , nestor , & rogers , 2007 ; pobric et al . this model holds that in addition to modality - specific sources of information ( spokes ) and their inter - connections , representation of conceptual knowledge requires an integrative hub . the hub uses information from the modality - specific spoke regions to develop modality - invariant , conceptual representations that capture deeper patterns of conceptual similarity across all sensory - motor and verbal modalities . these integrated representations are necessary because similarity in any particular sensory - motor domain is , at best , only a partial guide to conceptual similarity ( dilkina & lambon ralph , 2013 ; lambon ralph et al . the hub - and - spoke framework holds that the atl hub provides this critical aspect of conceptual representation through the formation of representations that integrate information from all sensory - motor - verbal domains . when this region is damaged , as in sd , the result is a breakdown in the complex boundaries that define different concepts , such that semantic decisions come to be made on the basis of superficial characteristics rather than their deeper conceptual properties . for example , sd patients may reject emu as an example of a bird but simultaneously over - extend the concept to accept butterfly previous work on the function of the ventrolateral atls has focused on their role in representing existing knowledge and its progressive deterioration as a result of atl atrophy in sd ( e.g. , the hub - and - spoke framework also predicts that the atls play a key role in the acquisition of novel concepts ( rogers & mcclelland , 2004 ) . the response properties of anterior inferior temporal neurons change as monkeys learn novel associations between visual stimuli , suggesting a role for this region in the acquisition of concepts ( albright , 2012 ) . in the present study , we tested this hypothesis in humans by studying acquisition of new conceptual knowledge in patients with sd . the hub - and - spoke model predicts that the atls are critical for integrating the various sensory features of an object into a unified , coherent conceptual representation that can be generalised to new exemplars . we tested this prediction by training sd patients to recognise novel visual stimuli as members of two categories . here , we tested the patients ' ability to acquire more complex category structures that could not be captured by a simple rule and when no information about the nature of the categories was supplied by the experimenter . the structure of the two categories ( shown in fig . 1a ) was designed such that optimal performance could only be achieved by acquiring integrated representations of the various typical characteristics of each category . the same was true for the number of shapes ( members of a usually contain one shape ) and the colour of the background square ( usually blue for a ) . this category structure , in which a number of features are associated with each category but no single feature is diagnostic , is termed a family resemblance structure and is characteristic of object categories in the real world ( rosch & mervis , 1975 ; smith & medin , 1981 ; wittgenstein , 1953 ) . optimum performance instead requires participants to form integrated representations that include second - order statistical information about the feature conjunctions that characterise each category , allowing them , for example , to correctly class an exemplar with two circles as a member of category b , even if it has a blue background . we predicted that forming such integrated representations is a key function of the atls and , therefore , that sd patients would be impaired in learning the categories . we deliberately selected an abstract , novel set of stimuli with little perceptual similarity to objects in the real word , to ensure that pre - existing conceptual knowledge would not influence the learning process . however , the novel stimuli 's underlying family resemblance structure meant that they shared several important attributes with real conceptual categories.1.items in a category shared a number of typical characteristics but no single feature was diagnostic of category membership ( e.g. our hypothesis was that the computational challenges posed by these complex , natural categories are met by the atls , which form integrated conceptual representations that allow us to categorise items based on the overall summation of their characteristics rather than relying on a single defining feature . other aspects of cognition were preserved in all but the most severe cases : patients were well - oriented in time and space and presented with fluent , grammatically correct speech . however , the case - series was intended to span the full range of severity in semantic performance and one of the most severe cases ( n.h . ) , while initially presenting with a selective semantic impairment , had begun to show signs of decline on other cognitive tasks at the time of the study . structural neuroimaging indicated bilateral atrophy of the anterior temporal region in each case ( see fig . conceptual knowledge was assessed using elements of the cambridge semantic battery ( bozeat et al . as expected , there was a broad range of impairment in conceptual knowledge from mild to very severe ( see table 1 ; patients are ordered from mild to severe based on word picture matching scores ) . we included both patients in the case - series in order to assess the effects of severe conceptual knowledge impairment on learning ; however , it is possible that concomitant deficits may have affected n.h . twenty - four abstract visual stimuli were created based on those used by waldron and ashby ( 2001 ) . a family resemblance structure was used to divide the stimuli into two categories , arbitrarily labelled a and b ( see fig . on each trial , they saw one of the 18 exemplars , presented in the centre of a laptop computer screen on a white background . the letters a and b were presented in bottom left and right corners of the screen and the patient was asked whether the exemplar was an a or a b. they were then presented with a green tick if they decided correctly or a red cross if they chose the wrong category . at no point were participants told which aspects of the stimuli to attend to or how to make their decisions . for the second session , the patients were told that they were continuing the task they started the previous day and that the identity of the a 's and b 's had not changed . to determine the degree to which participants were able to form integrated category representations , categorisation success during the second half of the second session was analysed in detail ( 72 trials ) . by this point , participants had completed 216 trials of the learning task , allowing them to form stable representations of the characteristics of each category . the generalisation test probed participants ' ability to apply their acquired knowledge of the categories to a new set of stimuli comprised the same features but in novel combinations . this allowed us to rule out an alternative basis for task performance : namely , that participants had used an episodic memory strategy and attempted to memorise the correct category for each individual stimulus , rather than learning the underlying properties that characterised the two categories . we reasoned that knowledge of the underlying category structure would generalise to a new set of stimuli that participants had not seen during learning . in the minimum ambiguity condition , the odd - one - out could be detected on the basis of a single stimulus dimension ( e.g. mean categorisation accuracy in the control group was 67% ( standard deviation = 9.7% ) , which indicates that learning the family resemblance category structure under experimental conditions was challenging even for healthy participants , as expected from previous studies ( medin , wattenmaker , & hampson , 1987 ) . this indicates that all of the patients understood the nature of the task ( i.e. 1a ; for example , she correctly classified most of the circle exemplars as b 's and the squares as a 's ) . the pattern in the patients was rather different and indicates that they were unable to form coherent representations that combined all three dimensions . ) learned about only one of the three critical dimensions , as indicated by strong differentiation and one dimension and a lack of discrimination on the other two dimensions . since different participants learned about different aspects of the stimuli ( e.g. because of the family resemblance structure employed here , each feature was only associated with its typical category on 78% of trials . scores higher than this indicate an over - extension of the learning in the strongest dimension , such that the information in this dimension was driving classification even for exemplars where the other two dimensions pointed towards a different category . this over - generalisation was present in four of the seven patients and is similar to the over - generalisation exhibited by sd patients when attempting to use their impaired conceptual knowledge of real objects ( see discussion ) . no patients demonstrated much learning in their second or weakest dimensions , in line with the prediction that they would be unable to form category representations that integrated all of the information required for optimum categorisation . as expected , there was a large disparity between the strongest dimension and the remaining two dimensions in sd , with a more balanced pattern of learning across the three dimensions in the control group . there was also a trend towards poorer learning on the second dimension in sd patients , relative to controls [ t(17 ) = 1.95 , p = .07 ] . while the general pattern in the patient group was towards strong , single - dimension learning , we did observe some variation across patients , with j.w . d scores in the sd group as a whole were also compared with those of the control group ( see fig . this suggests that they failed to integrate the various stimulus features into a coherent conceptual representation . as a strong test of this interpretation , we re - analysed categorisation accuracy but now specifically considered trials on which an over - reliance on learning in one dimension would cause participants to choose the wrong category . on these trials , participants could categorise correctly even if they had only acquired knowledge in a single dimension.2.inconsistent trials : due to the family resemblance structure , there were a minority of trials in which the feature in the participant 's strongest dimension did not indicate the correct category . inconsistent trials : due to the family resemblance structure , there were a minority of trials in which the feature in the participant 's strongest dimension did not indicate the correct category . this supports the hypothesis that patients were less able to form representations that included information from multiple dimensions and instead responded solely on the basis of their strongest dimension . the generalisation test probed participants ' ability to apply their acquired knowledge of the categories to novel stimuli . we also compared performance on the generalisation stimuli with performance in the final block of the learning task , to assess how successfully learning transferred to new exemplars . we excluded them because there were no equivalent stimuli in the generalisation set : all of the generalisation had at least one feature associated with the opposing category . instead , they attempted to form more general representations of the characteristics of each category , which allowed them to generalise to new exemplars . no patient showed a significant impairment in any of the conditions ( all t < 1.4 , p > .1 ) , indicating that their abnormal performance on the learning task was not due to difficulty in discriminating visually between the exemplars . the atls are thought to play a central role in the representation of conceptual knowledge ( lambon ralph et al . here , sd patients completed a category learning task , in which the category members conformed to a family resemblance structure designed to replicate the key computational challenges of acquiring real - world concepts . for optimal performance , it was necessary to integrate all three critical dimensions of the stimuli into a coherent representation . patients were unable to do this and instead based all of their category judgements on a single dimension . this deficit is consistent with the hub - and - spoke theory of conceptual knowledge and specifically with the theory that the atls act as a pan - modal representational hub , which integrates a concept 's disparate sensory - motor and verbal features into a single coherent representation ( lambon ralph et al . , 2010 ; with damage to the atls , sd patients largely retained the ability to associate individual stimulus features with novel categories but were unable to acquire the integrated feature structure necessary for optimal discrimination between the two categories . sd patients also demonstrated over - generalisation of the successful learning in their preferred dimension : information from one dimension dominated category decisions , even when the other features of the stimulus pointed towards an alternative response . over the course of the disease , patients become increasingly likely to over - extend category boundaries on the basis of superficial characteristics ( e.g. , 2011 ) , to use a single , highly familiar concept label to refer to a whole class of items ( e.g. in the present study , we were able to unmask one of the basic mechanisms underpinning this profound deterioration in conceptual representation : cerebral atrophy in sd affects integrated conceptual representations that bind together the various sources of information that characterise a particular set of items . without these coherent concepts , classification and identification of objects interestingly , another study indicates that sd patients can successfully make category judgements about novel items when they are not required to form integrated representations . one possibility for the difference between the two studies is that the application of explicit rules to determine category membership depends heavily on executive and attentional processes , while the acquisition of multi - dimensional feature structure is a more automatic process involving implicit learning mechanisms in temporal regions . crucially , an executively - demanding concurrent task slowed learning of the single - feature categories but had little effect on the categories that required integration . the authors suggested that learning a single - feature category involved using executive resources to extract an explicit rule that governs category membership . which regions within the atls are critically involved in acquiring and storing coherent concepts ? , 2004 ; converging evidence from other methodologies has also implicated the ventral and lateral aspects of the atls in the representation of conceptual knowledge ( binney et al . a parallel line of work has implicated medial anterior temporal regions , particularly the perirhinal cortex , in the perception and learning of novel feature conjunctions , both in humans ( barense et al . medial and ventrolateral temporal regions also appear to interact in the acquisition and representation of concepts . it is likely that medial temporal regions play a critical role in the perception and initial encoding of new conceptual information , while ventrolateral temporal cortex is necessary for longer - term storage of concepts ( albright , 2012 ; squire , stark , & clark , 2004 ) . sd patients were not generally amnesic for novel information , as would be expected in patients with medial temporal lobe dysfunction : they were able to learn single - feature information and maintain this between the two training sessions . , in contrast , their deficits stemmed from an inability to extract the underlying patterns of feature co - occurrence present over many trials to form representations of the two stimulus categories . however , a great deal more work is needed to determine precisely how different sub - regions within the atls work together to process complex feature conjunctions in a single experience and to integrate information acquired over many experiences into coherent concepts . the striatum and putamen are also involved in learning to classify stimuli when integration of two dimensions is required , particularly in the early stages of learning ( waldschmidt & ashby , 2011 ) . in this study , we focused on the integration of stimulus features within the visual modality . however , it is important to note that the atls play an important role in integrating conceptual knowledge across modalities : they are equally activated during conceptual processing of visual and auditory stimuli , both verbally and non - verbally ( binney et al . in the primate literature , the atls have been associated with associative learning both within the visual modality ( albright , 2012 ; messinger et al . indeed , the atls are strongly connected to visual , auditory and other sensory cortices ( moran , mufson , & mesulam , 1987 ; pandya & seltzer , 1982 ) , making this region a key area of polysensory or transmodal cortex ( mesulam , 1998 ; patterson et al . the hub - and - spoke model distinguishes between this transmodal cortex and spoke regions that are sensitive to structure in a single modality , though this distinction may be relative rather than absolute . recently , we have proposed that the anterior temporal region acts as a graded representational space ( plaut , 2002 ) , in which the type of information coded by each area of cortex is determined by the inputs it receives from sensory and unimodal association cortices ( binney et al . we have demonstrated that one key aspect of this process , the integration of individual features into coherent concepts , depends critically on the atls .

many of qsar studies are based on the assumption that molecules from the same chemical domain will behave in a similar manner , so that qsar models drawn up with the analogical molecules are hypothesized to exhibit better performance than that derived from miscellaneous data set . the traditional approach to qsars for acute toxicity of organic compounds to the fathead minnow is the modeling of the activity of homologous or congeneric series of chemicals such as nitroaromatics , alkylamines , halogenated hydrocarbons and phenols , and chlorobenzenes and chloroalinines . often , such chemicals have a single functional group or toxicophore and an alkyl moiety of variable size . some other studies [ 511 ] by using diverse molecule sets have usually relied on dividing a molecule set into subgroups ( chemical classes ) by clustering the molecules based on their mode of action . then , local qstrs built up for each subgroup are applicable only to certain mode of action . it is worthy mention here that there has been a successful effort to draw up a global qstr model by using a single descriptor , namely , logarithm of 1-octanol / water partition coefficient logp . this model is applicable to quite miscellaneous data set , but still counts quite a big number of molecules as outliers . hydrophobicity of a molecule is characterized by logp which is directly related to bio - uptake of chemicals by fish or many other organisms . it has been successfully used for the modeling of acute toxicity of chemicals with different modes and mechanism of toxic action to pimephales promelas , combined with additional parameters such as energy of lowest unoccupied molecular orbital ( elumo ) and maximum superdelocalizability ( smax ) , which is a molecular orbital parameter that quantifies the electro ( nucleo ) philicity of a molecule . developing a better qstr for the modeling of acute toxicity of diverse chemicals is a subject of interest due to its demand by the organizations such as oecd ( organization for economic co - operation and development ) or ec ( european communities ) to use the qstr model for regulatory purpose . the first one is to build qstr multiple regression model using quantum - mechanics - based molecular descriptors that correlate and predict the loglc50 value of acute toxicity of 48 compounds to the fathead minnow . lc50 ( mg / l ) , aquatic toxicity on pimephales promelas expressed as the chemical concentration at which 50% lethality is observed in a test batch of fish within a 96 h exposure period . molecules used in this study are quite a diverse set and were taken from a study . the second aim of this study is to compare the accuracy of semi - empirical and first principle methods for calculation of molecular descriptors . [ 16 , 17 ] are fast in computation , well suited to organic compounds , and belong to semi - empirical method family . these methods have been traditionally used to calculate the optimized 3d geometry and quantum mechanics descriptors of molecules in most of qsar studies . some previous comparative qsar works [ 1,1822 ] have shown that using descriptors calculated by hf [ 2325 ] or dft together with b3lyp hybrid function instead of semi - empirical am1 or pm3 methods improve the accuracy of the results that lead more reliable qsars . on the other hand , there is an interesting comparative qstr study relevant in this area . in that study , a huge molecule set ( 568 molecules ) these qstr models have been built up from descriptors which were calculated using two different theory levels namely am1 and dft / b3lyp ( 6 - 31 g * * ) . their study has shown that the choice of the precise but time - consuming dft / b3lyp method does not have an advantage over am1 method for the quality of the derived qstrs . for all molecules studied here , 3-d modeling and calculations were performed using the gaussian 03 quantum chemistry package . to save in computational time , initial geometry optimizations were carried out with the molecular mechanics ( mm ) method using amber force field . the lowest energy conformations of the molecules obtained by the mm method were further optimized by the dft method by employing becke s three - parameter hybrid functional ( b3lyp ) and the 6 - 31+g ( d ) basis set ; their fundamental vibrations were also calculated using the same method to check if there were true minima . all the computations were carried out for the ground states of these molecules as singlet state . the lowest energy conformations of the compounds obtained using dft were used as an input geometry for the calculations for hf/6 - 31+g , am1 and pm3 methods . ( codessa pro ) comprehensive descriptors for structural and statistical analysis , version 2.7.2 , was used for extracting descriptors of quantum mechanics and 3d geometry of the compounds from gaussian 03 output files . codessa pro enables the generation of hundreds of molecular descriptors ( constitutional , topological , and quantum mechanical ) from a loaded 3d geometry , and uses diverse statistical structure property / activity correlation techniques for the analysis of experimental data in combination with calculated molecular descriptors . a qsar / qstr model can be developed for a given set of molecules by using a various types of descriptors . sometimes , a model might have very good statistical parameters , but still not suffice to explore the mechanism of interaction between the ligand and receptor mechanistically . building a model with physically interpretable descriptors is an important task for value of a qsar / qstr work . in this study , we aimed to draw up a qstr model by using quantum mechanically calculated thermodynamical descriptors by virtue of which obtained models are usually mechanistically interpretable . about 50 thermodynamical descriptors depending on the number of atoms in a molecule were calculated using codessa pro and gaussian 03 packages . the heuristic method implemented in codessa pro was used to build up a multi - able regression model . by this method , a pre - selection of descriptors is accomplished . all descriptors are checked to ensure the following : ( a ) the values of each descriptor are available for each structure , and ( b ) there is a variation in these values . the descriptors for which values are not available for every structure in the data in question are discarded . descriptors having a constant value for all structures in the data set are also discarded . thereafter , the one - parameter correlation equations for each descriptor are calculated . to further reduce the number in the starting set of descriptors , the following criteria are applied and a descriptor is eliminated if any of the following conditions are met with : ( a ) the f - test s value for the one - parameter correlation with the descriptor is below 1.0 , ( b ) the squared correlation coefficient of the one - parameter equation is less than rmin by default 0.01 , ( c ) the parameter s t - value is less than t1 ( where rmin 0.1 by default and t1 1.5 by default are user defined values ) , ( d ) the descriptor is highly inter - correlated ( above rfull , where rfull is a user specified value by default 0.99 ) , with another descriptor . all the remaining descriptors are then listed in decreasing order according to the correlation coefficient of the corresponding one - parameter correlation equation . all two - parameter regression models with remaining descriptors are developed and ranked by the regression correlation coefficient r. a stepwise addition of the further descriptors scales is performed to find the best multi - parameter regression models with the optimum values of statistical criteria ( highest values of r , the cross - validated , rcv and the f value ) . rcv , the leave one out ( loo ) squared cross - validated coefficient , is a practical and reliable method for testing the predictive performance and stability of a regression model . loo approach consists in developing a number of models with one sample omitted at a time . after developing each model , the omitted data are predicted and the differences between the experimental and predicted activity values are calculated . then the rcv is calculated according to the following formula : where yi is the actual experimental activity , is the average actual experimental activity and i is the predicted activity of compound i computed by the new regression equation obtained each time after leaving out one datum point ( no . i ) . among the thermodynamical descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency were involved in the models that are presented in this study . this connection is based upon partitioning the total energy of a macroscopic system among the constituent molecules . translational entropy ( at 300 k ) is defined as ; where v is the volume of the system , n is the avogadro s number , h is the planck constant , m is the molecular mass , and kt is the boltzman temperature . highest normal mode of vibrational frequency and lowest normal mode of vibrational frequency are actually not frequencies ; they are wavenumbers ( in cm unit ) . it is customary to call normal modes of vibration of molecule as frequency in infrared and raman spectroscopy . definition of normal mode of vibration arises from quantum mechanical harmonic oscillator model of a diatomic molecule . in this model , energy of the vibrational states is given as , where h is the planck constant , is the vibrational quantum number ( 0 , 1 , 2 , ) , and v is the classical vibrational frequency given by , where k is the force constant of the chemical bond and is the reduced mass for nuclei of two atoms . more commonly , equation 5 is used as vibrational wavenumber ( ) form rather than frequency form , where where is the vibrational wavenumber , c is the velocity of light . final descriptor involved in our model is principal moment of inertia a ( ia ) that is obtained from the 3d - cooordinate of the atoms in the given molecule . its definition is given as , where mi are the atomic masses and rix denotes the distance of the i - th atomic nucleus from the main rotational axes , x. ia characterizes the mass distribution in the molecule . for all molecules studied here , 3-d modeling and calculations were performed using the gaussian 03 quantum chemistry package . to save in computational time , initial geometry optimizations were carried out with the molecular mechanics ( mm ) method using amber force field . the lowest energy conformations of the molecules obtained by the mm method were further optimized by the dft method by employing becke s three - parameter hybrid functional ( b3lyp ) and the 6 - 31+g ( d ) basis set ; their fundamental vibrations were also calculated using the same method to check if there were true minima . all the computations were carried out for the ground states of these molecules as singlet state . the lowest energy conformations of the compounds obtained using dft were used as an input geometry for the calculations for hf/6 - 31+g , am1 and pm3 methods . ( codessa pro ) comprehensive descriptors for structural and statistical analysis , version 2.7.2 , was used for extracting descriptors of quantum mechanics and 3d geometry of the compounds from gaussian 03 output files . codessa pro enables the generation of hundreds of molecular descriptors ( constitutional , topological , and quantum mechanical ) from a loaded 3d geometry , and uses diverse statistical structure property / activity correlation techniques for the analysis of experimental data in combination with calculated molecular descriptors . a qsar / qstr model can be developed for a given set of molecules by using a various types of descriptors . sometimes , a model might have very good statistical parameters , but still not suffice to explore the mechanism of interaction between the ligand and receptor mechanistically . building a model with physically interpretable descriptors is an important task for value of a qsar / qstr work . in this study , we aimed to draw up a qstr model by using quantum mechanically calculated thermodynamical descriptors by virtue of which obtained models are usually mechanistically interpretable . about 50 thermodynamical descriptors depending on the number of atoms in a molecule were calculated using codessa pro and gaussian 03 packages . the heuristic method implemented in codessa pro was used to build up a multi - able regression model . by this method , a pre - selection of descriptors is accomplished . all descriptors are checked to ensure the following : ( a ) the values of each descriptor are available for each structure , and ( b ) there is a variation in these values . the descriptors for which values are not available for every structure in the data in question are discarded . descriptors having a constant value for all structures in the data set are also discarded . thereafter , the one - parameter correlation equations for each descriptor are calculated . to further reduce the number in the starting set of descriptors , the following criteria are applied and a descriptor is eliminated if any of the following conditions are met with : ( a ) the f - test s value for the one - parameter correlation with the descriptor is below 1.0 , ( b ) the squared correlation coefficient of the one - parameter equation is less than rmin by default 0.01 , ( c ) the parameter s t - value is less than t1 ( where rmin 0.1 by default and t1 1.5 by default are user defined values ) , ( d ) the descriptor is highly inter - correlated ( above rfull , where rfull is a user specified value by default 0.99 ) , with another descriptor . all the remaining descriptors are then listed in decreasing order according to the correlation coefficient of the corresponding one - parameter correlation equation . all two - parameter regression models with remaining descriptors are developed and ranked by the regression correlation coefficient r. a stepwise addition of the further descriptors scales is performed to find the best multi - parameter regression models with the optimum values of statistical criteria ( highest values of r , the cross - validated , rcv and the f value ) . rcv , the leave one out ( loo ) squared cross - validated coefficient , is a practical and reliable method for testing the predictive performance and stability of a regression model . loo approach consists in developing a number of models with one sample omitted at a time . after developing each model , the omitted data are predicted and the differences between the experimental and predicted activity values are calculated . then the rcv is calculated according to the following formula : where yi is the actual experimental activity , is the average actual experimental activity and i is the predicted activity of compound i computed by the new regression equation obtained each time after leaving out one datum point ( no . among the thermodynamical descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency were involved in the models that are presented in this study . this connection is based upon partitioning the total energy of a macroscopic system among the constituent molecules . translational entropy ( at 300 k ) is defined as ; where v is the volume of the system , n is the avogadro s number , h is the planck constant , m is the molecular mass , and kt is the boltzman temperature . highest normal mode of vibrational frequency and lowest normal mode of vibrational frequency are actually not frequencies ; they are wavenumbers ( in cm unit ) . it is customary to call normal modes of vibration of molecule as frequency in infrared and raman spectroscopy . definition of normal mode of vibration arises from quantum mechanical harmonic oscillator model of a diatomic molecule . in this model , energy of the vibrational states is given as , where h is the planck constant , is the vibrational quantum number ( 0 , 1 , 2 , ) , and v is the classical vibrational frequency given by , where k is the force constant of the chemical bond and is the reduced mass for nuclei of two atoms . more commonly , equation 5 is used as vibrational wavenumber ( ) form rather than frequency form , where where is the vibrational wavenumber , c is the velocity of light . final descriptor involved in our model is principal moment of inertia a ( ia ) that is obtained from the 3d - cooordinate of the atoms in the given molecule . its definition is given as , where mi are the atomic masses and rix denotes the distance of the i - th atomic nucleus from the main rotational axes , x. ia characterizes the mass distribution in the molecule . the qsar / qstr method saves time and cost in determining the toxicity of a series of newly synthesized compounds with the help of toxicity of previously known compounds . forty - eight compounds have been taken in this study , and their toxicity ( loglc50 ) and calculated logarithm of 1-octanol / water partition coefficient ( logp ) values to fathead minnow have been taken from the literature and are given in table 1 . among the quantum mechanically calculated descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , and highest normal mode of vibrational frequency and lowest normal mode of vibrational frequency have been identified which are capable of modeling the toxicity and the structure of a molecule . the data matrix of these descriptors obtained from first principal ( hf and dft - b3lyp ) and semi - empirical ( am1 and pm3 ) methods calculations are shown in table 2 to5 . by using dft - based descriptors , several equations were generated by using all the variables and the statistically best model that we have obtained is four - parameters equation , which is as follows : where n is the number of compounds included in the model , r is the squared correlation coefficient , rcv is the squared cross - validation correlation coefficient , f is the fisher test for significance of the equation and s is the standard deviation of the regression . the statistical quality of the above equation is good as evident from its correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . the predicted toxicity of the compounds is given in table 2 by using equation 7 . in this dft - based model , compounds 23 , 38 , and 44 were selected as outliners due to the fact that during the calculation of their geometry and vibrational frequencies , all our attempts had failed to get all the frequencies as positive . this means that obtained structures of these molecules do not correspond to the global minima of potential energy surface . second , model based on other first principle method used in present study , namely , hf method is found as follows : statistical quality of this model is very close to the dft - based one . the predicted toxicity of the compounds by using equation 8 is given in table 3 . in this hf - based model , compounds 18 , 23 , and 30 were selected as outliers due to the same reason as dft - based model . by using am1-based descriptors , the statistically best model that we have obtained is as follows : second model , namely , pm3 based on other semi - empirical method used in present study was found as follow : statistical fit of equation 10 is similar to equation 9 . this result indicates that am1 and pm3 based models are similar statistical fit , but pm3 based model has a lower predictive power as is evident from its lower value of squared cross - validated coefficient ( rcv = 0.66 ) . finally , in order to elucidate the relationship between the hydrophobicity of compounds and their toxicity to fathead minnow , we have added logp value of compounds as an additional descriptor to the dft - based model ( equation 7 ) . influence of the logp to statistical fit of the equation 7 is as follows : as can be seen in equation 11 , statistical quality of dft - based model was increased dramatically by adding logp to the model . the above linear regression models obtained using the descriptors from dft and hf calculations are much better than those obtained from semi - empirical am1 and pm3 methods . introducing logp to dft - based model figure 1 gives the plots of observed loglc50 versus the predicted loglc50 by equations ( 7)(11 ) for compounds . qsar / qstr model quality depends on the reliability of the dataset ( i.e. uncertainty in toxicological and physicochemical and/or structural data ) . the authors usually have to rely on experimental toxicological data set that is taken from literature . the data are assumed to provide a uniform measure of toxicity for all of the compounds studied . when the uncertainties in physicochemical and/or structural data are considered , the accuracy of the descriptors is an important element for the qsar / qstrs . the computational level of theory is a major task for the accuracy of descriptor calculation . above presented results semi - empirical methods such as am1 and pm3 use the empirical or experimental parameters to deal with the schrdinger equation and omit some molecular integral calculations , so they are much faster than the first principle hf and dft - b3lyp schemes . therefore , they are utilized more widely in the calculations of molecular properties . based one with the correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . the second best equation was hf - based one , and in this model r is 0.84 and rcv is 0.78 . hf does not include the effects of an instant electronic correlation , whereas dft - b3lyp does , so that hf is inferior to dft in theory . the statistical quality of equations obtained from the semi - empirical methods was lower than that of equations obtained from the dft and hf methods as expected . am1 and pm3 based models have given similar statistical fits , but pm3 based model has a lower predictive power as evident from its lower value of rcv = 0.66 in contrast to that of am1 value of rcv = 0.72 . for all of the above mentioned models , the most representative descriptor with the highest coefficient is the translational entropy ( at 300 k ) str , which negatively correlates to loglc50 . it should be noted that all of the thermochemical properties of a molecule such as str arise from the energetics of vibrational frequencies . this connection is based upon partitioning of the total energy of a macroscopic system among the constituent molecules . other two descriptors involved in the models are lowest normal mode of vibrational frequency , l and highest normal mode of vibrational frequency , h . l correlates negatively to loglc50 in all the models , whereas h correlates positively to loglc50 in dft , hf , and dft with logp models . in several qsar studies they suggested that the eigen value ( eva ) descriptors are derived from fundamental ir and raman range of molecular vibrational frequencies . the idea behind the use of such data as descriptors was that a significant amount of information pertaining to molecular properties might be contained within the molecular vibration wave function . another descriptor involved in the models in this study is principal moment of inertia a , ( ia ) . it is a geometrical descriptor and originates from the rigid rotator approximation model of molecules . results of present study demonstrate that quantum mechanically calculated thermo chemical descriptors , str , h , and l jointly with geometrical descriptor , ia are capable of modeling the acute toxicity of the compounds to the fathead minnow . first principle dft and hf methods led to statistically better models than that of semi - empirical am1 and pm3 methods . this result is normal because dft and hf can calculate molecular properties such as optimized geometry and spectroscopic properties more accurately than semi - empirical methods . our results are in disagreement with the conclusions of the other comparative study which has concluded that the use of dft / b3lyp does not have an advantage over am1 for the quality of the derived qstrs . it may be due to the difference of the size of molecule sets between two studies . another possible reason is that the models were restricted to build up using only quantum chemical and thermodynamical descriptors in our study whereas natzeva et al . finally , logp has been inserted as an additional descriptor into the statistically best model ( dft - based one ) . this resulted in an increase of statistical quality of the model for the parameters ( r from 0.85 to 0.89 , f from 60.34 to 70.82 , and s from 0.27 to 0.19 ) . as mentioned in introduction section , logp itself has been used as single descriptor for many qstr models . narcosis is a general term that describes noncovalent interaction between xenobiotics and cellular membranes . whereas it is generally accepted that narcosis is the result of the accumulation of the compounds in cell membranes that disturbs their function , the exact mechanism is not known yet . logp characterizes the hydrophobicity of a molecule that is directly related to bio - uptake of chemicals by fish or many other organisms . results presented in this study demonstrates that quantum mechanically calculated thermo chemical descriptors in combination with logp are capable of modeling the acute toxicity of a quite diverse set of 48 compounds to the fathead minnow . this qstr study has been based on quantum mechanically calculated descriptors ( such as entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency ) and on the acute toxicity of the 48 organic compounds to the fathead minnow . the reliability of this study has been tested by four different methods , namely , am1 , mp3 , hf , and dft / b3lyp . a comparison of all the methods indicates that the dft / b3lyp method is more reliable than others and has a high predictive power . introduction of logp as an additional descriptor into dft - based model has resulted in an increase of statistical parameters of the model . the qsar / qstr method saves time and cost in determining the toxicity of a series of newly synthesized compounds with the help of toxicity of previously known compounds . forty - eight compounds have been taken in this study , and their toxicity ( loglc50 ) and calculated logarithm of 1-octanol / water partition coefficient ( logp ) values to fathead minnow have been taken from the literature and are given in table 1 . among the quantum mechanically calculated descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , and highest normal mode of vibrational frequency and lowest normal mode of vibrational frequency have been identified which are capable of modeling the toxicity and the structure of a molecule . the data matrix of these descriptors obtained from first principal ( hf and dft - b3lyp ) and semi - empirical ( am1 and pm3 ) methods calculations are shown in table 2 to5 . by using dft - based descriptors , several equations were generated by using all the variables and the statistically best model that we have obtained is four - parameters equation , which is as follows : where n is the number of compounds included in the model , r is the squared correlation coefficient , rcv is the squared cross - validation correlation coefficient , f is the fisher test for significance of the equation and s is the standard deviation of the regression . the statistical quality of the above equation is good as evident from its correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . the predicted toxicity of the compounds is given in table 2 by using equation 7 . in this dft - based model , compounds 23 , 38 , and 44 were selected as outliners due to the fact that during the calculation of their geometry and vibrational frequencies , all our attempts had failed to get all the frequencies as positive . this means that obtained structures of these molecules do not correspond to the global minima of potential energy surface . second , model based on other first principle method used in present study , namely , hf method is found as follows : statistical quality of this model is very close to the dft - based one . the predicted toxicity of the compounds by using equation 8 is given in table 3 . in this hf - based model , compounds 18 , 23 , and 30 were selected as outliers due to the same reason as dft - based model . by using am1-based descriptors , the statistically best model that we have obtained is as follows : second model , namely , pm3 based on other semi - empirical method used in present study was found as follow : statistical fit of equation 10 is similar to equation 9 . this result indicates that am1 and pm3 based models are similar statistical fit , but pm3 based model has a lower predictive power as is evident from its lower value of squared cross - validated coefficient ( rcv = 0.66 ) . finally , in order to elucidate the relationship between the hydrophobicity of compounds and their toxicity to fathead minnow , we have added logp value of compounds as an additional descriptor to the dft - based model ( equation 7 ) . influence of the logp to statistical fit of the equation 7 is as follows : as can be seen in equation 11 , statistical quality of dft - based model was increased dramatically by adding logp to the model . the above linear regression models obtained using the descriptors from dft and hf calculations are much better than those obtained from semi - empirical am1 and pm3 methods . introducing logp to dft - based model figure 1 gives the plots of observed loglc50 versus the predicted loglc50 by equations ( 7)(11 ) for compounds . qsar / qstr model quality depends on the reliability of the dataset ( i.e. uncertainty in toxicological and physicochemical and/or structural data ) . the authors usually have to rely on experimental toxicological data set that is taken from literature . the data are assumed to provide a uniform measure of toxicity for all of the compounds studied . when the uncertainties in physicochemical and/or structural data are considered , the accuracy of the descriptors is an important element for the qsar / qstrs . the computational level of theory is a major task for the accuracy of descriptor calculation . above presented results semi - empirical methods such as am1 and pm3 use the empirical or experimental parameters to deal with the schrdinger equation and omit some molecular integral calculations , so they are much faster than the first principle hf and dft - b3lyp schemes . therefore , they are utilized more widely in the calculations of molecular properties . based one with the correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . the second best equation was hf - based one , and in this model r is 0.84 and rcv is 0.78 . hf does not include the effects of an instant electronic correlation , whereas dft - b3lyp does , so that hf is inferior to dft in theory . the statistical quality of equations obtained from the semi - empirical methods was lower than that of equations obtained from the dft and hf methods as expected . am1 and pm3 based models have given similar statistical fits , but pm3 based model has a lower predictive power as evident from its lower value of rcv = 0.66 in contrast to that of am1 value of rcv = 0.72 . for all of the above mentioned models , the most representative descriptor with the highest coefficient is the translational entropy ( at 300 k ) str , which negatively correlates to loglc50 . it should be noted that all of the thermochemical properties of a molecule such as str arise from the energetics of vibrational frequencies . this connection is based upon partitioning of the total energy of a macroscopic system among the constituent molecules . other two descriptors involved in the models are lowest normal mode of vibrational frequency , l and highest normal mode of vibrational frequency , h . l correlates negatively to loglc50 in all the models , whereas h correlates positively to loglc50 in dft , hf , and dft with logp models . in several qsar studies , fundamental vibrational frequencies of molecules have been used as descriptors [ 3539 ] . they suggested that the eigen value ( eva ) descriptors are derived from fundamental ir and raman range of molecular vibrational frequencies . the idea behind the use of such data as descriptors was that a significant amount of information pertaining to molecular properties might be contained within the molecular vibration wave function . another descriptor involved in the models in this study is principal moment of inertia a , ( ia ) . it is a geometrical descriptor and originates from the rigid rotator approximation model of molecules . results of present study demonstrate that quantum mechanically calculated thermo chemical descriptors , str , h , and l jointly with geometrical descriptor , ia are capable of modeling the acute toxicity of the compounds to the fathead minnow . first principle dft and hf methods led to statistically better models than that of semi - empirical am1 and pm3 methods . this result is normal because dft and hf can calculate molecular properties such as optimized geometry and spectroscopic properties more accurately than semi - empirical methods . our results are in disagreement with the conclusions of the other comparative study which has concluded that the use of dft / b3lyp does not have an advantage over am1 for the quality of the derived qstrs . the disagreement between two studies may result from several reasons . it may be due to the difference of the size of molecule sets between two studies . another possible reason is that the models were restricted to build up using only quantum chemical and thermodynamical descriptors in our study whereas natzeva et al . finally , logp has been inserted as an additional descriptor into the statistically best model ( dft - based one ) . this resulted in an increase of statistical quality of the model for the parameters ( r from 0.85 to 0.89 , f from 60.34 to 70.82 , and s from 0.27 to 0.19 ) . as mentioned in introduction section , logp itself has been used as single descriptor for many qstr models . narcosis is a general term that describes noncovalent interaction between xenobiotics and cellular membranes . whereas it is generally accepted that narcosis is the result of the accumulation of the compounds in cell membranes that disturbs their function , the exact mechanism is not known yet . logp characterizes the hydrophobicity of a molecule that is directly related to bio - uptake of chemicals by fish or many other organisms . results presented in this study demonstrates that quantum mechanically calculated thermo chemical descriptors in combination with logp are capable of modeling the acute toxicity of a quite diverse set of 48 compounds to the fathead minnow . this qstr study has been based on quantum mechanically calculated descriptors ( such as entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency ) and on the acute toxicity of the 48 organic compounds to the fathead minnow . the reliability of this study has been tested by four different methods , namely , am1 , mp3 , hf , and dft / b3lyp . a comparison of all the methods indicates that the dft / b3lyp method is more reliable than others and has a high predictive power . introduction of logp as an additional descriptor into dft - based model has resulted in an increase of statistical parameters of the model .

several quantum - mechanics - based descriptors were derived for a diverse set of 48 organic compounds using am1 , pm3 , hf/6 - 31+g , and dft - b3lyp/6 - 31+g ( d ) level of the theory . lc50 values of acute toxicity of the compounds were correlated to the fathead minnow and predicted using calculated descriptors by employing comprehensive descriptors for structural and statistical analysis ( codessa ) program . the heuristic method , implemented in the codessa program for selecting the best regression model , was applied to a pre - selection of the most - representative descriptors by sequentially eliminating descriptors that did not satisfy a certain level of statistical criterion . first model , statistically , the most significant one has been drawn up with the help of dft calculations in which the squared correlation coefficient r2 is 0.85 , and the squared cross - validation correlation coefficient rcv2 is 0.79 . second model , which has been drawn up with the help of hf calculations , has its statistical quality very close to the dft - based one and in this model value of r2 is 0.84 and that of rcv2 is 0.78 . third and fourth models have been drawn up with the help of am1 and pm3 calculations , respectively . the values of r2 and rcv2 in the third case are correspondingly 0.79 and 0.66 , whereas in the fourth case they are 0.78 and 0.65 respectively . results of this study clearly demonstrate that for the calculations of descriptors in modeling of acute toxicity of organic compounds to the fathead minnow , first principal methods are much more useful than semi - empirical methods .

Introduction Procedures and Calculations Methods Computational details Theory Results and Discussion Results Discussion Conclusion

the traditional approach to qsars for acute toxicity of organic compounds to the fathead minnow is the modeling of the activity of homologous or congeneric series of chemicals such as nitroaromatics , alkylamines , halogenated hydrocarbons and phenols , and chlorobenzenes and chloroalinines . it has been successfully used for the modeling of acute toxicity of chemicals with different modes and mechanism of toxic action to pimephales promelas , combined with additional parameters such as energy of lowest unoccupied molecular orbital ( elumo ) and maximum superdelocalizability ( smax ) , which is a molecular orbital parameter that quantifies the electro ( nucleo ) philicity of a molecule . developing a better qstr for the modeling of acute toxicity of diverse chemicals is a subject of interest due to its demand by the organizations such as oecd ( organization for economic co - operation and development ) or ec ( european communities ) to use the qstr model for regulatory purpose . the first one is to build qstr multiple regression model using quantum - mechanics - based molecular descriptors that correlate and predict the loglc50 value of acute toxicity of 48 compounds to the fathead minnow . molecules used in this study are quite a diverse set and were taken from a study . the second aim of this study is to compare the accuracy of semi - empirical and first principle methods for calculation of molecular descriptors . [ 16 , 17 ] are fast in computation , well suited to organic compounds , and belong to semi - empirical method family . in that study , a huge molecule set ( 568 molecules ) these qstr models have been built up from descriptors which were calculated using two different theory levels namely am1 and dft / b3lyp ( 6 - 31 g * * ) . the lowest energy conformations of the molecules obtained by the mm method were further optimized by the dft method by employing becke s three - parameter hybrid functional ( b3lyp ) and the 6 - 31+g ( d ) basis set ; their fundamental vibrations were also calculated using the same method to check if there were true minima . the lowest energy conformations of the compounds obtained using dft were used as an input geometry for the calculations for hf/6 - 31+g , am1 and pm3 methods . ( codessa pro ) comprehensive descriptors for structural and statistical analysis , version 2.7.2 , was used for extracting descriptors of quantum mechanics and 3d geometry of the compounds from gaussian 03 output files . the heuristic method implemented in codessa pro was used to build up a multi - able regression model . by this method , a pre - selection of descriptors is accomplished . to further reduce the number in the starting set of descriptors , the following criteria are applied and a descriptor is eliminated if any of the following conditions are met with : ( a ) the f - test s value for the one - parameter correlation with the descriptor is below 1.0 , ( b ) the squared correlation coefficient of the one - parameter equation is less than rmin by default 0.01 , ( c ) the parameter s t - value is less than t1 ( where rmin 0.1 by default and t1 1.5 by default are user defined values ) , ( d ) the descriptor is highly inter - correlated ( above rfull , where rfull is a user specified value by default 0.99 ) , with another descriptor . all two - parameter regression models with remaining descriptors are developed and ranked by the regression correlation coefficient r. a stepwise addition of the further descriptors scales is performed to find the best multi - parameter regression models with the optimum values of statistical criteria ( highest values of r , the cross - validated , rcv and the f value ) . rcv , the leave one out ( loo ) squared cross - validated coefficient , is a practical and reliable method for testing the predictive performance and stability of a regression model . after developing each model , the omitted data are predicted and the differences between the experimental and predicted activity values are calculated . among the thermodynamical descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency were involved in the models that are presented in this study . in this model , energy of the vibrational states is given as , where h is the planck constant , is the vibrational quantum number ( 0 , 1 , 2 , ) , and v is the classical vibrational frequency given by , where k is the force constant of the chemical bond and is the reduced mass for nuclei of two atoms . the lowest energy conformations of the molecules obtained by the mm method were further optimized by the dft method by employing becke s three - parameter hybrid functional ( b3lyp ) and the 6 - 31+g ( d ) basis set ; their fundamental vibrations were also calculated using the same method to check if there were true minima . the lowest energy conformations of the compounds obtained using dft were used as an input geometry for the calculations for hf/6 - 31+g , am1 and pm3 methods . ( codessa pro ) comprehensive descriptors for structural and statistical analysis , version 2.7.2 , was used for extracting descriptors of quantum mechanics and 3d geometry of the compounds from gaussian 03 output files . the heuristic method implemented in codessa pro was used to build up a multi - able regression model . by this method , a pre - selection of descriptors is accomplished . to further reduce the number in the starting set of descriptors , the following criteria are applied and a descriptor is eliminated if any of the following conditions are met with : ( a ) the f - test s value for the one - parameter correlation with the descriptor is below 1.0 , ( b ) the squared correlation coefficient of the one - parameter equation is less than rmin by default 0.01 , ( c ) the parameter s t - value is less than t1 ( where rmin 0.1 by default and t1 1.5 by default are user defined values ) , ( d ) the descriptor is highly inter - correlated ( above rfull , where rfull is a user specified value by default 0.99 ) , with another descriptor . all two - parameter regression models with remaining descriptors are developed and ranked by the regression correlation coefficient r. a stepwise addition of the further descriptors scales is performed to find the best multi - parameter regression models with the optimum values of statistical criteria ( highest values of r , the cross - validated , rcv and the f value ) . rcv , the leave one out ( loo ) squared cross - validated coefficient , is a practical and reliable method for testing the predictive performance and stability of a regression model . after developing each model , the omitted data are predicted and the differences between the experimental and predicted activity values are calculated . among the thermodynamical descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency were involved in the models that are presented in this study . in this model , energy of the vibrational states is given as , where h is the planck constant , is the vibrational quantum number ( 0 , 1 , 2 , ) , and v is the classical vibrational frequency given by , where k is the force constant of the chemical bond and is the reduced mass for nuclei of two atoms . the qsar / qstr method saves time and cost in determining the toxicity of a series of newly synthesized compounds with the help of toxicity of previously known compounds . forty - eight compounds have been taken in this study , and their toxicity ( loglc50 ) and calculated logarithm of 1-octanol / water partition coefficient ( logp ) values to fathead minnow have been taken from the literature and are given in table 1 . among the quantum mechanically calculated descriptors , translational entropy ( at 300 k ) , principal moment of inertia a , and highest normal mode of vibrational frequency and lowest normal mode of vibrational frequency have been identified which are capable of modeling the toxicity and the structure of a molecule . the data matrix of these descriptors obtained from first principal ( hf and dft - b3lyp ) and semi - empirical ( am1 and pm3 ) methods calculations are shown in table 2 to5 . by using dft - based descriptors , several equations were generated by using all the variables and the statistically best model that we have obtained is four - parameters equation , which is as follows : where n is the number of compounds included in the model , r is the squared correlation coefficient , rcv is the squared cross - validation correlation coefficient , f is the fisher test for significance of the equation and s is the standard deviation of the regression . the statistical quality of the above equation is good as evident from its correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . in this dft - based model , compounds 23 , 38 , and 44 were selected as outliners due to the fact that during the calculation of their geometry and vibrational frequencies , all our attempts had failed to get all the frequencies as positive . second , model based on other first principle method used in present study , namely , hf method is found as follows : statistical quality of this model is very close to the dft - based one . in this hf - based model , compounds 18 , 23 , and 30 were selected as outliers due to the same reason as dft - based model . by using am1-based descriptors , the statistically best model that we have obtained is as follows : second model , namely , pm3 based on other semi - empirical method used in present study was found as follow : statistical fit of equation 10 is similar to equation 9 . this result indicates that am1 and pm3 based models are similar statistical fit , but pm3 based model has a lower predictive power as is evident from its lower value of squared cross - validated coefficient ( rcv = 0.66 ) . finally , in order to elucidate the relationship between the hydrophobicity of compounds and their toxicity to fathead minnow , we have added logp value of compounds as an additional descriptor to the dft - based model ( equation 7 ) . influence of the logp to statistical fit of the equation 7 is as follows : as can be seen in equation 11 , statistical quality of dft - based model was increased dramatically by adding logp to the model . the above linear regression models obtained using the descriptors from dft and hf calculations are much better than those obtained from semi - empirical am1 and pm3 methods . above presented results semi - empirical methods such as am1 and pm3 use the empirical or experimental parameters to deal with the schrdinger equation and omit some molecular integral calculations , so they are much faster than the first principle hf and dft - b3lyp schemes . therefore , they are utilized more widely in the calculations of molecular properties . based one with the correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . the second best equation was hf - based one , and in this model r is 0.84 and rcv is 0.78 . the statistical quality of equations obtained from the semi - empirical methods was lower than that of equations obtained from the dft and hf methods as expected . am1 and pm3 based models have given similar statistical fits , but pm3 based model has a lower predictive power as evident from its lower value of rcv = 0.66 in contrast to that of am1 value of rcv = 0.72 . for all of the above mentioned models , the most representative descriptor with the highest coefficient is the translational entropy ( at 300 k ) str , which negatively correlates to loglc50 . results of present study demonstrate that quantum mechanically calculated thermo chemical descriptors , str , h , and l jointly with geometrical descriptor , ia are capable of modeling the acute toxicity of the compounds to the fathead minnow . first principle dft and hf methods led to statistically better models than that of semi - empirical am1 and pm3 methods . this result is normal because dft and hf can calculate molecular properties such as optimized geometry and spectroscopic properties more accurately than semi - empirical methods . our results are in disagreement with the conclusions of the other comparative study which has concluded that the use of dft / b3lyp does not have an advantage over am1 for the quality of the derived qstrs . finally , logp has been inserted as an additional descriptor into the statistically best model ( dft - based one ) . this resulted in an increase of statistical quality of the model for the parameters ( r from 0.85 to 0.89 , f from 60.34 to 70.82 , and s from 0.27 to 0.19 ) . results presented in this study demonstrates that quantum mechanically calculated thermo chemical descriptors in combination with logp are capable of modeling the acute toxicity of a quite diverse set of 48 compounds to the fathead minnow . this qstr study has been based on quantum mechanically calculated descriptors ( such as entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency ) and on the acute toxicity of the 48 organic compounds to the fathead minnow . the reliability of this study has been tested by four different methods , namely , am1 , mp3 , hf , and dft / b3lyp . the qsar / qstr method saves time and cost in determining the toxicity of a series of newly synthesized compounds with the help of toxicity of previously known compounds . forty - eight compounds have been taken in this study , and their toxicity ( loglc50 ) and calculated logarithm of 1-octanol / water partition coefficient ( logp ) values to fathead minnow have been taken from the literature and are given in table 1 . the data matrix of these descriptors obtained from first principal ( hf and dft - b3lyp ) and semi - empirical ( am1 and pm3 ) methods calculations are shown in table 2 to5 . by using dft - based descriptors , several equations were generated by using all the variables and the statistically best model that we have obtained is four - parameters equation , which is as follows : where n is the number of compounds included in the model , r is the squared correlation coefficient , rcv is the squared cross - validation correlation coefficient , f is the fisher test for significance of the equation and s is the standard deviation of the regression . the statistical quality of the above equation is good as evident from its correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . in this dft - based model , compounds 23 , 38 , and 44 were selected as outliners due to the fact that during the calculation of their geometry and vibrational frequencies , all our attempts had failed to get all the frequencies as positive . second , model based on other first principle method used in present study , namely , hf method is found as follows : statistical quality of this model is very close to the dft - based one . in this hf - based model , compounds 18 , 23 , and 30 were selected as outliers due to the same reason as dft - based model . by using am1-based descriptors , the statistically best model that we have obtained is as follows : second model , namely , pm3 based on other semi - empirical method used in present study was found as follow : statistical fit of equation 10 is similar to equation 9 . this result indicates that am1 and pm3 based models are similar statistical fit , but pm3 based model has a lower predictive power as is evident from its lower value of squared cross - validated coefficient ( rcv = 0.66 ) . finally , in order to elucidate the relationship between the hydrophobicity of compounds and their toxicity to fathead minnow , we have added logp value of compounds as an additional descriptor to the dft - based model ( equation 7 ) . influence of the logp to statistical fit of the equation 7 is as follows : as can be seen in equation 11 , statistical quality of dft - based model was increased dramatically by adding logp to the model . the above linear regression models obtained using the descriptors from dft and hf calculations are much better than those obtained from semi - empirical am1 and pm3 methods . above presented results semi - empirical methods such as am1 and pm3 use the empirical or experimental parameters to deal with the schrdinger equation and omit some molecular integral calculations , so they are much faster than the first principle hf and dft - b3lyp schemes . therefore , they are utilized more widely in the calculations of molecular properties . based one with the correlation coefficient r value = 0.85 and a cross - validation coefficient rcv value = 0.79 . the second best equation was hf - based one , and in this model r is 0.84 and rcv is 0.78 . the statistical quality of equations obtained from the semi - empirical methods was lower than that of equations obtained from the dft and hf methods as expected . am1 and pm3 based models have given similar statistical fits , but pm3 based model has a lower predictive power as evident from its lower value of rcv = 0.66 in contrast to that of am1 value of rcv = 0.72 . for all of the above mentioned models , the most representative descriptor with the highest coefficient is the translational entropy ( at 300 k ) str , which negatively correlates to loglc50 . results of present study demonstrate that quantum mechanically calculated thermo chemical descriptors , str , h , and l jointly with geometrical descriptor , ia are capable of modeling the acute toxicity of the compounds to the fathead minnow . first principle dft and hf methods led to statistically better models than that of semi - empirical am1 and pm3 methods . this result is normal because dft and hf can calculate molecular properties such as optimized geometry and spectroscopic properties more accurately than semi - empirical methods . our results are in disagreement with the conclusions of the other comparative study which has concluded that the use of dft / b3lyp does not have an advantage over am1 for the quality of the derived qstrs . finally , logp has been inserted as an additional descriptor into the statistically best model ( dft - based one ) . this resulted in an increase of statistical quality of the model for the parameters ( r from 0.85 to 0.89 , f from 60.34 to 70.82 , and s from 0.27 to 0.19 ) . results presented in this study demonstrates that quantum mechanically calculated thermo chemical descriptors in combination with logp are capable of modeling the acute toxicity of a quite diverse set of 48 compounds to the fathead minnow . this qstr study has been based on quantum mechanically calculated descriptors ( such as entropy ( at 300 k ) , principal moment of inertia a , highest normal mode of vibrational frequency , and lowest normal mode of vibrational frequency ) and on the acute toxicity of the 48 organic compounds to the fathead minnow . the reliability of this study has been tested by four different methods , namely , am1 , mp3 , hf , and dft / b3lyp . introduction of logp as an additional descriptor into dft - based model has resulted in an increase of statistical parameters of the model .

the provision of high quality drinking water is a priority for many regions of the globe [ 13 ] . in particular , arsenic contamination of drinking water supplies is a public health issue of growing importance . in some of the rural parts of bangladesh , arsenic contamination has been an ongoing public health scourge , causing economic harm in the form of reduced supply of household labor . bangladeshis are exposed to arsenic by consuming water from wells which tap into contaminated aquifers . in addition to bangladesh , arsenic - contaminated aquifers exist in many parts of the globe , including china , sri lanka , argentina , and several regions in the us . in the us , the growing awareness and concern over arsenic contamination have prompted regional water leaders and water managers to investigate this issue and identify strategies that may be effective in reducing risks to public health [ 1114 ] . the health - related harms associated with arsenic exposure include increased risks of a wide variety of diseases and outcomes , including skin lesions , cancers , and increased mortality rates . some of these health risks may be intensified when coupled with other known cancer - causing behaviors , such as cigarette smoking . contact and ingestion of arsenic - contaminated water can be avoided through public and/or community - level activities , such as enhancing water treatment processes at the point of distribution . these top - down , public - good efforts may require buy - in and implementation across regions , across levels of government , and may involve complex cost - sharing arrangements . health - risk aversion activities can also occur at the household level , where such activities may be the private response of local residents and families or parts of a larger publically funded campaign or project . effective point - of - use treatment systems in the household for arsenic contamination can include reverse osmosis systems , ceramic filters specifically designed for arsenic removal , or even the purchase and consumption of arsenic - free bottled water . this study investigates health - risk reduction and aversion activities at the household level in the context of several us arsenic - contaminated locations . previous research on the analysis of household water use as it relates to health and water quality issues can be divided into two categories , focusing on either valuations of water improvements or household behaviors . valuation measurements have been estimated by eliciting a willingness to pay for improvements in water quality or reductions in water - related health risks [ 24 , 25 ] . the second category of studies that focus on behavior , specifically household - level decisions , has investigated the nature and content of informational messages [ 26 , 27 ] or the particular health - risk circumstances and the types of environmental contaminants affecting the household 's health - risk aversion decision . the present study falls within the behavior - focused category and adds to this growing literature by investigating the associations of different sources of water - related health - risk information may have with household - level health - risk aversion behavior . this paper proposes a two - stage model to separate the effect of information from a household 's propensity to acquire health - related information . for water resource planners and managers , the optimal portfolio of water - related , health - risk reducing strategies is likely to contain a mixture of public and private actions . theoretically , ignoring the private actions in such circumstances may result in unnecessary and excessive spending by the public sector . more practically , understanding the aversion behaviors of residents allows water planners and managers to implement more precise and effective public outreach programs to achieve a community 's water - related public health objectives . the objective of this study is to implement a propensity - score matching analysis and to gain a better understanding of the characteristics of water consumers who chose to engage in private health - risk reducing activities in response to knowledge about regional arsenic contamination . supposing that , first , a household acquires water - related health - risk information and then decides whether or not to engage in an activity that averts some or all of the health risks , the effect of information at the household level on the decision to engage in a water - contamination aversion activity is modeled as a two - stage process . in the first stage , the household 's propensity to acquire water - related health information is estimated . using the propensity scores estimated in the first stage to weight observations , the second stage estimates the associations between a household 's decision to implement a point - of - use filtration device and the household 's relevant characteristics as well as the source of water - contamination information . this paper proceeds with a description of the methods and the results and concludes with a discussion of results . the conceptual model subsection describes the concern for the endogenous determination of health - risk information and aversion behavior in a household . the description of data highlights some of the sample characteristics , study limitations , and assumptions that are required to map the components of the dataset with the components identified in the conceptual model . beyond the intuitive appeal of the two - stage model , the empirical benefits of modeling the aversion decision as a two - stage process are demonstrated by comparing estimation results from the two - stage model with estimation results from a reduced form ( or nave ) model , which does not attempt to control for the endogeneity of acquiring health - risk information and averting a health risk . the distribution of health - related risk information is one of the more readily available , implementable , and potentially less expensive tools in the toolkit held by planners and managers looking to affect desired public health objectives within a water supply and public health system . two broad categories of such factors may include the means of the information being transmitted through the population and the responsiveness of the population to information . the responsiveness of the local population is likely to depend on a number of factors . some of those factors may include familiarity with water treatment processes , health issues , the particular contaminant involved , or the level trust and believability in the information source held by local residents and business owners . once a household is informed about a water - related health issue , conditional on the value of the information along with other household characteristics , the household decision - making agent or decision - making process chooses whether or not to engage in some form of health - risk aversion activity . the objective of this study is to isolate the effect of information and , specifically , the source of information on the household 's decision to avert . for this study , information is defined as self - reported prior knowledge of arsenic contamination in the area and aversion is defined as self - reported use of a water treatment device in the home . conceptually , prior knowledge and the decision to avert are correlated with several location and household characteristics . the discovery of a more extensive level of arsenic contamination in a region is likely to induce greater public awareness as well as higher rates of aversion activity adoption . households that are predisposed to be aware of public health issues may be more likely to encounter public health - related information in addition to being more likely to adopt practices that reduce household health risks . this study implements a two - stage model to represent this decision - making process ( figure 1 ) . the outcome of the second stage is the household 's aversion decision , controlling for prior knowledge . the second stage controls for prior knowledge by using prior knowledge as a covariate in the aversion decision equation and also by weighting household observations by their estimated propensity to acquire prior knowledge . more details on the background and methodological description of using propensity scores and propensity - based regression weighting to estimate treatment effects can be found in the literature [ 2931 ] . the dataset used for this analysis has been used in several previous papers that address water consumption and health - risk behaviors in the context of arsenic - contaminated groundwater [ 19 , 22 , 32 ] , where more extensive descriptions of the data and survey methods can be found . to summarize , survey respondents were randomly selected from within several arsenic contamination these areas include albuquerque , new mexico ; fernley , nevada ; oklahoma city , oklahoma ; and appleton and outagamie county , wisconsin . for this paper , some observations were dropped from the complete survey dataset because of missing values , mostly in the income field . these characteristics of the data constitute sample biases , which should be considered when making inferences from the results of this study to a broader population . arsenic contamination aversion activities are captured by poufilter , an indicator for participation equal to one if the household uses any type of pou filtration device . unfortunately , the specific type of pou device is not known . this means potentially that the pou device in service at a household may not be appropriate for the effective treatment and removal of arsenic . initial analysis showed no measurable association with bottled water drinking and information about arsenic contamination . as goods that are potentially consumed to reduce health risk , bottled water may be considered a substitute ( or a complement ) to pou filtration and as such may also be subject to the same codetermined endogeneity as household - level use of a pou device . still , bottled water consumption has many other attributes . in particular , the effect on consumption from the convenience of bottled water may dwarf any perceived health - related benefits so as to make the health - related effect difficult or impossible to be measured in the current dataset . for this reason , bottled water is omitted from the presented analysis . in robustness checks , bottled water was considered as a covariate . in general , the relationship between bottled water consumption and use of a pou filter was negative , suggesting they may be substitutes in this sample . but this neither changed the direction nor substantially changed the statistical significance of the other estimates in the results of the two - stage model . like the aversion activity , prior knowledge is also captured by an indicator variable , descriptively named priorknow , and is equal to one if the respondent self - reports knowledge of the local arsenic contamination issue . the self - reported sources of any prior knowledge are captured by indicator variables as well : friend , utility , media , and othersource . these responses varied between the following : indicating arsenic contamination information was common knowledge , the information was received from a real estate professional , the information was acquired due to the respondent 's employment in a water supply - related business , or the information was received from a previous water - quality test at the household . the other explanatory variables used in this study capture various characteristics of the households facing the arsenic aversion decision . the variables age and schooling are , respectively , the self - reported age in years of the respondent and the approximate highest grade level of schooling completed by the respondent . the variable haschildren is an indicator variable equal to one if children are present in the household , where children are defined as persons who are under the age of 18 . the descriptive statistics are presented for a pooled sample as well as two subsamples that represent , for the purposes of this study , the subsample of nonaverters and the subsample of averters . the nonaverters correspond to poufilter = 0 and the averters correspond to poufilter = 1 . just less than half ( 48.5% ) of the households are averters . and , in the pooled sample , approximately 62% ( or 166 respondents ) reported knowing about the local arsenic contamination . only 9% ( or 25 respondents ) of the pooled sample reported knowledge of the arsenic contamination and that a friend is a source of that knowledge . comparing averters to nonaverters , a larger percentage of averters report having obtained their prior knowledge from a friend or othersource . the averting subsample has higher average income levels and a higher proportion of households with children present ( haschildren ) . the two subsamples also have different geographic distributions . proportionally , more averting households are found in nevada and wisconsin , with proportionally fewer found in new mexico and oklahoma . the exact reasons for these differences in the geographic distribution of the subsamples are unknown but could be due to the heterogeneous influence and efforts of the local water supply system managers , health departments , the pervasiveness of arsenic - contamination - related knowledge , and the availability of goods and services that might be useful in aversion activities . the statistical or empirical approach of this study investigates which of the four potential sources of prior knowledge has the greatest , or any measurable , effect on the household decision to avert arsenic - related health risks . initially , all four sources of information are considered within two reduced form , or nave , probit models . the first of these reduced form models , called the basic model , contains only the source of information as covariates . the second of these models , called the expanded model , contains information sources along with the full suite of available household characteristics . , in the basic reduced form specification , the entirety of the variation in poufilter is attributed to the source of information and an error term . since no other covariates are included , relative to the other statistical models in the study , the basic reduced form model is the one most likely to measure some statistically significant effect on an information source , naively , due to confounding from one of many potentially omitted variables . therefore , any statistically significant effect that is measured in the basic model requires additional investigation to determine if the effect is more likely to be causal or to be a measurement of correlated effects from other omitted covariates . the expanded reduced form model adds these additional covariates into the set of repressors to investigate if any of the effects measured in the basic model is reduced or reversed or made unmeasurable by the introduction of previously omitted variables . controlling for these other covariates , any effects among the information sources that remain statistically significant in the expanded model suggest an information source that is more likely to reflect an actual causal association and more likely to produce measurable effects in later analyses . the second purpose of the reduced form model is to underscore the usefulness of using the two - stage approach . the two - stage model is more conceptually intuitive and , as will be shown in the results section , the two - stage model identifies more empirical effects which are in line with intuitive expectations about household behavior and averting health risks . the available data and variable names used in the empirical analysis are presented alongside the associated components of the conceptual model in table 2 . both equations of the two - stage model are estimated using a probit procedure . in the first stage , the independent variables capture geographic and household characteristics by including geographic , state - level indicator variables ( nm , nv , and wi ) , and household - level variables likely to be correlated with the household 's social group and education level ( edu , age , and income ) . after the propensity scores from the first stage estimation are recorded , a gaussian kernel matching procedure is performed to identify the matched control sample . to achieve statistical balance between the matched control and treatment samples among the determinants of friend propensity , quadratic terms of the household characteristics are also included as explanatory variables in the first stage . in robustness checks of the two - stage and the reduced form models , the logit procedure was used in place of the probit procedure , finding no qualitative differences in results . as with the first stage , the selection of independent variables reflects the best attempt to represent the second stage of the conceptual model with the available data ( table 2 ) . as in the first stage , the geographic characteristics are once again represented by group of state - level indicator variables . household characteristics in the second stage are represented by the household - level variables income and haschildren . the results are presented in three sections : the reduced form model , the first stage of the two - stage model , and the second stage . the statistical significance of friend in both the basic and the expanded reduced form models motivates the later focus of this study on information from a friend . in the basic reduced form model , the set of explanatory variables is limited to only the potential sources of information about arsenic contamination events ( i.e. , only friend , utility , media , and othersource ) . in so doing , relative to the other models presented in this study , the basic model attributes the most dependent variable variation to the information sources . under these generous assumptions , as the name implies , the expanded reduced form model expands the basic model by including the full set of potential cofactors . these results ( table 3 ) first , they demonstrate the source of information is correlated with the other potential covariates ( those covariates that are omitted from the basic model ) . in particular , the geographic indicator variables seem particularly influential , with statistically significant effects measured for nv , nm , and wi . with the alternative covariates included in the expanded model , the effect on othersource is no longer measurable . the effect measured on friend in the expanded reduced form model may be suggestive of a measurable causal relationship , which is more explicitly represented in the two - stage model . this raises concern that the reduced form model misspecifies the household - aversion decision , since past findings in the literature have indicated that both income and the presence of children [ 33 , 34 ] influence valuation of water supply improvements . in the first stage , the objective of the matching step ( or first stage ) is to obtain two samples ( the treated and the matched control ) that are balanced ( or equal ) in the values of the observed variables . no statistically significant differences are found among the treatment and matched control groups ; however , the treated group only consists of 25 observations , which may provide insufficient power to identify a difference between the treatment and matched control samples . while their differences are not statistically significant , table 4 shows that the two groups are not identical , particularly among the geographic indicator variables , where the treated group contains a higher proportion of observations from wisconsin than from new mexico , nevada , and oklahoma . table 5 presents the results from the aversion stage with and without propensity - score weights . after adjustment for the propensity to be informed of arsenic contamination by a friend , the information from the friend exhibits a positive association with the probability of adopting an aversion practice . in addition to measuring a significant positive effect on friend , the propensity - weighted model brings to light previously unmeasured associations . in contrast to the reduced form results of table 3 , the propensity - weighted model measures a significant positive effect on haschildren and income . the effect of haschildren comports with previous findings in the literature [ 23 , 33 ] . with other things being equal , households with children are more likely to perceive and respond to household health risks . both the weighted and the nonweighted regressions measure a small effect for income , suggesting that reductions in health risk behave like normal goods . overall results presented in this study underscore the ability for households to privately pursue reductions to their water - related health risks . in particular , this research investigates whether or not any particular source of information seems more influential than another in motivating aversion behaviors . the primary finding of this study is that water - contamination information from a friend is found to have the strongest association with the adoption of an aversion activity , which in this case is defined as the implementation of a point - of - use filtration device in the household . additional findings include positive and statistically significant associations between aversion and household income as well as aversion and the presence of children in the household . for the purposes practically affecting the aversion behavior of individuals exposed to arsenic contamination , the number of children and the income belonging to a household are not generally considered within the purview of environmental and public health groups . however , in the future , efforts to communicate health - risk information may be calibrated to take advantage of the most effective communication mediums and may be directed towards individuals , locations , or institutions with relatively extensive social networks . the reduced form probit regressions served largely an exploratory role . since the reduced form analysis pointed to knowledge from a friend as bearing the largest association with adoption of aversion practices , the subsequent analyses focused exclusively on prior knowledge from a friend . in robustness checks , the other sources of information were subjected to the same stage 1 and stage 2 analyses with no statistically significant result measured for their effects . while these results are generated from a propensity - score matching process that produced no statistically significant differences between the treated and matched control samples , the qualification remains that the number of treatment observations is relatively small , making any difference between the treated and match control groups relatively less likely to be statistically significant . another limitation worth discussion is the appropriateness of state - level indicator variables serving as proxy for the geographically correlated contributing factors in the 1st and 2nd stages . first , recall that respondents are surveyed from only one locale within each state ; so , while these variables may be referred to as state - level , they actually capture a spatial scale closer to a city or county . in the 1st stage , geographic indicators proxy for the level of arsenic contamination across a region or a city and local utility and media networks across a region . in the 2nd stage , the state - level indicators proxy for the level of arsenic contamination , prices of aversion activities , and availability of aversion activities and alternatives . of the two stages , the assumptions in 2nd stage seem less appropriate than in the 1st stage because , in particular , the availability and relative prices of aversion activities and aversion activity substitutes and complements seem more likely to be heterogeneous across households within a given region than the other variables ( media and utility networks ) . considered another way , local media and utility systems are more likely to be homogeneous across households within a given region ; so , regional indicators are more appropriate . more generally , these results are qualified by several contextual conditions , some of which were directly addressed in the modeling and others not directly addressed due to the unavailability of data . the dataset is not drawn from a statistically representative sample of any particular state , region , or the us ; so , making inferences to larger populations is not warranted . since the data is self - reported and from a survey , standard concerns over recall accuracy , reporting accuracy , and potential sample bias are applicable and therefore any inferences and generalizations to other populations are not recommended . price data was not collected as part of the original survey and could not be reliably collected from other sources ex post . presumably , these prices are somewhat consistent across given locations ; so , while being not ideal , the price effect is assumed to be captured by the geographic indicator variables . another potentially relevant socioeconomic factor that is similarly omitted includes the reliability and ease of access to retail locations for bottled water and pou devices . the use of pou devices as well as the consumption of bottled water is likely substitute goods ( but could conceivably even be complement goods ) for the purposes of avoiding health risks from arsenic contamination of groundwater . as such , a model that ignores the reciprocating relationship between two related goods is likely to be biased in some way , but for this study the data could not support a more rigorous approach . in a robustness check , an indicator for bottled water consumption is added as a covariate variable in the propensity - weighted stage - two equation . a significant effect is found for bottled water consumption , but this did not substantially change the effect found on friend . in general , the small size of the dataset and particularly the small number of observations associated with the treatment variable friend prohibit drawing stronger conclusions from any of the findings in this paper . this paper primarily provides a workable illustration of propensity - score matching for the purposes of estimating the relative effectiveness of health - risk communication methods .

this paper provides a demonstration of propensity - score matching estimation methods to evaluate the effectiveness of health - risk communication efforts . this study develops a two - stage regression model to investigate household and respondent characteristics as they contribute to aversion behavior to reduce exposure to arsenic - contaminated groundwater . the aversion activity under study is a household - level point - of - use filtration device . since the acquisition of arsenic contamination information and the engagement in an aversion activity may be codetermined , a two - stage propensity - score model is developed . in the first stage , the propensity for households to acquire arsenic contamination information is estimated . then , the propensity scores are used to weight observations in a probit regression on the decision to avert the arsenic - related health risk . of four potential sources of information , utility , media , friend , or others , information received from a friend appears to be the source of information most associated with aversion behavior . other statistically significant covariates in the household 's decision to avert contamination include reported household income , the presence of children in household , and region - level indicator variables . these findings are primarily illustrative and demonstrate the usefulness of propensity - score methods to estimate health - risk communication effectiveness . they may also be suggestive of areas for future research .

1. Introduction 2. Methods 3. Results 4. Discussion

in particular , arsenic contamination of drinking water supplies is a public health issue of growing importance . in some of the rural parts of bangladesh , arsenic contamination has been an ongoing public health scourge , causing economic harm in the form of reduced supply of household labor . in addition to bangladesh , arsenic - contaminated aquifers exist in many parts of the globe , including china , sri lanka , argentina , and several regions in the us . in the us , the growing awareness and concern over arsenic contamination have prompted regional water leaders and water managers to investigate this issue and identify strategies that may be effective in reducing risks to public health [ 1114 ] . the health - related harms associated with arsenic exposure include increased risks of a wide variety of diseases and outcomes , including skin lesions , cancers , and increased mortality rates . some of these health risks may be intensified when coupled with other known cancer - causing behaviors , such as cigarette smoking . contact and ingestion of arsenic - contaminated water can be avoided through public and/or community - level activities , such as enhancing water treatment processes at the point of distribution . health - risk aversion activities can also occur at the household level , where such activities may be the private response of local residents and families or parts of a larger publically funded campaign or project . effective point - of - use treatment systems in the household for arsenic contamination can include reverse osmosis systems , ceramic filters specifically designed for arsenic removal , or even the purchase and consumption of arsenic - free bottled water . this study investigates health - risk reduction and aversion activities at the household level in the context of several us arsenic - contaminated locations . valuation measurements have been estimated by eliciting a willingness to pay for improvements in water quality or reductions in water - related health risks [ 24 , 25 ] . the second category of studies that focus on behavior , specifically household - level decisions , has investigated the nature and content of informational messages [ 26 , 27 ] or the particular health - risk circumstances and the types of environmental contaminants affecting the household 's health - risk aversion decision . the present study falls within the behavior - focused category and adds to this growing literature by investigating the associations of different sources of water - related health - risk information may have with household - level health - risk aversion behavior . this paper proposes a two - stage model to separate the effect of information from a household 's propensity to acquire health - related information . for water resource planners and managers , the optimal portfolio of water - related , health - risk reducing strategies is likely to contain a mixture of public and private actions . more practically , understanding the aversion behaviors of residents allows water planners and managers to implement more precise and effective public outreach programs to achieve a community 's water - related public health objectives . the objective of this study is to implement a propensity - score matching analysis and to gain a better understanding of the characteristics of water consumers who chose to engage in private health - risk reducing activities in response to knowledge about regional arsenic contamination . supposing that , first , a household acquires water - related health - risk information and then decides whether or not to engage in an activity that averts some or all of the health risks , the effect of information at the household level on the decision to engage in a water - contamination aversion activity is modeled as a two - stage process . in the first stage , the household 's propensity to acquire water - related health information is estimated . using the propensity scores estimated in the first stage to weight observations , the second stage estimates the associations between a household 's decision to implement a point - of - use filtration device and the household 's relevant characteristics as well as the source of water - contamination information . this paper proceeds with a description of the methods and the results and concludes with a discussion of results . the conceptual model subsection describes the concern for the endogenous determination of health - risk information and aversion behavior in a household . the description of data highlights some of the sample characteristics , study limitations , and assumptions that are required to map the components of the dataset with the components identified in the conceptual model . beyond the intuitive appeal of the two - stage model , the empirical benefits of modeling the aversion decision as a two - stage process are demonstrated by comparing estimation results from the two - stage model with estimation results from a reduced form ( or nave ) model , which does not attempt to control for the endogeneity of acquiring health - risk information and averting a health risk . the distribution of health - related risk information is one of the more readily available , implementable , and potentially less expensive tools in the toolkit held by planners and managers looking to affect desired public health objectives within a water supply and public health system . two broad categories of such factors may include the means of the information being transmitted through the population and the responsiveness of the population to information . some of those factors may include familiarity with water treatment processes , health issues , the particular contaminant involved , or the level trust and believability in the information source held by local residents and business owners . once a household is informed about a water - related health issue , conditional on the value of the information along with other household characteristics , the household decision - making agent or decision - making process chooses whether or not to engage in some form of health - risk aversion activity . the objective of this study is to isolate the effect of information and , specifically , the source of information on the household 's decision to avert . for this study , information is defined as self - reported prior knowledge of arsenic contamination in the area and aversion is defined as self - reported use of a water treatment device in the home . conceptually , prior knowledge and the decision to avert are correlated with several location and household characteristics . the discovery of a more extensive level of arsenic contamination in a region is likely to induce greater public awareness as well as higher rates of aversion activity adoption . households that are predisposed to be aware of public health issues may be more likely to encounter public health - related information in addition to being more likely to adopt practices that reduce household health risks . this study implements a two - stage model to represent this decision - making process ( figure 1 ) . the outcome of the second stage is the household 's aversion decision , controlling for prior knowledge . the second stage controls for prior knowledge by using prior knowledge as a covariate in the aversion decision equation and also by weighting household observations by their estimated propensity to acquire prior knowledge . more details on the background and methodological description of using propensity scores and propensity - based regression weighting to estimate treatment effects can be found in the literature [ 2931 ] . the dataset used for this analysis has been used in several previous papers that address water consumption and health - risk behaviors in the context of arsenic - contaminated groundwater [ 19 , 22 , 32 ] , where more extensive descriptions of the data and survey methods can be found . to summarize , survey respondents were randomly selected from within several arsenic contamination these areas include albuquerque , new mexico ; fernley , nevada ; oklahoma city , oklahoma ; and appleton and outagamie county , wisconsin . for this paper , some observations were dropped from the complete survey dataset because of missing values , mostly in the income field . arsenic contamination aversion activities are captured by poufilter , an indicator for participation equal to one if the household uses any type of pou filtration device . unfortunately , the specific type of pou device is not known . this means potentially that the pou device in service at a household may not be appropriate for the effective treatment and removal of arsenic . as goods that are potentially consumed to reduce health risk , bottled water may be considered a substitute ( or a complement ) to pou filtration and as such may also be subject to the same codetermined endogeneity as household - level use of a pou device . in particular , the effect on consumption from the convenience of bottled water may dwarf any perceived health - related benefits so as to make the health - related effect difficult or impossible to be measured in the current dataset . in general , the relationship between bottled water consumption and use of a pou filter was negative , suggesting they may be substitutes in this sample . but this neither changed the direction nor substantially changed the statistical significance of the other estimates in the results of the two - stage model . like the aversion activity , prior knowledge is also captured by an indicator variable , descriptively named priorknow , and is equal to one if the respondent self - reports knowledge of the local arsenic contamination issue . the self - reported sources of any prior knowledge are captured by indicator variables as well : friend , utility , media , and othersource . these responses varied between the following : indicating arsenic contamination information was common knowledge , the information was received from a real estate professional , the information was acquired due to the respondent 's employment in a water supply - related business , or the information was received from a previous water - quality test at the household . the other explanatory variables used in this study capture various characteristics of the households facing the arsenic aversion decision . the variables age and schooling are , respectively , the self - reported age in years of the respondent and the approximate highest grade level of schooling completed by the respondent . the variable haschildren is an indicator variable equal to one if children are present in the household , where children are defined as persons who are under the age of 18 . the descriptive statistics are presented for a pooled sample as well as two subsamples that represent , for the purposes of this study , the subsample of nonaverters and the subsample of averters . and , in the pooled sample , approximately 62% ( or 166 respondents ) reported knowing about the local arsenic contamination . only 9% ( or 25 respondents ) of the pooled sample reported knowledge of the arsenic contamination and that a friend is a source of that knowledge . comparing averters to nonaverters , a larger percentage of averters report having obtained their prior knowledge from a friend or othersource . the exact reasons for these differences in the geographic distribution of the subsamples are unknown but could be due to the heterogeneous influence and efforts of the local water supply system managers , health departments , the pervasiveness of arsenic - contamination - related knowledge , and the availability of goods and services that might be useful in aversion activities . the statistical or empirical approach of this study investigates which of the four potential sources of prior knowledge has the greatest , or any measurable , effect on the household decision to avert arsenic - related health risks . initially , all four sources of information are considered within two reduced form , or nave , probit models . the first of these reduced form models , called the basic model , contains only the source of information as covariates . , in the basic reduced form specification , the entirety of the variation in poufilter is attributed to the source of information and an error term . since no other covariates are included , relative to the other statistical models in the study , the basic reduced form model is the one most likely to measure some statistically significant effect on an information source , naively , due to confounding from one of many potentially omitted variables . therefore , any statistically significant effect that is measured in the basic model requires additional investigation to determine if the effect is more likely to be causal or to be a measurement of correlated effects from other omitted covariates . the expanded reduced form model adds these additional covariates into the set of repressors to investigate if any of the effects measured in the basic model is reduced or reversed or made unmeasurable by the introduction of previously omitted variables . controlling for these other covariates , any effects among the information sources that remain statistically significant in the expanded model suggest an information source that is more likely to reflect an actual causal association and more likely to produce measurable effects in later analyses . the second purpose of the reduced form model is to underscore the usefulness of using the two - stage approach . the two - stage model is more conceptually intuitive and , as will be shown in the results section , the two - stage model identifies more empirical effects which are in line with intuitive expectations about household behavior and averting health risks . both equations of the two - stage model are estimated using a probit procedure . in the first stage , the independent variables capture geographic and household characteristics by including geographic , state - level indicator variables ( nm , nv , and wi ) , and household - level variables likely to be correlated with the household 's social group and education level ( edu , age , and income ) . after the propensity scores from the first stage estimation are recorded , a gaussian kernel matching procedure is performed to identify the matched control sample . to achieve statistical balance between the matched control and treatment samples among the determinants of friend propensity , quadratic terms of the household characteristics are also included as explanatory variables in the first stage . in robustness checks of the two - stage and the reduced form models , the logit procedure was used in place of the probit procedure , finding no qualitative differences in results . as with the first stage , the selection of independent variables reflects the best attempt to represent the second stage of the conceptual model with the available data ( table 2 ) . as in the first stage , the geographic characteristics are once again represented by group of state - level indicator variables . household characteristics in the second stage are represented by the household - level variables income and haschildren . the results are presented in three sections : the reduced form model , the first stage of the two - stage model , and the second stage . the statistical significance of friend in both the basic and the expanded reduced form models motivates the later focus of this study on information from a friend . in the basic reduced form model , the set of explanatory variables is limited to only the potential sources of information about arsenic contamination events ( i.e. , only friend , utility , media , and othersource ) . in so doing , relative to the other models presented in this study , the basic model attributes the most dependent variable variation to the information sources . these results ( table 3 ) first , they demonstrate the source of information is correlated with the other potential covariates ( those covariates that are omitted from the basic model ) . in particular , the geographic indicator variables seem particularly influential , with statistically significant effects measured for nv , nm , and wi . with the alternative covariates included in the expanded model , the effect on othersource is no longer measurable . the effect measured on friend in the expanded reduced form model may be suggestive of a measurable causal relationship , which is more explicitly represented in the two - stage model . this raises concern that the reduced form model misspecifies the household - aversion decision , since past findings in the literature have indicated that both income and the presence of children [ 33 , 34 ] influence valuation of water supply improvements . in the first stage , the objective of the matching step ( or first stage ) is to obtain two samples ( the treated and the matched control ) that are balanced ( or equal ) in the values of the observed variables . no statistically significant differences are found among the treatment and matched control groups ; however , the treated group only consists of 25 observations , which may provide insufficient power to identify a difference between the treatment and matched control samples . while their differences are not statistically significant , table 4 shows that the two groups are not identical , particularly among the geographic indicator variables , where the treated group contains a higher proportion of observations from wisconsin than from new mexico , nevada , and oklahoma . table 5 presents the results from the aversion stage with and without propensity - score weights . after adjustment for the propensity to be informed of arsenic contamination by a friend , the information from the friend exhibits a positive association with the probability of adopting an aversion practice . in addition to measuring a significant positive effect on friend , the propensity - weighted model brings to light previously unmeasured associations . in contrast to the reduced form results of table 3 , the propensity - weighted model measures a significant positive effect on haschildren and income . both the weighted and the nonweighted regressions measure a small effect for income , suggesting that reductions in health risk behave like normal goods . overall results presented in this study underscore the ability for households to privately pursue reductions to their water - related health risks . in particular , this research investigates whether or not any particular source of information seems more influential than another in motivating aversion behaviors . the primary finding of this study is that water - contamination information from a friend is found to have the strongest association with the adoption of an aversion activity , which in this case is defined as the implementation of a point - of - use filtration device in the household . additional findings include positive and statistically significant associations between aversion and household income as well as aversion and the presence of children in the household . for the purposes practically affecting the aversion behavior of individuals exposed to arsenic contamination , the number of children and the income belonging to a household are not generally considered within the purview of environmental and public health groups . however , in the future , efforts to communicate health - risk information may be calibrated to take advantage of the most effective communication mediums and may be directed towards individuals , locations , or institutions with relatively extensive social networks . since the reduced form analysis pointed to knowledge from a friend as bearing the largest association with adoption of aversion practices , the subsequent analyses focused exclusively on prior knowledge from a friend . in robustness checks , the other sources of information were subjected to the same stage 1 and stage 2 analyses with no statistically significant result measured for their effects . while these results are generated from a propensity - score matching process that produced no statistically significant differences between the treated and matched control samples , the qualification remains that the number of treatment observations is relatively small , making any difference between the treated and match control groups relatively less likely to be statistically significant . another limitation worth discussion is the appropriateness of state - level indicator variables serving as proxy for the geographically correlated contributing factors in the 1st and 2nd stages . first , recall that respondents are surveyed from only one locale within each state ; so , while these variables may be referred to as state - level , they actually capture a spatial scale closer to a city or county . in the 1st stage , geographic indicators proxy for the level of arsenic contamination across a region or a city and local utility and media networks across a region . in the 2nd stage , the state - level indicators proxy for the level of arsenic contamination , prices of aversion activities , and availability of aversion activities and alternatives . of the two stages , the assumptions in 2nd stage seem less appropriate than in the 1st stage because , in particular , the availability and relative prices of aversion activities and aversion activity substitutes and complements seem more likely to be heterogeneous across households within a given region than the other variables ( media and utility networks ) . the dataset is not drawn from a statistically representative sample of any particular state , region , or the us ; so , making inferences to larger populations is not warranted . since the data is self - reported and from a survey , standard concerns over recall accuracy , reporting accuracy , and potential sample bias are applicable and therefore any inferences and generalizations to other populations are not recommended . presumably , these prices are somewhat consistent across given locations ; so , while being not ideal , the price effect is assumed to be captured by the geographic indicator variables . the use of pou devices as well as the consumption of bottled water is likely substitute goods ( but could conceivably even be complement goods ) for the purposes of avoiding health risks from arsenic contamination of groundwater . as such , a model that ignores the reciprocating relationship between two related goods is likely to be biased in some way , but for this study the data could not support a more rigorous approach . in a robustness check , an indicator for bottled water consumption is added as a covariate variable in the propensity - weighted stage - two equation . in general , the small size of the dataset and particularly the small number of observations associated with the treatment variable friend prohibit drawing stronger conclusions from any of the findings in this paper . this paper primarily provides a workable illustration of propensity - score matching for the purposes of estimating the relative effectiveness of health - risk communication methods .

systemic lupus erythematosus ( sle ) is an intriguing auto - immune disease which constitutes a complex interaction between the innate and adaptive immune system . conventional belief has cornered sle to be primarily a disease of autoantibodies and immune complex deposition . however , mounting evidence has implied that cytokines are also key players in the pathogenesis . cytokines are soluble factors which play a pivotal role in the differentiation , maturation , and activation of various immune cells . they are not only involved in immune dysregulation of sle , but also in the local inflammatory response which ultimately leads to tissue injury . the knowledge of cytokines not only provides new insight into pathogenesis of sle , but also sheds light on various clinical applications . in addition , the manipulation of these cytokines may become potential therapeutic targets for the treatment of sle . in this context , il-6 is a pleiotropic cytokine synthesized predominantly by monocytes , fibroblasts , and endothelial cells , although its secretion may also be found in t- and b - lymphocytes . its production is triggered by il-1 , il-2 , and tnf- but dampened by il-4 , il-10 , and il-13 . one of the most important effects of il-6 is to induce the maturation of b lymphocytes into plasma cells and augment the immunoglobulin secretion [ 2 , 3 ] . other actions include the up - regulation of il-2 and its receptor expression , stimulation of platelet production from megakaryocytes , differentiation of macrophage and osteoclast as well as the production of acute phase reactants . il-6 binds to the il-6 receptors which belong to the type 1 cytokine receptor superfamily that consists of two subunits , namely the il-6 r and the gp 130 . the interaction between il-6 and its receptor results in the dimerization of gp130 , activation of jak1 and subsequent tyrosine phosphorylation of gp130 . the pivotal role of il-6 in the pathogenesis of sle had been supported by both murine and human experiments . in mrl / lpr mice , there exists an age - related elevation of serum il-6 levels , soluble il6-r and aberrant expression of the il-6 r [ 4 , 5 ] . it should be highlighted that no other lymphokine studies have been shown to be capable of directly inducing the igg anti - dna antibodies . exogenous administration of recombinant human il-6 accelerated glomerulonephritis in nzb / w mice . in il-6 deficient mrl / lpr mice , there was significant reduction of infiltrating macrophages in the kidney , a decrease in renal igg and c3 deposition , and a diminution of cd4 and cd8 lymphocytes with the absence of il-6 . the renal parenchymal expression of adhesion molecule vcam-1 was also found to be down - regulated in mrl - fas(lpr ) il-6/ compared to il-6-intact mice . these data support the notion that il-6 is a strong promoter of lupus nephritis and may be a promising new therapeutic target in the treatment of human lupus nephritis . in fact , il-6 blockade modulated the age - related raise in anti - ds dna levels , retarded proteinuria and significantly improved mortality in nzb / w mice [ 8 , 9 ] . in b6.sle1.yaa mice , il-6 levels were elevated and the increase was coupled with the loss of cd19 b cells and more primitive b - lymphoid progenitors in bone marrow . stimulation by il-6 prompted these uncommitted progenitor cells to express transcription factors which inhibited lymphopoiesis and promoted myelopoiesis in sle . another mechanism of how il-6 may affect the b cell survival is via the recombination - activation gene ( rag ) machinery which are crucial for the revision of rearranged immunoglobulin v ( d ) j genes . il-6 favors the expression of rags and hence facilitates the rescue of autoreactive b cells from apoptosis . in jun b(delaep ) mice , the development of sle phenotype was linked to increased epidermal il-6 secretion and intercrosses with il-6 deficient mice could rescue the sle phenotype . these studies suggest a possible role of il-6 in the generation of autoantibodies and the development of various clinical manifestations in animal models . in human lupus patients , accentuated il-6 levels correlated with the disease activity and anti - dna levels [ 13 , 14 ] . lymphoblastoid cells isolated from lupus subjects expressed high levels of il-6 and il-6 antagonism resulted in reduction of anti - ds dna in vitro . in contrast to healthy subjects , b lymphocytes from lupus patients spontaneously generate heightened quantity of immunoglobulins ( ig ) . il-6 blockade significantly abolished this spontaneous immunoglobulin synthesis which was restored with exogenous il-6 administration . it had been shown that b - lymphocytes from lupus patients secreted anti - ds dna spontaneously and this autoantibody production ex vivo was predominantly caused by low density b lymphocytes . it is worthwhile to note that il-6 can facilitate these low density b cells from active lupus subjects to differentiate directly into ig - secreting cells . cd5 expression suppressed bcr signaling in sle b cells and il-6 down - regulated cd5 expression via dna methylation and hence promoted the activation and expansion of auto - reactive b cells in sle patients . apart from its systemic effects , il-6 was shown to have a particularly close link with the renal manifestation of sle . several studies demonstrated elevated urinary il-6 excretion in patients with active lupus nephritis ( who class iii / iv lupus nephritis ) who also had high titers of anti - ds dna antibodies [ 18 , 19 ] . the urinary level of il-6 in patients with active lupus nephritis was higher than that in patients with quiescent renal disease . moreover , there was enhanced in situ expression of il-6 along the glomeruli and tubules in lupus nephritis kidneys . apart from the kidneys , neuropsychiatric manifestation was associated with an excessive il-6 levels in the cerebrospinal fluid of lupus patients . these findings of raised il-6 in local tissues have suggested the pathogenetic role of il-6 in mediating local inflammation and tissue insults . il-6 and its receptors may be useful biomarkers to monitor disease activity and treatment response . il-6 release from pbmc was associated with disease activity and treatment response in lupus nephritis patients . other studies have also revealed an increased expression of il-6 agonistic receptor gp130 on peripheral lymphocytes in sle patients and its level correlated with the disease activity . the success of il-6 antagonism in murine models has prompted research into the therapeutic application of il-6 in human lupus . tocilizumab ( anti - il6 r antagonist ) when used in mild to moderate lupus patient has demonstrated preliminary success and good tolerability in phase i trials . it impedes the activation of antigen presenting cells ( apc ) , down - regulates the expression of co - stimulatory molecules and , thereby , blunts t cell activation and tnf- secretion . il-10 boosts b cell proliferation and immunoglobulin class switching resulting in enhanced antibody secretion with the capacity to enter extravascular compartments and promote inflammation in sle . several stimuli , including anti - ds dna antibodies and immune complexes when bound to fcrii , are potent triggers of il-10 [ 27 , 28 ] . prominent staining of il-10 in intra - renal cells was detected in kidneys of lupus patients , although the data suggested that the ifn/il-10 ratio is of more clinical relevance . elevated ifn/il-10 ratio was observed in active class iv lupus nephritis , while class v nephritis may behave oppositely in this regard . apparently , the increased il-10 was of pathogenic bearing in nzb / w mice where the effects of il-10 were largely opposite to those of tnf- . in fact , anti - il10 therapy led to increased tnf- and its beneficial effects were abrogated when tnf- was blocked . on the other hand , il-10 appeared to play a role in the regulatory t cell commitment and action , which might be beneficial in sle . the observed down - regulation of t cell activation in peripheral blood mononuclear cells from sle patients was consistent with such effects . in animal models of lupus nephritis , anti - il 10 blockade offered some benefits in limiting renal damage . preliminary data have shown that anti - il-10 monoclonal antibody improved cutaneous lesions , joint symptoms , and sledai in lupus patients . il-17 is a type i transmembrane protein isolated initially from a rodent cd4 t cell cdna library . it is a potent pro - inflammatory cytokine produced by activated t lymphocytes , with the th17 cells being the most vibrant producer . these th17 cells are in fact a subset of cd4 t lymphocytes named after its hallmark cytokine il-17 . th17 cells are regarded as a distinct t helper cell subset because they originate from nave t cells and only exhibit a characteristic cytokine release profile when appropriately primed by exclusive transcription factors . there are a total of six family members ( il-17 a to f ) and five receptors ( il-17r a to e ) in the il-17 family . il-17 has great potency to recruit monocytes and neutrophils , facilitate t cell infiltration , and up - regulate adhesion molecule expressions [ 37 , 38 ] . several important cytokines including il-6 , il-21 , and il-23 are in intimate association with il-17 . the combination of il-6 and transforming growth factor ( tgf) was shown to induce the differentiation of murine nave t cells into th17 cells [ 39 , 40 ] . these observations implied that the presence of an inflammatory signal is required to transform the nave t cells to become pro - inflammatory . , il-21 is produced by the th17 cells as well as the t - follicular helper cells but not by antigen presenting cells and , hence , being postulated to act as an auto - amplifier of th17 response . moreover , it is important to note the effect of il-23 on a pathogenic th17 response in nave wild - type mice upon t cell adoptive transfer . , these findings suggest the presence of a novel t helper functional axis ( il-23/il-17 axis ) which may be important in the pathogenesis of autoimmune disorders . although nave cd4 t cells can differentiate into th1 , th2 , or th17 effector subsets , the cytokine milieu characteristic of sle patients ( il-2 poor but il-6 and il-21 rich ) favors th17 expansion . th17 cells can also serve as an independent t helper effector cell subset promoting inflammation through cytokine secretion . the signature cytokines secreted by the th17 cells include il-17a , il-17f , il-21 , and il-22 . this array of cytokines can stimulate b lymphocytes , and set off local inflammation and tissue injury leading to the pathogenesis of sle . evidences supporting the role of il-17 in sle pathogenesis are found in both animal and human lupus . in mrl / lpr mice , enhanced il-17 mediated tissue injury was observed after ischemic reperfusion of the gut . in il-12r deficient bxd2 mice , there was diminished splenic germinal centre formation as well as lower anti - dna and antihistone antibodies levels . furthermore , splenocytes from snf1 mice secreted higher level of il-17 than non - autoimmune b6 mice . cd3cd4cd8 t cells from mrl / lpr mice produced copious il-17 and the expression of il-17 and il-23 receptors in the lymphocytes from these mice increased as the disease progressed . these lymphoid cells from mrl / lpr mice , after treatment with il-23 in vitro and transferred to non - autoimmune species , can induce nephritis . these data indicate that an aberrantly active il-23/il-17 axis is responsible for the development of nephritis in lupus - prone mice . in human subjects , sle patients have raised serum levels of il-17 and il-23 and the plasma level of il-17 correlates with disease activity . recent data have suggested that a significant portion of il-17 in sle patients is contributed by the tcr-cd4cd8 t lymphocytes . these cr-cd4cd8 t cells and th17 cells are also found in renal biopsies from lupus nephritis patients , supporting their pathogenic role in renal lupus . b lymphocyte stimulator ( blys ) is a member of the tumor necrosis factor ligand family . being a type - ii transmembrane protein , it is cleaved at the cell surface by furin protease that results in the release of a soluble , biologically active molecule . expression of blys is highly confined to myeloid lineage cells ( e.g. , monocytes , macrophages , dendritic cells and activated neutrophils ) and its release is upregulated by interferon- and il-10 . blys couples intensely to b lymphocytes and is a vital factor for b lymphocyte proliferation and immunoglobulin secretion as suggested by recent data . in blys - deficient mice , there is significant shrinkage in numbers of mature b cells , reduced baseline serum immunoglobulin levels and impaired immunoglobulin response to t cell dependent and independent antigens . three types of blys receptor are found , namely , baffr , bcma , and taci receptors . blys can bind to these three receptors on b cells , whereas a proliferation - inducing ligand ( april ) can only engage to taci and bcma . among these three receptors , mice deficient in bcma and taci receptors exhibit no discernible phenotypic or functional abnormalities [ 52 , 54 ] . in contrast , a / wysnj mice ( which bear a mutated baffr gene ) display a reduction in mature b cell numbers and antibody level reminiscent of blys - deficient mice . blys - triggered intracellular events are complex mainly mediating through the interaction of blys receptors and several tnf receptor associated factors ( trafs ) . docking of blys with its receptors activates phospholipase c-2 and subsequently the nf-b pathways . this results in the inhibition of b cell apoptosis and hence prolonged b lymphocyte survival . in murine models , there are several distinct observations which suggest the contribution of blys to the pathogenesis of sle . in blys transgenic mice ( blys - tg mice ) , overproduction of blys not only leads to polyclonal hypergammaglobulinemia but also elevated titres of multiple autoantibodies ( including anti - ds dna ) , circulating immune complexes and renal immunoglobulin deposition . these mice develop autoimmune disorders similar to sle and sjogren syndrome . in sle - prone mice such as the ( nzb / w ) f1 mice and mrl - lpr / lpr mice , treatment of nzb / wf1 mice with soluble taci - ig fusion protein prevented the development of proteinuria and prolonged the survival of the animals . these findings highlight the involvement of blys and its receptors in the development of sle and indirectly suggest taci - ig as a promising treatment for human autoimmune disease . furthermore , mice treated with exogenous blys showed increased numbers of antichromatin b cells and augmented production of anti - ds dna antibodies . deletion of either blys or br3 severely impaired b cell development beyond the transitional developmental stages [ 52 , 55 , 59 , 61 ] . t - cell - deficient baff transgenic ( tg ) mice developed sle similar to t - cell - sufficient baff tg mice , and these features were associated with innate activation of b cells and release of pro - inflammatory autoantibodies . these data suggest that a dysregulated innate activation of b cells alone can drive disease independently of the t cells . circulating level of blys was raised in human lupus patients and the level correlated with the anti - ds dna level . in a survey which measured the serum blys level and disease activities , healthy controls uniformly displayed a normal serum blys level over time , whereas escalated blys level was observed in sle patients ( persistent elevation in 25% and intermittent elevation in another 25% of patients ) . although the aforementioned observations make blys a promising therapeutic target , data regarding blys - targeted therapy are not as encouraging as expected in lupus patients . in a phase i trial of belimumab ( a fully humanized monoclonal antibody ( igg1 ) that binds to soluble blys and hence inhibits its binding to taci , bcma , and baffr ) , there was no significant improvement in disease activity despite a reduction in the cd20 b cells . trial of atacicept ( a fusion protein against the taci receptor ) showed a trend toward clinical improvement in moderate sle , yet phase ii trial of this drug in lupus nephritis was suspended due to high risk of severe infections . interferons ( ifn ) are proteins with the capacity to suppress viral replications : the types i ifns are regarded the most important ones in human lupus . viral dna and rna are classical triggers of type i ifn and the signals are mediated via the toll - like receptors ( tlr ) or the retinoic acid - inducible gene i ( rig - i ) like receptors . double - stranded rna stimulates ifn release via tlr3 while single stranded rna induces ifn via tlr7/8 and the cytosine - phosphate - guanine ( cpg ) rich dna via tlr9 . type i ifns are produced by all leucocytes with plasmacytoid dendritic cells ( pdc ) as the most active secretor in response to tlr7 or tlr9 activation . several mechanisms of how ifns may contribute to the pathogenesis of sle have been proposed . immune complexes generated from autoantibodies and autoantigens lead to dendritic cell activation and hence enhanced antigen presentation and increased ifn secretion . ifns can increase the expression of autoantigen such as ro52 and also the release of autoantigens by translocation of ro52 to the nucleus with subsequent induction of apoptosis . other actions include the promotion of dendritic cell maturation and upregulation of cell surface molecules ( mhc class i and ii , costimulatory molecules ) . in addition , type i ifns also enhance antibody production and class switching , decrease the selectivity of b cells for cpg - rich dna , and permit stimulation by even non - cpg dna [ 74 , 75 ] . elevation of type i ifn in lupus patients was one of the first described cytokine abnormalities in autoimmune diseases . the association of ifn levels and disease activity , anti - ds dna levels , and clinical manifestations supports the role of ifn in sle pathogenesis . although pdc population is lower in the peripheral blood of lupus patients , they are found in the dermal lesions and are responsible for the continuous ifn release . pdc can also accumulate in active lupus nephritis and the migration of these cells to the glomeruli is thought to be influenced by il-18 . findings from experimental lupus model suggested that ifn drive the lupus nephritis and end organ damage . autoantibodies with the ability to form very potent interferogenic immune complexes together with rna - containing autoantigens were detected in the cerebrospinal fluid of patients with cerebral lupus . gene expression profiling showed that sle patients expressed ifn inducible genes in pbmc and the expression correlated with disease activities . these findings demonstrate that raised ifn levels are capable of inducing alterations in gene expression in active lupus patients and support the notion that ifns play an important pathogenic role in sle . in the contrary , deficiency of the ifn / receptor resulted in attenuation of autoantibody production and amelioration of disease in nzb / w mice . moreover , patients receiving anti - ifn therapy for other disease may develop autoantibodies and sle - like syndrome . the causal role of ifn in sle is further supported by genetic studies . transcription factor irf5 was the first identified gene directly involved in ifn production and was associated with increased risk of lupus . lupus patients with a risk haplotype of irf5 had higher serum ifn activity when compared to patients lacking this risk genotype and the effect was most prominent in patients with autoantibodies against either rna - binding proteins or double - stranded dna . another example is the signal transducer and activator of transcription 4 ( stat4 ) which interacts with the cytoplasmic part of the ifnar and variants of stat4 have been shown to be strongly associated with lupus . a link between the ifn response and sle had also been established for patients with polymorphism of jak tyk2 . clinical applications of the ifn include the use as a biomarker as well as a therapeutic target . healthy first degree relatives of lupus patients have increased serum ifn activity and the levels are more pronounced in younger individuals . a combination of risk alleles in the type i signaling pathway ( e.g. , stat4 and irf5 ) may imply an additive risk of disease . it can be inferred that the use of genetic mapping may help predicting the development and severity of disease in the future . ifn regulated chemokines may also be employed to monitor disease activity and organ damage [ 90 , 91 ] . a phase i clinic trial of anti - ifn monoclonal antibodies showed a dose - dependent inhibition of type i ifn - inducible genes in both the peripheral blood and skin biopsies in parallel with reduction in disease activity . tumor necrosis factor - alpha ( tnf- ) is expressed as a trimer on cell surface and in soluble form after the activation of macrophages and dendritic cells . the significance of tnf- in the pathogenesis of sle remains controversial since it has been depicted as both protective and detrimental in different murine models . / w mice , tnf- deficiency is an important trigger for lupus - like autoimmunity . while tnf- deficient nzb / w mice develop severe disease manifestations , tnf- competent nzb / w mice only show modest lupus activity . in contrast , tnf- concentration was high in both sera and renal tissue of mrl / lpr lupus mice and the levels of tnf- correlated with the severity of kidney disease . moreover , even in nzb / w mice , renal expression of tnf- is elevated in conjunction with kidney inflammation . in mrl / lpr mice , anti - tnf therapy offered improvement of joint and lung manifestations in these animal models [ 97 , 98 ] . there is substantial evidence to show that tnf- may play a similar pro - inflammatory role in human sle . serum levels of tnf- in active sle patients closely correlated with disease activity and abundant tnf- expression was demonstrated in lupus nephritis kidneys . nevertheless , conflicting results were observed in patients with other autoimmune diseases receiving anti - tnf- therapy [ 100 , 101 ] . these patients developed antinuclear factors , anti - ds dna and anticardiolipin antibodies as well as a lupus - like syndrome . yet these symptoms and autoantibodies resolved following discontinuation of tnf- blocking therapy . given the findings of elevated serum tnf- in active sle and over - expression of tnf- in active lupus nephritis [ 23 , 102 ] , tnf- antagonism still appears to be an attractive option for the treatment of active lupus disease . however , evidence for therapeutic efficacy of tnf- blockade in sle is still limited [ 103 , 104 ] and larger prospective studies are needed to evaluate their effectiveness and side effects profile . this review has discussed an array of information on a possible pathogenic link between the various cytokines and sle ( figure 1 ) . there are still many facets of inflammatory cascades elicited by these cytokines to be elucidated . a more in - depth understanding of these cytokines may be of clinical significance in the context of devising biomarkers or therapeutic agents ( table 1 ) . most of the recent trials have addressed the use of cytokinetargeted agents in the induction phase of severe disease manifestation or in refractory cases . these new therapies may offer an advantage to achieve rapid disease control and to minimize the corticosteroid usage . however , the role of these anticytokine treatments in the maintenance phase still remains undefined . one must also appreciate that interactions between the cytokine milieus are complex and the attenuation of one cytokine may have potentiated more profound effects than expected . the long - term use of these cytokines as a therapeutic target may appear attractive , yet one must be alerted to the possible complications such as infection and malignancy .

systemic lupus erythematosus ( sle ) is a complex auto - immune disorder which involves various facets of the immune system . in addition to autoantibody production and immune complex deposition , emerging evidences suggest that cytokines may act as key players in the immunopathogenesis of sle . these cytokines assume a critical role in the differentiation , maturation and activation of cells and also participate in the local inflammatory processes that mediate tissue insults in sle . certain cytokines such as the il-6 , il-10 , il-17 , blys , type i interferons ( ifn ) and tumor necrosis factor- ( tnf- ) are closely linked to pathogenesis of sle . the delineation of the role played by these cytokines not only fosters our understanding of this disease but also provides a sound rationale for various therapeutic approaches . in this context , this review focuses on selected cytokines which exert significant effect in the pathogenesis of sle and their possible clinical applications .

1. Introduction 2. Interleukin 6 (IL-6) 3. Interleukin 10 (IL-10) 4. Interleukin 17 (IL-17) 5. B-Lymphocytes Stimulators (BLys) 6. Type I Interferons (Type I IFN) 7. Tumor Necrosis Factor- 8. Concluding Remarks

systemic lupus erythematosus ( sle ) is an intriguing auto - immune disease which constitutes a complex interaction between the innate and adaptive immune system . conventional belief has cornered sle to be primarily a disease of autoantibodies and immune complex deposition . however , mounting evidence has implied that cytokines are also key players in the pathogenesis . cytokines are soluble factors which play a pivotal role in the differentiation , maturation , and activation of various immune cells . they are not only involved in immune dysregulation of sle , but also in the local inflammatory response which ultimately leads to tissue injury . the knowledge of cytokines not only provides new insight into pathogenesis of sle , but also sheds light on various clinical applications . in addition , the manipulation of these cytokines may become potential therapeutic targets for the treatment of sle . in this context , il-6 is a pleiotropic cytokine synthesized predominantly by monocytes , fibroblasts , and endothelial cells , although its secretion may also be found in t- and b - lymphocytes . its production is triggered by il-1 , il-2 , and tnf- but dampened by il-4 , il-10 , and il-13 . one of the most important effects of il-6 is to induce the maturation of b lymphocytes into plasma cells and augment the immunoglobulin secretion [ 2 , 3 ] . other actions include the up - regulation of il-2 and its receptor expression , stimulation of platelet production from megakaryocytes , differentiation of macrophage and osteoclast as well as the production of acute phase reactants . il-6 binds to the il-6 receptors which belong to the type 1 cytokine receptor superfamily that consists of two subunits , namely the il-6 r and the gp 130 . the interaction between il-6 and its receptor results in the dimerization of gp130 , activation of jak1 and subsequent tyrosine phosphorylation of gp130 . the pivotal role of il-6 in the pathogenesis of sle had been supported by both murine and human experiments . in mrl / lpr mice , there exists an age - related elevation of serum il-6 levels , soluble il6-r and aberrant expression of the il-6 r [ 4 , 5 ] . it should be highlighted that no other lymphokine studies have been shown to be capable of directly inducing the igg anti - dna antibodies . in il-6 deficient mrl / lpr mice , there was significant reduction of infiltrating macrophages in the kidney , a decrease in renal igg and c3 deposition , and a diminution of cd4 and cd8 lymphocytes with the absence of il-6 . these data support the notion that il-6 is a strong promoter of lupus nephritis and may be a promising new therapeutic target in the treatment of human lupus nephritis . in b6.sle1.yaa mice , il-6 levels were elevated and the increase was coupled with the loss of cd19 b cells and more primitive b - lymphoid progenitors in bone marrow . stimulation by il-6 prompted these uncommitted progenitor cells to express transcription factors which inhibited lymphopoiesis and promoted myelopoiesis in sle . another mechanism of how il-6 may affect the b cell survival is via the recombination - activation gene ( rag ) machinery which are crucial for the revision of rearranged immunoglobulin v ( d ) j genes . in jun b(delaep ) mice , the development of sle phenotype was linked to increased epidermal il-6 secretion and intercrosses with il-6 deficient mice could rescue the sle phenotype . these studies suggest a possible role of il-6 in the generation of autoantibodies and the development of various clinical manifestations in animal models . lymphoblastoid cells isolated from lupus subjects expressed high levels of il-6 and il-6 antagonism resulted in reduction of anti - ds dna in vitro . it had been shown that b - lymphocytes from lupus patients secreted anti - ds dna spontaneously and this autoantibody production ex vivo was predominantly caused by low density b lymphocytes . cd5 expression suppressed bcr signaling in sle b cells and il-6 down - regulated cd5 expression via dna methylation and hence promoted the activation and expansion of auto - reactive b cells in sle patients . apart from its systemic effects , il-6 was shown to have a particularly close link with the renal manifestation of sle . the urinary level of il-6 in patients with active lupus nephritis was higher than that in patients with quiescent renal disease . apart from the kidneys , neuropsychiatric manifestation was associated with an excessive il-6 levels in the cerebrospinal fluid of lupus patients . these findings of raised il-6 in local tissues have suggested the pathogenetic role of il-6 in mediating local inflammation and tissue insults . other studies have also revealed an increased expression of il-6 agonistic receptor gp130 on peripheral lymphocytes in sle patients and its level correlated with the disease activity . it impedes the activation of antigen presenting cells ( apc ) , down - regulates the expression of co - stimulatory molecules and , thereby , blunts t cell activation and tnf- secretion . il-10 boosts b cell proliferation and immunoglobulin class switching resulting in enhanced antibody secretion with the capacity to enter extravascular compartments and promote inflammation in sle . several stimuli , including anti - ds dna antibodies and immune complexes when bound to fcrii , are potent triggers of il-10 [ 27 , 28 ] . prominent staining of il-10 in intra - renal cells was detected in kidneys of lupus patients , although the data suggested that the ifn/il-10 ratio is of more clinical relevance . elevated ifn/il-10 ratio was observed in active class iv lupus nephritis , while class v nephritis may behave oppositely in this regard . apparently , the increased il-10 was of pathogenic bearing in nzb / w mice where the effects of il-10 were largely opposite to those of tnf- . in fact , anti - il10 therapy led to increased tnf- and its beneficial effects were abrogated when tnf- was blocked . on the other hand , il-10 appeared to play a role in the regulatory t cell commitment and action , which might be beneficial in sle . the observed down - regulation of t cell activation in peripheral blood mononuclear cells from sle patients was consistent with such effects . il-17 is a type i transmembrane protein isolated initially from a rodent cd4 t cell cdna library . it is a potent pro - inflammatory cytokine produced by activated t lymphocytes , with the th17 cells being the most vibrant producer . th17 cells are regarded as a distinct t helper cell subset because they originate from nave t cells and only exhibit a characteristic cytokine release profile when appropriately primed by exclusive transcription factors . there are a total of six family members ( il-17 a to f ) and five receptors ( il-17r a to e ) in the il-17 family . several important cytokines including il-6 , il-21 , and il-23 are in intimate association with il-17 . the combination of il-6 and transforming growth factor ( tgf) was shown to induce the differentiation of murine nave t cells into th17 cells [ 39 , 40 ] . these observations implied that the presence of an inflammatory signal is required to transform the nave t cells to become pro - inflammatory . , il-21 is produced by the th17 cells as well as the t - follicular helper cells but not by antigen presenting cells and , hence , being postulated to act as an auto - amplifier of th17 response . moreover , it is important to note the effect of il-23 on a pathogenic th17 response in nave wild - type mice upon t cell adoptive transfer . , these findings suggest the presence of a novel t helper functional axis ( il-23/il-17 axis ) which may be important in the pathogenesis of autoimmune disorders . although nave cd4 t cells can differentiate into th1 , th2 , or th17 effector subsets , the cytokine milieu characteristic of sle patients ( il-2 poor but il-6 and il-21 rich ) favors th17 expansion . the signature cytokines secreted by the th17 cells include il-17a , il-17f , il-21 , and il-22 . this array of cytokines can stimulate b lymphocytes , and set off local inflammation and tissue injury leading to the pathogenesis of sle . evidences supporting the role of il-17 in sle pathogenesis are found in both animal and human lupus . in mrl / lpr mice , enhanced il-17 mediated tissue injury was observed after ischemic reperfusion of the gut . in il-12r deficient bxd2 mice , there was diminished splenic germinal centre formation as well as lower anti - dna and antihistone antibodies levels . furthermore , splenocytes from snf1 mice secreted higher level of il-17 than non - autoimmune b6 mice . cd3cd4cd8 t cells from mrl / lpr mice produced copious il-17 and the expression of il-17 and il-23 receptors in the lymphocytes from these mice increased as the disease progressed . these lymphoid cells from mrl / lpr mice , after treatment with il-23 in vitro and transferred to non - autoimmune species , can induce nephritis . in human subjects , sle patients have raised serum levels of il-17 and il-23 and the plasma level of il-17 correlates with disease activity . recent data have suggested that a significant portion of il-17 in sle patients is contributed by the tcr-cd4cd8 t lymphocytes . these cr-cd4cd8 t cells and th17 cells are also found in renal biopsies from lupus nephritis patients , supporting their pathogenic role in renal lupus . b lymphocyte stimulator ( blys ) is a member of the tumor necrosis factor ligand family . being a type - ii transmembrane protein , it is cleaved at the cell surface by furin protease that results in the release of a soluble , biologically active molecule . , monocytes , macrophages , dendritic cells and activated neutrophils ) and its release is upregulated by interferon- and il-10 . blys couples intensely to b lymphocytes and is a vital factor for b lymphocyte proliferation and immunoglobulin secretion as suggested by recent data . blys can bind to these three receptors on b cells , whereas a proliferation - inducing ligand ( april ) can only engage to taci and bcma . among these three receptors , mice deficient in bcma and taci receptors exhibit no discernible phenotypic or functional abnormalities [ 52 , 54 ] . in contrast , a / wysnj mice ( which bear a mutated baffr gene ) display a reduction in mature b cell numbers and antibody level reminiscent of blys - deficient mice . this results in the inhibition of b cell apoptosis and hence prolonged b lymphocyte survival . in murine models , there are several distinct observations which suggest the contribution of blys to the pathogenesis of sle . in blys transgenic mice ( blys - tg mice ) , overproduction of blys not only leads to polyclonal hypergammaglobulinemia but also elevated titres of multiple autoantibodies ( including anti - ds dna ) , circulating immune complexes and renal immunoglobulin deposition . these mice develop autoimmune disorders similar to sle and sjogren syndrome . in sle - prone mice such as the ( nzb / w ) f1 mice and mrl - lpr / lpr mice , treatment of nzb / wf1 mice with soluble taci - ig fusion protein prevented the development of proteinuria and prolonged the survival of the animals . these findings highlight the involvement of blys and its receptors in the development of sle and indirectly suggest taci - ig as a promising treatment for human autoimmune disease . furthermore , mice treated with exogenous blys showed increased numbers of antichromatin b cells and augmented production of anti - ds dna antibodies . t - cell - deficient baff transgenic ( tg ) mice developed sle similar to t - cell - sufficient baff tg mice , and these features were associated with innate activation of b cells and release of pro - inflammatory autoantibodies . these data suggest that a dysregulated innate activation of b cells alone can drive disease independently of the t cells . in a survey which measured the serum blys level and disease activities , healthy controls uniformly displayed a normal serum blys level over time , whereas escalated blys level was observed in sle patients ( persistent elevation in 25% and intermittent elevation in another 25% of patients ) . although the aforementioned observations make blys a promising therapeutic target , data regarding blys - targeted therapy are not as encouraging as expected in lupus patients . in a phase i trial of belimumab ( a fully humanized monoclonal antibody ( igg1 ) that binds to soluble blys and hence inhibits its binding to taci , bcma , and baffr ) , there was no significant improvement in disease activity despite a reduction in the cd20 b cells . trial of atacicept ( a fusion protein against the taci receptor ) showed a trend toward clinical improvement in moderate sle , yet phase ii trial of this drug in lupus nephritis was suspended due to high risk of severe infections . interferons ( ifn ) are proteins with the capacity to suppress viral replications : the types i ifns are regarded the most important ones in human lupus . viral dna and rna are classical triggers of type i ifn and the signals are mediated via the toll - like receptors ( tlr ) or the retinoic acid - inducible gene i ( rig - i ) like receptors . type i ifns are produced by all leucocytes with plasmacytoid dendritic cells ( pdc ) as the most active secretor in response to tlr7 or tlr9 activation . several mechanisms of how ifns may contribute to the pathogenesis of sle have been proposed . ifns can increase the expression of autoantigen such as ro52 and also the release of autoantigens by translocation of ro52 to the nucleus with subsequent induction of apoptosis . other actions include the promotion of dendritic cell maturation and upregulation of cell surface molecules ( mhc class i and ii , costimulatory molecules ) . in addition , type i ifns also enhance antibody production and class switching , decrease the selectivity of b cells for cpg - rich dna , and permit stimulation by even non - cpg dna [ 74 , 75 ] . elevation of type i ifn in lupus patients was one of the first described cytokine abnormalities in autoimmune diseases . the association of ifn levels and disease activity , anti - ds dna levels , and clinical manifestations supports the role of ifn in sle pathogenesis . although pdc population is lower in the peripheral blood of lupus patients , they are found in the dermal lesions and are responsible for the continuous ifn release . findings from experimental lupus model suggested that ifn drive the lupus nephritis and end organ damage . autoantibodies with the ability to form very potent interferogenic immune complexes together with rna - containing autoantigens were detected in the cerebrospinal fluid of patients with cerebral lupus . gene expression profiling showed that sle patients expressed ifn inducible genes in pbmc and the expression correlated with disease activities . these findings demonstrate that raised ifn levels are capable of inducing alterations in gene expression in active lupus patients and support the notion that ifns play an important pathogenic role in sle . in the contrary , deficiency of the ifn / receptor resulted in attenuation of autoantibody production and amelioration of disease in nzb / w mice . moreover , patients receiving anti - ifn therapy for other disease may develop autoantibodies and sle - like syndrome . the causal role of ifn in sle is further supported by genetic studies . transcription factor irf5 was the first identified gene directly involved in ifn production and was associated with increased risk of lupus . another example is the signal transducer and activator of transcription 4 ( stat4 ) which interacts with the cytoplasmic part of the ifnar and variants of stat4 have been shown to be strongly associated with lupus . clinical applications of the ifn include the use as a biomarker as well as a therapeutic target . a combination of risk alleles in the type i signaling pathway ( e.g. it can be inferred that the use of genetic mapping may help predicting the development and severity of disease in the future . a phase i clinic trial of anti - ifn monoclonal antibodies showed a dose - dependent inhibition of type i ifn - inducible genes in both the peripheral blood and skin biopsies in parallel with reduction in disease activity . tumor necrosis factor - alpha ( tnf- ) is expressed as a trimer on cell surface and in soluble form after the activation of macrophages and dendritic cells . the significance of tnf- in the pathogenesis of sle remains controversial since it has been depicted as both protective and detrimental in different murine models . / w mice , tnf- deficiency is an important trigger for lupus - like autoimmunity . moreover , even in nzb / w mice , renal expression of tnf- is elevated in conjunction with kidney inflammation . there is substantial evidence to show that tnf- may play a similar pro - inflammatory role in human sle . serum levels of tnf- in active sle patients closely correlated with disease activity and abundant tnf- expression was demonstrated in lupus nephritis kidneys . nevertheless , conflicting results were observed in patients with other autoimmune diseases receiving anti - tnf- therapy [ 100 , 101 ] . given the findings of elevated serum tnf- in active sle and over - expression of tnf- in active lupus nephritis [ 23 , 102 ] , tnf- antagonism still appears to be an attractive option for the treatment of active lupus disease . however , evidence for therapeutic efficacy of tnf- blockade in sle is still limited [ 103 , 104 ] and larger prospective studies are needed to evaluate their effectiveness and side effects profile . this review has discussed an array of information on a possible pathogenic link between the various cytokines and sle ( figure 1 ) . there are still many facets of inflammatory cascades elicited by these cytokines to be elucidated . a more in - depth understanding of these cytokines may be of clinical significance in the context of devising biomarkers or therapeutic agents ( table 1 ) . most of the recent trials have addressed the use of cytokinetargeted agents in the induction phase of severe disease manifestation or in refractory cases . however , the role of these anticytokine treatments in the maintenance phase still remains undefined . the long - term use of these cytokines as a therapeutic target may appear attractive , yet one must be alerted to the possible complications such as infection and malignancy .

in a wide range of research fields , including heterogeneous catalysis , photocatalysis , or energy conversion , tailored functional materials play an ever increasing role . in most cases , this involves nanoparticles dispersed on a variety of structurally different supports , prepared via typical physical or chemical vapor deposition techniques . for electrocatalytically active perovskite materials , recent studies indicate a very elegant pathway of generating well dispersed catalytically active 3d metal nanoparticles , or structures by efficient control of perovskite oxygen anion nonstoichiometry . exsolution of metal particles from various perovskites such as la0.6sr0.4feo3 ( lsf ) or la0.3sr0.7 fe0.7cr0.3o3 is a well - documented phenomenon and has been shown to have significant impact on the physicochemical properties of the materials under question . however , only by controlling the intrinsic material properties ( nonstoichiometry or dopants ) or extrinsic experimental parameters ( oxygen partial pressure , reduction conditions , gas atmosphere ) , a well - defined system of dispersed nanoparticles can be obtained in situ . of special importance in electrocatalysis is the precipitation , segregation , and exsolution of iron from iron - rich perovskite materials , occurring under sufficiently reducing conditions . in most cases , reversible antisegregation is again observed upon reoxidation , e.g. , as outlined in ref ( 3 ) . for lsf , this iron metal segregation , already induced by mild reductive cathodic polarization in a h2/h2o mixture , has been shown to lead to strongly enhanced electrochemical water - splitting kinetics . also in this case , subsequent anodic polarization leads to full reversal of the phenomenon . however , despite the important observations , key structural and morphological features of fe(0 ) segregation remained unclear : the presence of fe(0 ) was derived from corresponding near - ambient x - ray photoelectron spectroscopy data , which showed a strong fe(0 ) contribution in fe 2p peaks and , in parallel , a strong decrease in total iron intensity during cathodic polarization . this observation was in turn linked to the formation of fe(0 ) nanoparticles or , more generally , of 3d nanostructures of fe(0 ) exceeding the inelastic mean free path of the photoelectrons . a model picture of this type can in principle explain the observed decrease of xps - accessible fe area via exsolution of fe from near - surface regions . of equal importance of the understanding of exsolution it is well - known that pure perovskites are promising anode materials in solid - oxide fuel cells , but their catalytic performance can eventually be significantly improved by the addition of catalytically active metals . the latter , e.g. , would include small attached ni particles for improved methanation reaction performance . however , this has some significant strings attached , which are connected with high - temperature treatment in hydrogen and the associated stability issues and complex structural segregation behavior . hydrogen treatment is usually necessary to enter the catalytically active metallic ni state , and , of equal importance , hydrogen is a reactant in methanation reaction mixtures ( e.g. , co or co2 and h2 ) and product of methane reforming . possible effects might include , but are not limited to , exsolution of solid iron nanoparticles or ni fe alloy formation . as the exsolution of iron nanoparticles occurs also on the ni - free fe - containing perovskites under distinct reduction conditions , the addition of ni particles , due to dissociative activation of hydrogen , might offer different pathways of perovskite reduction and the subsequent favored exsolution of other elements such as la or sr in eventually different morphologies . in the present study , we provide direct electron microscopic insights into the chemically driven exsolution of different metallic and oxidic species of distinct morphology at the atomic level , following controlled reduction treatments of pure lsf and ni - lsf materials . this involves treatments in hydrogen reaction mixtures of different reduction potentials to narrow the possible decomposition pathways of the perovskite materials . eventually , the obtained results will , on the one hand , possibly offer an explanation for the so far unknown fe(0 ) morphology and the associated xps intensity effects following cathodic polarization of pure lsf ( under comparable reduction conditions ) ; on the other hand , especially the experiments on ni - lsf will set the stage for an improved understanding of the structural complexity of those materials , especially under reducing conditions , and its possible influence on catalytic performance . to induce the formation of fe(0 ) , commercial lsf powder ( sigma - aldrich , powder < 0.5 m particle size ) was treated either in flowing ( dry ) or static ( moist ) hydrogen at a temperature of 600 c . the treatment in dry h2 at 600 c exceeds the thermodynamic stability limit of lsf , therefore facilitating the precipitation of fe(0 ) . static reduction was performed in a home - built quartz reactor setup including furnace ( 13 ml volume ; about 150 mg sample mass ) by treatment in moist hydrogen at 600 c ( 1 h , corresponding to a saturation pressure at 300 k of 24 mbar water , 1 bar h2 ) . corresponding dry treatments were conducted in a comparable quartz reactor setup in flowing hydrogen using a linn furnace ( frv-25/150/1100 ) for heating ( 1 ml s , sample mass about 150 mg ) . ni - lsf samples were prepared by a standard impregnation technique using ni acetylacetonate ( ni(acac)2 ) as precursor material . the latter was necessary to avoid aquatic impregnation , which might lead to destruction of the perovskite lattice by hydrolysis of alkaline oxides ( la2o3 , sro ) . in detail , ni(acac)2 was dissolved in acetone , and the resulting solution poured over the lsf powder . the latter solution was subsequently stirred for 30 min . as a last step , the powder was dried at 100 c for 1 h and calcined at 600 c in pure oxygen for 2 h. aberration - corrected transmission electron microscopes ( fei titan 80 - 300 and tem titan 80 - 300 stem ) and operated 300 kv , as well as a fei tecnai g2 f20 operated at 200 kv , were used for imaging . edx maps were acquired using a fei titan 80 - 200 chemistem with four super - x silicon drift detectors , operated at 200 kv . the structure of the pure lsf sample treated under flowing conditions ( 1 ml s ) is shown in figure 1 . all tem images exhibit rod - like fe features of different length up to 100 nm and more or less uniform thickness of 20 nm . all rods exhibit some specific internal contrast along the rod contours , appearing as a shell surrounding the particle cores . the thickness of these shells is in the range of a few nanometers . transmission electron micrographs of lsf after reduction in flowing hydrogen at 600 c ( 1 h ) , highlighting the rod - like morphology of exsolved iron ( main panel and inset ) . the basis of this analysis is edx mapping using the specific electronic transitions of individual elements : energy region fe it is immediately clear , that the rods almost entirely consist of iron ( subpanel a1/2 ) . oxygen is found within the perovskite bulk ( a2 ) , but also appears enriched at the rod edges . this indicates partial oxidation of originally metallic iron in air during transport to the electron microscope . most importantly , neither strontium ( a1 ) nor lanthanum ( a4 ) is detected within the rods . as edx mapping indicates some variation of the oxidation state of iron within the rods , the detailed chemical state of iron is further highlighted in panels b and c. eel spectra ( left side of panel b ) were taken along the rod axis perpendicular to the perovskite surface , following the color - coded spots shown in panel c. the color code indicates the respective eel spectra and thus the exact spot , where the spectrum was taken . while the la m4,5 intensity ( and also the ratio of the m4 and m5 peaks ) does not change within the perovskite , the fe l2,3 peak shows considerable changes from the rod edge toward its center . in fact , the intensity ratio of the fe l3/l2 peaks can be used as an indicator of the fe oxidation state . high ratios ( 57 ) indicate fe in higher oxidation states ( + ii+iv ) ; low ratios ( 4 and below ) reduced / metallic fe . the fe l3/l2 peak ratio along the profile is therefore highlighted in the right side of panel b. oxidized iron is thus only found at the particle edges ( and within the perovskite bulk ) , metallic iron in the rod center . note that this can be also indirectly derived from the internal haadf contrast of the iron rod : as the haadf intensity is mostly dominated by the average atom number , oxygen - rich areas are darker than iron - rich ones , and in turn , the oxygen concentration is highest along the rod edge . l , and la m edges to highlight the spatial distribution of the individual elements ( a ) . the chemical state of iron within the rod is highlighted in panel b ( individual eel spectra taken along the spot profile shown in panel c as well as the fe l3/l2 intensity ratio ) and panel c ( eels spot profile perpendicular to the lsf surface parallel to the rod ) . the presence of metallic and oxidized iron is further corroborated by high - resolution imaging ( figure 3 ) . three images of representative rod areas are shown in panels a ( rod tip ) , b ( rod center ) , and c ( intergrowth area of lsf grain and rod ) . from these images , a high crystallinity of the rods can be deduced . to improve the visibility , fast fourier transforms have been created , and individual colored spots , unambiguously attributable to iron in different oxidation states and lsf , have been used to color - code the corresponding lattice fringes in the hrtem images . thus , metallic iron is found mainly in the center , whereas the tip of the rod consists of fe3o4 . note that magnetite is the only iron oxide that is found in substantial amounts within the rods . most interesting , with respect to a possible mechanism of iron exsolution , is the atom - resolved intergrowth area of lsf and exsolved fe rod , shown in panel c. here , two lsf grains in the upper left and right center region of the image are easily seen ; the former has its ( 006)/(202 ) lattice spacings color - coded in blue . as these two lattice fringes do not overlap with one of those of fe3o4 , the boundary region of lsf and oxidized fe rod can be particularly well distinguished . although one has to take into account the two - dimensional projection of the structure in tem images , it appears that the upper lsf grain is the origin of the fe rod and the interface and that the latter is not in highly dynamic state : interdiffusion phenomena or structural defects are largely absent . ( a ) rod tip , ( b ) rod center , and ( c ) intergrowth area of lsf and fe rod . for better visibility , selected spots in the fast fourier transform have been used to color - code specific fe , fe3o4 , and lsf lattice spacings in the hrtem image . the upper lsf grain is blue - colored with its 006/202 lattice fringes . to restrict the experimental parameter space that leads to iron rod formation , connecting experiments under static reduction conditions , i.e. , at a strongly increased water partial pressure of 24 mbar , figure 4 in turn shows a representative overview tem image of lsf treated under static conditions . without exception , the images show agglomerated lsf grains of varying contrast , probably due to thickness variations , but exsolved iron particles or rod - like features have never been observed following this treatment . transmission electron micrographs of lsf after reduction in static hydrogen at 600 c ( 1 h ) . corroborating these findings , figure 5a in turn shows that indeed no iron segregation or rod formation is observable after this static treatment under otherwise identical experimental conditions ( upper panel ) . the eel spectra collected along the line shown in figure 5 ( lower panel ) do not show any changes . furthermore , atom - resolved haadf images ( panel b ) only show the terminating sr o perovskite surface , but no iron segregation . in summary , the comparison of harsh ( dry hydrogen ; flowing ) and comparably mild reduction conditions ( moist hydrogen ; static ) already indicate a variable pathway of cation exsolution from the lsf lattice . before focusing in detail on the exact mechanism , we note the data provide a possible structural explanation for the spectroscopic fingerprint of fe 2p intensity trends in near - ambient x - ray photoelectron spectra ( which were already raised in the introduction section ) . as studies on lsf model electrodes showed enhanced electrochemical water - splitting kinetics following cathodic polarization in humid reducing h2 atmospheres ( i.e. , under reducing cathodic conditions ) and the associated appearance of fe(0 ) in xp spectra , the question was raised on the exact iron morphology formed during reduction . this was deemed especially important since following cathodic polarization , a strong decrease of the total iron intensity was observed , which could be only explained by exsolution of iron and the corresponding suspected formation of isolated iron nanoparticles on the iron - depleted perovskite surface . the presented microscopy results suggest that this could be connected to rod - like features . in this case , the xps - accessible fe area would also decrease as observed in the experiment . ( a ) haadf image of lsf after static reduction in moist hydrogen at 600 c ( 1 h , 24 mbar water , 1 bar h2 ) with a eels line profile perpendicular to the surface alongside the corresponding eel spectra . ( b ) corresponding atom - resolved haadf image of lsf . for a direct comparison of exsolution phenomena , the following figures ( highlighting the state of the material after flowing reduction in hydrogen at 600 c ) , both the edx maps and the bright - field image of a single ni particle ( panel a , lower right corner ) reveal that the ni particle diameters are typically in the range of 50100 nm ( ni is colored in green ) . more importantly , also in this case rod - like features are clearly observable ( two rods emanating from a single ni particle can be seen in the bright - field image in panel a , lower right corner ) . interestingly , these rods only contain sr and o. this is derived from the edx maps ( the sr k edge in red ) and from the high - resolution image ( upper right inset in panel a ) . note that on ni - lsf , no fe - containing rods are observed , although the sro rod morphology is similar . rod lengths are in the range of 50 nm , with diameters of about 5 nm . in fact , sro rod formation is exclusively observed on ni particles , not directly on lsf grains and also not on additionally exsolved iron nanoparticles . panel b is particularly interesting because it shows a sro rod growing partially from a ni particle ( lower image ) but also an irregularly shaped sro particle obviously growing from a lsf grain . this directly reveals that in the present case obviously only ni can act as growth template for sro rods . already at this point , it is clear that perovskite decomposition and exsolution of metal particles takes a different pathway on lsf and ni - lsf . predominant sro exsolution and the importance of ni is also seen in panel a , revealing the enrichment of sr(o ) at the lsf - ni interface as increased bright yellow intensity ( marked by black arrows ) and sro covering the accessible ni area ( marked by white arrows ) . l intensity to highlight the spatial distribution of the individual elements ( after treatment of ni - lsf in flowing hydrogen at 600 c ( 1 h ) ) ( a , b ) . the insets in panel a shows a high - resolution hrtem image of a single sro rod ( upper inset ) and an overview tem image of a ni particle with two attached sro rods ( lower inset ) . the black and white arrows indicate the enrichment of sro at the interface and on the ni surface , respectively . the subsequent figures 7 and 8 reveal two exsolution phenomena , which have not been observed on the pure lsf material , but as for pure lsf , under static and flowing reduction conditions strongly depend on the strength of reduction . flowing reduction yields , as shown in figure 7 , in addition to sro rod formation , isolated exsolved iron particles , mostly with a pronounced oxide shell around the particle core ( oxygen k - edge intensity in red ; oxidation most likely happens during transport in air ) . under static reduction conditions , iron exsolution is also observed , but single iron particles are very rarely observed . the presence of exsolved iron manifests itself in alloyed ni fe particles , resulting from diffusion of fe(0 ) into the ni particle . figure 8 highlights such a particle ( panel b ) , alongside an overview tem image ( panel a ) . the presence of iron within the particle is usually verified by surface oxidation of iron - rich regions of the alloyed particles . note that in principle the mere existence of fe3o4 patches on metallic particles could also arise from some kind of strong metal such interaction is usually the result of the reduction of an oxidic support in hydrogen at elevated temperatures and leads to loss of active metal area by overgrowth of substoichiometric oxides . this has been verified for a number of noble metal particles and a range of oxides , including fe3o4 on pt particles . however , in the present case , such structural features of overgrown metal particles are clearly absent . l edge intensity to highlight the spatial distribution of the individual elements within a single exsolved iron particle with oxidized shell , obtained after treatment of ni - lsf in flowing hydrogen at 600 c ( 1 h ) . ( a ) transmission electron micrographs of ni - lsf after reduction in static hydrogen at 600 c ( 1 h ) . ( b ) high - resolution tem images of different areas of a single alloyed ni / nio particle after static reduction in moist hydrogen at 600 c ( 1 h , 24 mbar water , 1 bar h2 ) . for better visibility , selected spots in the fast fourier transform have been used to color - code specific nio , lsf , and fe3o4 lattice spacings in the hrtem image . undesired iron whisker formation is a well - documented phenomenon in metallurgical research , which gives valuable hints toward the mechanism of iron exsolution and rod growth also in perovskites : during reduction of iron ore , whisker formation sometimes causes catastrophic swelling of the oxide pellets , leading to disintegration , degradation , and gas permeability changes . generation of iron metal dust is also frequently observed , which bears some resemblance to ni dusting phenomona in sofc - related research , causing similar problems of electrode degredation . both reduction in co and h2 leads to pellet irregularities and especially during the reduction of feo ( wstite ) to fe in co atmosphere , whisker formation is observed . the morphology of the resulting iron metal is subsequently steered by different mechanisms : if iron ( cation ) transport through the solid is rate determining , this leads to a large density of nuclei , finally merging to a dense layer of iron ( scheme 1 , upper panel ) . under pure oxygen ( anion ) transport control to the surface , iron is reported to be fed down a steep gradient to the nucleus , leaving no time for significant removal of oxygen around the nucleus , leading to outward growth and whisker formation . under mixed control , , the documented appearance of rods proves that the reduction of lsf proceeds primarily via oxygen anion transport control ( scheme 1 , lower panel ; dark blue indicates more oxidized , light blue more reduced iron ) : after creating a supersaturated region with iron beneath the surface and reaching a critical value for nucleation , the first nucleus is formed . rod formation then takes place only without effective oxygen removal around the nucleus , if the transport of iron from the supersaturated region is much faster than oxygen removal itself . in this case , only one nucleus is formed and acts as a iron drain . oxygen removal therefore is the rate - limiting step and leaves the nucleus no other way than to grow outward . exactly this situation appears to be fulfilled for lsf under flowing reduction in hydrogen and explains also the situation , why under static conditions no rod formation is observed : reduction is simply too inefficient in terms of oxygen removal . the question remains to be answered , how the exsolution phenomena observed on the ni - lsf samples occur mechanistically . in essence , the presence of ni obviously suppresses the pathway to fe rods effectively , but opens another pathway to the similar formation of sro rods ( and , additionally , fe and feni nanoparticles ) . in due course , two conclusions can be immediately drawn : first , the absence of fe rods indicates that the exsolution does not proceed via strict oxygen removal control , but a more complex mechanism must be assumed . second , the presence of metallic ni appears to be of even more crucial importance . as ni is capable of dissociative hydrogen activation , the reduction kinetics around the ni particles are likely faster ; this , as confirmed by the tem experiments , finally leads to sro exsolution . unfortunately , while exsolution of b - site dopants is a common feature in sofc research and as such , well documented , not only the exact mechanism of sr / sro exsolution itself but especially that of sro rod formation is unclear up to now . one might speculate about a similar mechanism that leads to fe rod formation : sro rod formation must therefore also proceed via strict oxygen removal control ; otherwise , continuous sro layers would be expected , based on the previously discussed model similar to iron whisker formation as iron layer growth on pure lsf . however , as this would corroborate the obvious catalytic action of ni for sro rod growth , one might want to stress analogies to the model of catalytic carbon nanowire growth on ni . in this model , after catalytic decomposition of the carbon fuel , carbon dissolves and diffuses through the ni particle or on the surface . subsequently , it precipitates as graphite and nanowires . in cases , where bulk diffusion is limited or blocked , nanowire growth controlled by bulk diffusion pushes the ni particles out , and thus , the ni particle can then be found at the tip of the nanowire . in some cases , however , this nanoparticle is missing and so - called this happens , according to a model by baker and harris , when surface diffusion is dominating and the catalytic ni particle is not detached . revisiting figure 6a again , marked by white arrows , thus , we might infer that for sro rod growth , surface diffusion is dominating . a possible mechanism is based on two reasonable assumptions : ( i ) sro can not be reduced to metallic sr under the chosen reduction conditions and ( ii ) sro can not be dissolved in the ni particle . the observed sro rod growth then is the result of a delicate balance between the obvious high interfacial energy of lsf and sro ( derived from the high stability of the sro termination ) on the ni - free perovskite and the obviously dominating cohesive energy of sro on the ni particles . the effective transport species might also be related to a hydroxylated sr(oh)x species , resulting from dissociative h2 adsorption on ni and the subsequent diffusion of h to the ni - stf interface . upon transport of these species to ni , the hydroxy species likely decompose under the chosen reduction conditions and finally lead to sro rod growth . a schematic picture of the reduction process , leading to a sro extrusion rod , is depicted in scheme 2 . most importantly , the results of this study show how information from quite diametral research areas , such as in the present case , metallurgy , and solid state electrochemistry / fuel cell technology , can be jointly used for improved physicochemical understanding . although perovskites in general and lsf in particular are important candidates for electrodes in sofcs and their reduction behavior is well - understood , the structural and application - oriented consequences of deep reduction of these complex oxide systems have so far not been described in detail . , consequences are severe : whisker formation might have beneficial ( electro)catalytic effects , such as the potential enhancement of water - splitting activity by in situ formed fe(0 ) nano - electrodes , or detrimental structural effects , leading to partial fracture of the lsf structure . combined studies on pure lsf and ni - lsf materials directly revealed how this collapse of the lsf structure can give rise to different pathways of metal and oxide exsolution : this includes fe nanorods without simultaneous fe nanoparticles ( pure lsf , flowing h2 ) , ni fe alloy particles ( ni - lsf , static h2 ) , or sro nanorods and fe nanoparticles , but no fe nanorods ( ni - lsf , flowing h2 ) . hence , under strict control of the experimental parameters the presented data open an attractive chemically driven pathway to metal and metal oxide nanoarchitectures beyond the formation of simple nanoparticles . further experimental in situ microscopic studies are definitely needed to fully clarify the exact mechanism of whisker formation in complex oxide systems , as so far , information is deduced from chemically similar , but not identical , materials only . this would also include dedicated studies on the exact control of the reduction / cathodic potential in h2/h2o mixtures of varying composition to induce or possibly suppress rod formation under water electrolysis conditions .

formation of uniform fe and sro rods as well as nanoparticles following controlled reduction of la0.6sr0.4feo3 ( lsf ) and ni - lsf samples in dry and moist hydrogen is studied by aberration - corrected electron microscopy . metallic fe and sro precipitate from the perovskite lattice as rods of several tenths of nm and thicknesses up to 20 nm . based on a model of fe whisker growth following reduction of pure iron oxides , fe rod exsolution from lsf proceeds via rate - limiting lattice oxygen removal . this favors the formation of single iron metal nuclei at the perovskite surface , subsequently growing as isolated rods . the latter is only possible upon efficient removal of reduction - induced water and , subsequently , reduction of fe + iii/+iv to fe(0 ) . if water remains in the system , no reduction or rod formation occurs . in contrast , formation of sro rods following reduction in dry hydrogen is a catalytic process aided by ni particles . it bears significant resemblance to surface diffusion - controlled carbon whisker growth on ni , leading to similar extrusion rods and filaments . in addition to sro rod growth , the exsolution of fe nanoparticles and , subsequently , ni fe alloy particles is observed . the latter have also been observed under static hydrogen reduction . under strict control of the experimental parameters , the presented data therefore open an attractive chemically driven pathway to metal nanoarchitectures beyond the formation of simple nanoparticles .

Introduction Experimental Section Results Discussion Conclusions

exsolution of metal particles from various perovskites such as la0.6sr0.4feo3 ( lsf ) or la0.3sr0.7 fe0.7cr0.3o3 is a well - documented phenomenon and has been shown to have significant impact on the physicochemical properties of the materials under question . however , only by controlling the intrinsic material properties ( nonstoichiometry or dopants ) or extrinsic experimental parameters ( oxygen partial pressure , reduction conditions , gas atmosphere ) , a well - defined system of dispersed nanoparticles can be obtained in situ . however , despite the important observations , key structural and morphological features of fe(0 ) segregation remained unclear : the presence of fe(0 ) was derived from corresponding near - ambient x - ray photoelectron spectroscopy data , which showed a strong fe(0 ) contribution in fe 2p peaks and , in parallel , a strong decrease in total iron intensity during cathodic polarization . this observation was in turn linked to the formation of fe(0 ) nanoparticles or , more generally , of 3d nanostructures of fe(0 ) exceeding the inelastic mean free path of the photoelectrons . a model picture of this type can in principle explain the observed decrease of xps - accessible fe area via exsolution of fe from near - surface regions . hydrogen treatment is usually necessary to enter the catalytically active metallic ni state , and , of equal importance , hydrogen is a reactant in methanation reaction mixtures ( e.g. possible effects might include , but are not limited to , exsolution of solid iron nanoparticles or ni fe alloy formation . as the exsolution of iron nanoparticles occurs also on the ni - free fe - containing perovskites under distinct reduction conditions , the addition of ni particles , due to dissociative activation of hydrogen , might offer different pathways of perovskite reduction and the subsequent favored exsolution of other elements such as la or sr in eventually different morphologies . in the present study , we provide direct electron microscopic insights into the chemically driven exsolution of different metallic and oxidic species of distinct morphology at the atomic level , following controlled reduction treatments of pure lsf and ni - lsf materials . this involves treatments in hydrogen reaction mixtures of different reduction potentials to narrow the possible decomposition pathways of the perovskite materials . eventually , the obtained results will , on the one hand , possibly offer an explanation for the so far unknown fe(0 ) morphology and the associated xps intensity effects following cathodic polarization of pure lsf ( under comparable reduction conditions ) ; on the other hand , especially the experiments on ni - lsf will set the stage for an improved understanding of the structural complexity of those materials , especially under reducing conditions , and its possible influence on catalytic performance . to induce the formation of fe(0 ) , commercial lsf powder ( sigma - aldrich , powder < 0.5 m particle size ) was treated either in flowing ( dry ) or static ( moist ) hydrogen at a temperature of 600 c . the treatment in dry h2 at 600 c exceeds the thermodynamic stability limit of lsf , therefore facilitating the precipitation of fe(0 ) . ni - lsf samples were prepared by a standard impregnation technique using ni acetylacetonate ( ni(acac)2 ) as precursor material . the latter was necessary to avoid aquatic impregnation , which might lead to destruction of the perovskite lattice by hydrolysis of alkaline oxides ( la2o3 , sro ) . as a last step , the powder was dried at 100 c for 1 h and calcined at 600 c in pure oxygen for 2 h. aberration - corrected transmission electron microscopes ( fei titan 80 - 300 and tem titan 80 - 300 stem ) and operated 300 kv , as well as a fei tecnai g2 f20 operated at 200 kv , were used for imaging . all tem images exhibit rod - like fe features of different length up to 100 nm and more or less uniform thickness of 20 nm . as edx mapping indicates some variation of the oxidation state of iron within the rods , the detailed chemical state of iron is further highlighted in panels b and c. eel spectra ( left side of panel b ) were taken along the rod axis perpendicular to the perovskite surface , following the color - coded spots shown in panel c. the color code indicates the respective eel spectra and thus the exact spot , where the spectrum was taken . while the la m4,5 intensity ( and also the ratio of the m4 and m5 peaks ) does not change within the perovskite , the fe l2,3 peak shows considerable changes from the rod edge toward its center . the fe l3/l2 peak ratio along the profile is therefore highlighted in the right side of panel b. oxidized iron is thus only found at the particle edges ( and within the perovskite bulk ) , metallic iron in the rod center . note that this can be also indirectly derived from the internal haadf contrast of the iron rod : as the haadf intensity is mostly dominated by the average atom number , oxygen - rich areas are darker than iron - rich ones , and in turn , the oxygen concentration is highest along the rod edge . the chemical state of iron within the rod is highlighted in panel b ( individual eel spectra taken along the spot profile shown in panel c as well as the fe l3/l2 intensity ratio ) and panel c ( eels spot profile perpendicular to the lsf surface parallel to the rod ) . thus , metallic iron is found mainly in the center , whereas the tip of the rod consists of fe3o4 . most interesting , with respect to a possible mechanism of iron exsolution , is the atom - resolved intergrowth area of lsf and exsolved fe rod , shown in panel c. here , two lsf grains in the upper left and right center region of the image are easily seen ; the former has its ( 006)/(202 ) lattice spacings color - coded in blue . as these two lattice fringes do not overlap with one of those of fe3o4 , the boundary region of lsf and oxidized fe rod can be particularly well distinguished . although one has to take into account the two - dimensional projection of the structure in tem images , it appears that the upper lsf grain is the origin of the fe rod and the interface and that the latter is not in highly dynamic state : interdiffusion phenomena or structural defects are largely absent . to restrict the experimental parameter space that leads to iron rod formation , connecting experiments under static reduction conditions , i.e. without exception , the images show agglomerated lsf grains of varying contrast , probably due to thickness variations , but exsolved iron particles or rod - like features have never been observed following this treatment . corroborating these findings , figure 5a in turn shows that indeed no iron segregation or rod formation is observable after this static treatment under otherwise identical experimental conditions ( upper panel ) . furthermore , atom - resolved haadf images ( panel b ) only show the terminating sr o perovskite surface , but no iron segregation . in summary , the comparison of harsh ( dry hydrogen ; flowing ) and comparably mild reduction conditions ( moist hydrogen ; static ) already indicate a variable pathway of cation exsolution from the lsf lattice . before focusing in detail on the exact mechanism , we note the data provide a possible structural explanation for the spectroscopic fingerprint of fe 2p intensity trends in near - ambient x - ray photoelectron spectra ( which were already raised in the introduction section ) . , under reducing cathodic conditions ) and the associated appearance of fe(0 ) in xp spectra , the question was raised on the exact iron morphology formed during reduction . this was deemed especially important since following cathodic polarization , a strong decrease of the total iron intensity was observed , which could be only explained by exsolution of iron and the corresponding suspected formation of isolated iron nanoparticles on the iron - depleted perovskite surface . in this case , the xps - accessible fe area would also decrease as observed in the experiment . for a direct comparison of exsolution phenomena , the following figures ( highlighting the state of the material after flowing reduction in hydrogen at 600 c ) , both the edx maps and the bright - field image of a single ni particle ( panel a , lower right corner ) reveal that the ni particle diameters are typically in the range of 50100 nm ( ni is colored in green ) . interestingly , these rods only contain sr and o. this is derived from the edx maps ( the sr k edge in red ) and from the high - resolution image ( upper right inset in panel a ) . note that on ni - lsf , no fe - containing rods are observed , although the sro rod morphology is similar . in fact , sro rod formation is exclusively observed on ni particles , not directly on lsf grains and also not on additionally exsolved iron nanoparticles . this directly reveals that in the present case obviously only ni can act as growth template for sro rods . already at this point , it is clear that perovskite decomposition and exsolution of metal particles takes a different pathway on lsf and ni - lsf . predominant sro exsolution and the importance of ni is also seen in panel a , revealing the enrichment of sr(o ) at the lsf - ni interface as increased bright yellow intensity ( marked by black arrows ) and sro covering the accessible ni area ( marked by white arrows ) . l intensity to highlight the spatial distribution of the individual elements ( after treatment of ni - lsf in flowing hydrogen at 600 c ( 1 h ) ) ( a , b ) . the insets in panel a shows a high - resolution hrtem image of a single sro rod ( upper inset ) and an overview tem image of a ni particle with two attached sro rods ( lower inset ) . the black and white arrows indicate the enrichment of sro at the interface and on the ni surface , respectively . the subsequent figures 7 and 8 reveal two exsolution phenomena , which have not been observed on the pure lsf material , but as for pure lsf , under static and flowing reduction conditions strongly depend on the strength of reduction . flowing reduction yields , as shown in figure 7 , in addition to sro rod formation , isolated exsolved iron particles , mostly with a pronounced oxide shell around the particle core ( oxygen k - edge intensity in red ; oxidation most likely happens during transport in air ) . the presence of exsolved iron manifests itself in alloyed ni fe particles , resulting from diffusion of fe(0 ) into the ni particle . l edge intensity to highlight the spatial distribution of the individual elements within a single exsolved iron particle with oxidized shell , obtained after treatment of ni - lsf in flowing hydrogen at 600 c ( 1 h ) . ( a ) transmission electron micrographs of ni - lsf after reduction in static hydrogen at 600 c ( 1 h ) . ( b ) high - resolution tem images of different areas of a single alloyed ni / nio particle after static reduction in moist hydrogen at 600 c ( 1 h , 24 mbar water , 1 bar h2 ) . undesired iron whisker formation is a well - documented phenomenon in metallurgical research , which gives valuable hints toward the mechanism of iron exsolution and rod growth also in perovskites : during reduction of iron ore , whisker formation sometimes causes catastrophic swelling of the oxide pellets , leading to disintegration , degradation , and gas permeability changes . generation of iron metal dust is also frequently observed , which bears some resemblance to ni dusting phenomona in sofc - related research , causing similar problems of electrode degredation . both reduction in co and h2 leads to pellet irregularities and especially during the reduction of feo ( wstite ) to fe in co atmosphere , whisker formation is observed . the morphology of the resulting iron metal is subsequently steered by different mechanisms : if iron ( cation ) transport through the solid is rate determining , this leads to a large density of nuclei , finally merging to a dense layer of iron ( scheme 1 , upper panel ) . under pure oxygen ( anion ) transport control to the surface , iron is reported to be fed down a steep gradient to the nucleus , leaving no time for significant removal of oxygen around the nucleus , leading to outward growth and whisker formation . under mixed control , , the documented appearance of rods proves that the reduction of lsf proceeds primarily via oxygen anion transport control ( scheme 1 , lower panel ; dark blue indicates more oxidized , light blue more reduced iron ) : after creating a supersaturated region with iron beneath the surface and reaching a critical value for nucleation , the first nucleus is formed . rod formation then takes place only without effective oxygen removal around the nucleus , if the transport of iron from the supersaturated region is much faster than oxygen removal itself . oxygen removal therefore is the rate - limiting step and leaves the nucleus no other way than to grow outward . exactly this situation appears to be fulfilled for lsf under flowing reduction in hydrogen and explains also the situation , why under static conditions no rod formation is observed : reduction is simply too inefficient in terms of oxygen removal . the question remains to be answered , how the exsolution phenomena observed on the ni - lsf samples occur mechanistically . in essence , the presence of ni obviously suppresses the pathway to fe rods effectively , but opens another pathway to the similar formation of sro rods ( and , additionally , fe and feni nanoparticles ) . in due course , two conclusions can be immediately drawn : first , the absence of fe rods indicates that the exsolution does not proceed via strict oxygen removal control , but a more complex mechanism must be assumed . as ni is capable of dissociative hydrogen activation , the reduction kinetics around the ni particles are likely faster ; this , as confirmed by the tem experiments , finally leads to sro exsolution . unfortunately , while exsolution of b - site dopants is a common feature in sofc research and as such , well documented , not only the exact mechanism of sr / sro exsolution itself but especially that of sro rod formation is unclear up to now . one might speculate about a similar mechanism that leads to fe rod formation : sro rod formation must therefore also proceed via strict oxygen removal control ; otherwise , continuous sro layers would be expected , based on the previously discussed model similar to iron whisker formation as iron layer growth on pure lsf . however , as this would corroborate the obvious catalytic action of ni for sro rod growth , one might want to stress analogies to the model of catalytic carbon nanowire growth on ni . in cases , where bulk diffusion is limited or blocked , nanowire growth controlled by bulk diffusion pushes the ni particles out , and thus , the ni particle can then be found at the tip of the nanowire . in some cases , however , this nanoparticle is missing and so - called this happens , according to a model by baker and harris , when surface diffusion is dominating and the catalytic ni particle is not detached . revisiting figure 6a again , marked by white arrows , thus , we might infer that for sro rod growth , surface diffusion is dominating . the observed sro rod growth then is the result of a delicate balance between the obvious high interfacial energy of lsf and sro ( derived from the high stability of the sro termination ) on the ni - free perovskite and the obviously dominating cohesive energy of sro on the ni particles . the effective transport species might also be related to a hydroxylated sr(oh)x species , resulting from dissociative h2 adsorption on ni and the subsequent diffusion of h to the ni - stf interface . upon transport of these species to ni , the hydroxy species likely decompose under the chosen reduction conditions and finally lead to sro rod growth . a schematic picture of the reduction process , leading to a sro extrusion rod , is depicted in scheme 2 . although perovskites in general and lsf in particular are important candidates for electrodes in sofcs and their reduction behavior is well - understood , the structural and application - oriented consequences of deep reduction of these complex oxide systems have so far not been described in detail . , consequences are severe : whisker formation might have beneficial ( electro)catalytic effects , such as the potential enhancement of water - splitting activity by in situ formed fe(0 ) nano - electrodes , or detrimental structural effects , leading to partial fracture of the lsf structure . combined studies on pure lsf and ni - lsf materials directly revealed how this collapse of the lsf structure can give rise to different pathways of metal and oxide exsolution : this includes fe nanorods without simultaneous fe nanoparticles ( pure lsf , flowing h2 ) , ni fe alloy particles ( ni - lsf , static h2 ) , or sro nanorods and fe nanoparticles , but no fe nanorods ( ni - lsf , flowing h2 ) . hence , under strict control of the experimental parameters the presented data open an attractive chemically driven pathway to metal and metal oxide nanoarchitectures beyond the formation of simple nanoparticles . this would also include dedicated studies on the exact control of the reduction / cathodic potential in h2/h2o mixtures of varying composition to induce or possibly suppress rod formation under water electrolysis conditions .

the current standard of care for neovascular age - related macular degeneration ( namd ) is treatment with agents ( such as ranibizumab ) that inhibit vascular endothelial growth factor ( vegf ) , a potent angiogenic mediator associated with age - dependent choroidal neovascularization.1 ranibizumab was approved for use in namd largely due to the findings from two pivotal clinical studies.2,3 in these studies , monthly ranibizumab ( 0.3 or 0.5 mg ) was shown to be superior to sham or photodynamic therapy for the 12-month primary efficacy outcome , and visual and anatomical benefits were sustained over a 24-month follow - up.24 based on these studies , monthly dosing with ranibizumab was recommended.5 however , this approach can be unsustainable in many clinical practices . less frequent dosing is now the norm , and the label for all the currently available anti - vegf agents recommends an initial loading phase followed by as - needed ( prn ) dosing with frequent monitoring.5,6 however , it is acknowledged that prn or quarterly dosing may be less effective compared with monthly dosing , and optimal regimens need to be established.711 some studies show that the visual benefit declines during the maintenance phase despite an initial marked improvement in acuity at the completion of the loading phase.7,8,10,11 in contrast , the sustain study found that ranibizumab prn was comparable with monthly dosing.12 the aura study was undertaken to monitor ranibizumab use and treatment outcomes in a real - life setting . aura was a large , retrospective study conducted in eight countries ( canada , france , germany , ireland , italy , the netherlands , the uk , and venezuela ) that evaluated the real - life clinical use of ranibizumab treatment for patients with namd who were diagnosed and treated by their own physicians.13 in the global population ( all eight included countries ) , there was a mean increase in visual acuity of + 2.4 letters during year 1 , which decreased to + 0.6 letters during year 2 . patients received approximately seven injections over a 2-year period , but there was variability between countries . the findings also indicated that monitoring strategies and retreatment frequencies varied between countries , which may explain the poorer visual outcomes in the overall aura population . the aim of the present report is to describe the outcomes of the uk cohort of aura and to compare with the global aura cohort ( canada , france , germany , ireland , italy , the netherlands , and venezuela ) . the study design has been reported elsewhere.13 in brief , patients who were diagnosed with namd and given ranibizumab injections during january 1 , 2009 to august 31 , 2009 were eligible ; the diagnosis and subsequent decision to treat was made by the patient s own physician . for inclusion , patients were required to have received one or more ranibizumab injections with documented follow - up to the end of their treatment and/or monitoring , or until august 31 , 2011 , whichever came first ( figure s1 ) . patients who switched to bevacizumab or pegaptanib sodium , but received ranibizumab at some point , remained in the study . only one eye was included per patient , which was defined as the eye treated at the start of ranibizumab therapy . in patients with bilateral disease patients taking part in an investigational study of any other drug or device were excluded . approval from the country - specific independent ethics committees or institutional review boards was received based on the requirements of local law and/or regulations , and written consent was obtained from each patient prior to inclusion . we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research . this paper reports the findings from the uk cohort only , but makes explicit comparisons with the global results . all data , including demographics , were collected retrospectively from patients medical records ( paper or electronic ) , which included results from examinations performed during routine clinical practice . the primary assessment was the mean change in visual acuity over the 2-year period ( with key assessments at year 1 [ day 360 ] and year 2 [ day 720 ] ) after the start of ranibizumab treatment . as the study was observational , visual acuity was measured using a variety of vision charts , but was converted to a standardized visual acuity scoring system ( letter count ) . the conversion chart used is provided in the online supplementary material for the primary manuscript.13 secondary assessments included resource utilization during the 2-year period ( ie , the number of clinic visits , use of optical coherence tomography [ oct ] and visual acuity tests , and the number of injections ) . the mean change in visual acuity over the 2-year period was calculated for patients who received one or more doses of ranibizumab treatment and had one or more post - baseline assessments of visual acuity for the treated eye . this was defined as the effectiveness analysis set , and it was estimated that a guiding sample size of 399 subjects per country was required to estimate the change from baseline in the standardized visual acuity score within a 95% confidence interval ( ci ) of 1.5 letters . mean change in visual acuity was assessed using a last observation carried forward ( locf ) analysis to account for missing data . secondary assessments were analyzed using frequency tables or summary statistics and were also performed using the effectiveness analysis set . the study design has been reported elsewhere.13 in brief , patients who were diagnosed with namd and given ranibizumab injections during january 1 , 2009 to august 31 , 2009 were eligible ; the diagnosis and subsequent decision to treat was made by the patient s own physician . for inclusion , patients were required to have received one or more ranibizumab injections with documented follow - up to the end of their treatment and/or monitoring , or until august 31 , 2011 , whichever came first ( figure s1 ) . patients who switched to bevacizumab or pegaptanib sodium , but received ranibizumab at some point , remained in the study . only one eye was included per patient , which was defined as the eye treated at the start of ranibizumab therapy . in patients with bilateral disease patients taking part in an investigational study of any other drug or device were excluded . approval from the country - specific independent ethics committees or institutional review boards was received based on the requirements of local law and/or regulations , and written consent was obtained from each patient prior to inclusion . we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research . this paper reports the findings from the uk cohort only , but makes explicit comparisons with the global results . all data , including demographics , were collected retrospectively from patients medical records ( paper or electronic ) , which included results from examinations performed during routine clinical practice . the primary assessment was the mean change in visual acuity over the 2-year period ( with key assessments at year 1 [ day 360 ] and year 2 [ day 720 ] ) after the start of ranibizumab treatment . as the study was observational , visual acuity was measured using a variety of vision charts , but was converted to a standardized visual acuity scoring system ( letter count ) . the conversion chart used is provided in the online supplementary material for the primary manuscript.13 secondary assessments included resource utilization during the 2-year period ( ie , the number of clinic visits , use of optical coherence tomography [ oct ] and visual acuity tests , and the number of injections ) . the mean change in visual acuity over the 2-year period was calculated for patients who received one or more doses of ranibizumab treatment and had one or more post - baseline assessments of visual acuity for the treated eye . this was defined as the effectiveness analysis set , and it was estimated that a guiding sample size of 399 subjects per country was required to estimate the change from baseline in the standardized visual acuity score within a 95% confidence interval ( ci ) of 1.5 letters . mean change in visual acuity was assessed using a last observation carried forward ( locf ) analysis to account for missing data . secondary assessments were analyzed using frequency tables or summary statistics and were also performed using the effectiveness analysis set . a total of 774 patients were screened , and 461 of these were enrolled from 13 centers in the uk . a total of 313 patients were excluded for the following reasons : participation in an investigational study ( n=40 ) , no diagnosis of namd ( n=3 ) , no informed consent form signed ( n=257 ) , and start of ranibizumab therapy outside inclusion dates ( n=13 ) . of the enrolled patients , 410 received one or more doses of ranibizumab , had one or more post - baseline measurements of visual acuity for the treated eye , and were included in the effectiveness analysis set ( thus meeting the prespecified sample size of 399 subjects ) . the baseline characteristics of patients included in the effectiveness analysis sets in the uk and global cohorts are shown in table 1 . the cohorts were well - matched with regard to age , sex , oct volume ( center point thickness ) , and mean visual acuity ( letter count ) at baseline . although the aim of the study was to follow patients receiving ranibizumab treatment , five patients received at least one injection of bevacizumab and one patient received one injection of pegaptanib sodium the majority of patients ( 99.3% overall : 99.8% at year 1 and 99.5% at year 2 ) did not receive any treatment change . three patients switched from ranibizumab to bevacizumab due to nonresponse ( n=2 ) and reason not known ( n=1 ) . visual acuity ( letter count ) improved from baseline following initiation of ranibizumab treatment , peaking at day 270 with a mean ( standard deviation [ sd ] ) gain in visual acuity of + 6.5 ( 14.4 ) letters in the overall uk cohort . the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 , but there was a small decline in visual acuity over time , and the mean ( sd ) change was + 4.1 ( 16.9 ) at year 2 in the uk cohort ( figure 1a ) . the majority of patients in the uk cohort ( 355/410 ; 86.6% ) received a loading phase ( ie , first three ranibizumab injections within 90 days ) . the mean change in visual acuity was greater in these patients compared with patients who did not receive a loading phase ; however , a small decline in visual acuity was observed during the follow - up in both groups ( figure 1a ) . the mean change in visual acuity from baseline was + 6.8 ( 15.5 ) letters in year 1 and + 4.8 ( 17.1 ) letters in year 2 in patients receiving a loading phase , and + 0.5 ( 13.7 ) and 0.2 ( 14.3 ) in patients who did not receive a loading phase . the mean improvement in visual acuity was also greater in the uk compared with the global population ( figure 1b ) ; there was a decline in both the uk and global populations , but this occurred earlier in the global population ( from approximately day 120 onward ) and was more marked . compared with the other countries enrolled in aura , the uk demonstrated the greatest visual acuity improvements over time , followed by the netherlands ( figure 1c ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was higher for the uk than for the global population ( figure 2 ) . in the uk , mean ( sd ) improvement in visual acuity was also higher in patients with a baseline score of < 35 letters ( + 15.1 [ 16.9 ] at year 1 and + 11.6 [ 16.9 ] at year 2 ) ( figure 2 ) . overall , 43.4% of patients in the uk cohort received ranibizumab treatment within 2 weeks of receiving their namd diagnosis ( figure 3 ) , and the mean ( sd ) treatment duration was 634.7 ( 292.4 ) days . in the uk cohort , the mean ( sd ) number of visits to the ophthalmologist was 18.4 ( 5.0 ) over the full 2-year observation period ( figure 4a ) . there was a higher mean ( sd ) number of visits in the first year ( 10.4 [ 2.2 ] ) than in the second year ( 8.0 [ 3.3 ] ) . similar findings were observed for the mean ( sd ) number of visual acuity tests ( 10.0 [ 2.4 ] in year 1 and 7.8 [ 3.3 ] in year 2 ) and oct tests ( 9.2 [ 2.4 ] in year 1 and 7.4 [ 3.0 ] in year 2 ) ( figure 4a ) . patients from the uk received a mean ( sd ) of 9.0 ( 4.7 ) injections during the full 2-year period ( 5.8 [ 2.4 ] in year 1 and 3.2 [ 2.8 ] in year 2 ) ( figure 4a ) . there were more visits , tests , and injections in the uk compared with the global population during year 1 and year 2 ( figure 4b and c ) . the mean number of injections in the uk compared with the global population ( excluding the uk ) was 5.8 versus 4.8 in year 1 and 3.2 versus 2.0 in year 2 . in the global cohort , 80.2% ( n=1,786/2,227 ) completed a 3-month loading phase , and 19.9% ( 355/1,786 ) of these patients were from the uk . the mean ( sd ) time from the end of the loading phase to the next injection ( maintenance phase ) was 102.8 ( 121.4 ) days in the uk and 134.0 ( 141.2 ) days in the global cohort ( excluding the uk ) . the mean ( sd ) duration between visits in the maintenance phase was 42.1 ( 11.7 ) days in the uk compared with 59.3 ( 51.0 ) in the global cohort ( excluding the uk ) . the mean ( sd ) duration between injections in the maintenance phase was 100.0 ( 80.3 ) in the uk cohort versus 104.1 ( 98.7 ) days in the global cohort ( excluding the uk ) . a total of 365 patients ( 89.0% ) completed the observation period ( august 31 , 2011 ) . the remaining patients ( n=45 ; 11.0% ) discontinued the study prior to this for a number of reasons , including permanent discontinuation of anti - vegf ( n=34 ; 8.3% ) ( due to treatment failure [ n=22 ] , patient decision [ n=2 ] , stable disease [ n=6 ] , and other reason [ n=4 ] ) , change of treating physician ( n=5 ; 1.2% ) , withdrawal by patient ( n=1 ; 0.2% ) , lost to follow - up ( n=1 ; 0.2% ) , other reason ( n=3 ; 0.7% ) , and missing ( n=1 ; 0.2% ) . a total of 774 patients were screened , and 461 of these were enrolled from 13 centers in the uk . a total of 313 patients were excluded for the following reasons : participation in an investigational study ( n=40 ) , no diagnosis of namd ( n=3 ) , no informed consent form signed ( n=257 ) , and start of ranibizumab therapy outside inclusion dates ( n=13 ) . of the enrolled patients , 410 received one or more doses of ranibizumab , had one or more post - baseline measurements of visual acuity for the treated eye , and were included in the effectiveness analysis set ( thus meeting the prespecified sample size of 399 subjects ) . the baseline characteristics of patients included in the effectiveness analysis sets in the uk and global cohorts are shown in table 1 . the cohorts were well - matched with regard to age , sex , oct volume ( center point thickness ) , and mean visual acuity ( letter count ) at baseline . although the aim of the study was to follow patients receiving ranibizumab treatment , five patients received at least one injection of bevacizumab and one patient received one injection of pegaptanib sodium the majority of patients ( 99.3% overall : 99.8% at year 1 and 99.5% at year 2 ) did not receive any treatment change . three patients switched from ranibizumab to bevacizumab due to nonresponse ( n=2 ) and reason not known ( n=1 ) . visual acuity ( letter count ) improved from baseline following initiation of ranibizumab treatment , peaking at day 270 with a mean ( standard deviation [ sd ] ) gain in visual acuity of + 6.5 ( 14.4 ) letters in the overall uk cohort . the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 , but there was a small decline in visual acuity over time , and the mean ( sd ) change was + 4.1 ( 16.9 ) at year 2 in the uk cohort ( figure 1a ) . the majority of patients in the uk cohort ( 355/410 ; 86.6% ) received a loading phase ( ie , first three ranibizumab injections within 90 days ) . the mean change in visual acuity was greater in these patients compared with patients who did not receive a loading phase ; however , a small decline in visual acuity was observed during the follow - up in both groups ( figure 1a ) . the mean change in visual acuity from baseline was + 6.8 ( 15.5 ) letters in year 1 and + 4.8 ( 17.1 ) letters in year 2 in patients receiving a loading phase , and + 0.5 ( 13.7 ) and 0.2 ( 14.3 ) in patients who did not receive a loading phase . the mean improvement in visual acuity was also greater in the uk compared with the global population ( figure 1b ) ; there was a decline in both the uk and global populations , but this occurred earlier in the global population ( from approximately day 120 onward ) and was more marked . compared with the other countries enrolled in aura , the uk demonstrated the greatest visual acuity improvements over time , followed by the netherlands ( figure 1c ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was higher for the uk than for the global population ( figure 2 ) . in the uk , mean ( sd ) improvement in visual acuity was also higher in patients with a baseline score of < 35 letters ( + 15.1 [ 16.9 ] at year 1 and + 11.6 [ 16.9 ] at year 2 ) ( figure 2 ) . overall , 43.4% of patients in the uk cohort received ranibizumab treatment within 2 weeks of receiving their namd diagnosis ( figure 3 ) , and the mean ( sd ) treatment duration was 634.7 ( 292.4 ) days . in the uk cohort , the mean ( sd ) number of visits to the ophthalmologist was 18.4 ( 5.0 ) over the full 2-year observation period ( figure 4a ) . there was a higher mean ( sd ) number of visits in the first year ( 10.4 [ 2.2 ] ) than in the second year ( 8.0 [ 3.3 ] ) . similar findings were observed for the mean ( sd ) number of visual acuity tests ( 10.0 [ 2.4 ] in year 1 and 7.8 [ 3.3 ] in year 2 ) and oct tests ( 9.2 [ 2.4 ] in year 1 and 7.4 [ 3.0 ] in year 2 ) ( figure 4a ) . patients from the uk received a mean ( sd ) of 9.0 ( 4.7 ) injections during the full 2-year period ( 5.8 [ 2.4 ] in year 1 and 3.2 [ 2.8 ] in year 2 ) ( figure 4a ) . there were more visits , tests , and injections in the uk compared with the global population during year 1 and year 2 ( figure 4b and c ) . the mean number of injections in the uk compared with the global population ( excluding the uk ) was 5.8 versus 4.8 in year 1 and 3.2 versus 2.0 in year 2 . in the global cohort , 80.2% ( n=1,786/2,227 ) completed a 3-month loading phase , and 19.9% ( 355/1,786 ) of these patients were from the uk . the mean ( sd ) time from the end of the loading phase to the next injection ( maintenance phase ) was 102.8 ( 121.4 ) days in the uk and 134.0 ( 141.2 ) days in the global cohort ( excluding the uk ) . the mean ( sd ) duration between visits in the maintenance phase was 42.1 ( 11.7 ) days in the uk compared with 59.3 ( 51.0 ) in the global cohort ( excluding the uk ) . the mean ( sd ) duration between injections in the maintenance phase was 100.0 ( 80.3 ) in the uk cohort versus 104.1 ( 98.7 ) days in the global cohort ( excluding the uk ) . a total of 365 patients ( 89.0% ) completed the observation period ( august 31 , 2011 ) . the remaining patients ( n=45 ; 11.0% ) discontinued the study prior to this for a number of reasons , including permanent discontinuation of anti - vegf ( n=34 ; 8.3% ) ( due to treatment failure [ n=22 ] , patient decision [ n=2 ] , stable disease [ n=6 ] , and other reason [ n=4 ] ) , change of treating physician ( n=5 ; 1.2% ) , withdrawal by patient ( n=1 ; 0.2% ) , lost to follow - up ( n=1 ; 0.2% ) , other reason ( n=3 ; 0.7% ) , and missing ( n=1 ; 0.2% ) . patients from aura who were diagnosed with namd and treated with ranibizumab by their own ophthalmologists according to routine practice were retrospectively monitored to determine long - term effectiveness of ranibizumab from a uk perspective . compared with the global population , the uk - specific outcomes were better and may have been achieved by higher monitoring and retreatment rates . also , most patients in the uk ( 86.6% ) received a loading phase ( ie , three injections within 90 days ) , which is more than was observed in the global population ( 80.2% ) . the improvement in visual acuity was higher in patients who received a loading phase in both populations , but there was still decline over time , which was more marked in the global population . the mean number of ophthalmology visits in years 1 and 2 was 10.4 and 8.0 in the uk compared with 8.6 and 4.9 in the global population , respectively . the mean number of injections was also higher in the uk ( 5.8 in year 1 and 3.2 in year 2 ) compared with the global population ( 5.0 in year 1 and 2.2 in year 2 ) . these findings reflect how ranibizumab injection frequency and long - term monitoring affect visual outcomes in real - life , and are an indication of how the uk situation compares with the outcomes from other countries . although less frequent ranibizumab dosing is not as effective as monthly dosing , four to five injections in the 9 months following a 3-month loading phase is still expected to maintain visual acuity based on clinical study outcomes . the catt study showed that the mean change in visual acuity was + 8.8 letters with monthly ranibizumab ( mean number of injections was 22.4 ) and + 6.7 letters with prn ranibizumab ( mean number of injections was 12.6 ) over a 2-year period.14 in the anchor study , ranibizumab 0.5 mg monthly was associated with a mean change in visual acuity of + 11.3 letters in year 1 and + 10.7 letters in year 2.2,15 the corresponding values in the marina study were + 7.2 letters in year 1 and + 6.6 in year 2.3 in comparison , the current observational study showed that the mean change in visual acuity was + 6.8 letters in year 1 and + 4.8 letters in year 2 ( with a loading phase ) , with an average of nine injections over a 2-year period . the emr study , a national namd database study of ranibizumab in the uk that included information from 11,135 patients ( 12,951 eyes ) and more than 300,000 clinic visits showed that mean visual acuity only improved by + 2 letters at the end of year 1 and by + 1 letter at the end of year 2 ( the mean baseline visual acuity was 55 letters).16 however , the median number of visits and injections were similar to those observed in the current study ( 9.2 and five in year 1 and 8.2 and four in year 2 , respectively ) . the differences in the improvement in visual acuity between the present study and the national amd database study may be attributed to differences in the clinical characteristics of the sites providing data for the two studies and/or the exclusion criteria applied in aura , as 40.4% of the cases were excluded from the original screened sample in the present study , contributing to selection bias . a comparison of the uk results with the other individual countries in the aura study ( figure 1c ) showed notable differences in visual acuity over time , including better outcomes with the uk cohort . in the uk , treatment of namd with appropriate imaging is undertaken to specified standards that are agreed with the local national health service provider , while in other countries , there may be limits in terms of the numbers of treatments that may be administered , and monitoring may also occur less frequently . the national institute for health and care excellence criteria17 may have also helped select the most appropriate patients for treatment , based on guidance ta155 , to receive intravitreal anti - vegf injections : patients must have best - corrected visual acuity between 6/12 and 6/96 , with no permanent structural damage to the central fovea . royal college of ophthalmologists clinical guidelines also specify the need for frequent monitoring and appropriate treatment.6 uk clinical commissioning groups conduct regular audits , and it is likely that this results in improved adherence with protocols . consequently , monthly monitoring and relatively frequent treatment are likely to be more prevalent in the uk , resulting in better functional outcomes compared with other countries . it was moderately sized , employed a stringent approach to extraction of data ( ensuring that all clinical information was captured at every visit ) , and involved multiple centers , which were geographically dispersed throughout the uk . it may also better reflect real - world outcomes than randomized studies , and could help inform policy and provide benchmarks for uk audits . limitations include its retrospective nature , observational design , use of different methods for assessing visual acuity outcomes ( although they were standardized ) , and its conduct between 2009 and 2011 ; it is likely that with increasing experience of anti - vegf therapy and the addition of other therapeutic agents , treatment of namd will have changed in recent years . the study also used an locf approach , and thus excluded data from patients with poorer or declining visual outcomes ( ie , those who discontinued early due to treatment failure or for reasons not stated ) . however , observational data ( also reported in the global paper ) showed a similar pattern in visual acuity outcomes compared with the locf analysis . in the uk cohort , visual acuity changed from 55.0 letters ( n=408 ) at baseline to 62.1 letters ( + 7.1 ) at year 1 ( n=263 ) and to 60.8 letters ( + 5.8 letters ) at year 2 ( n=209 ) using observational data ( data not shown ) . if patients had bilateral disease , then the eye with the worst visual acuity was included into the analysis . in the uk cohort , there are also limitations associated with evaluating the uk subgroup independently of the global population . one of the major drawbacks of the uk aura cohort is that the 13 centers that were included may not be representative of uk practice as a whole . it is likely that only better - organized centers with excellent record keeping and adequate research staff were recruited to the study . the relatively good results seen with ranibizumab in the uk cohort does not exclude the possibility that results with ranibizumab over the whole of the uk may be closer to those seen in larger real - world studies such as the emr study , which found that real world outcomes did not match the results in randomized studies.16 the ongoing luminous study ( nct01318941 ) , which includes patients from a larger number of uk centers , may also provide additional information from a real - world perspective . in summary , visual outcomes in real - life clinical experience in the uk cohort of aura were only slightly lower than those initially observed in clinical studies such as catt and marina,3,14 but there was still some decline over time . this study also showed that anti - vegf agents are effective in patients with poor baseline visual acuity , which is similar to what was reported in another uk study.18 the findings in the uk also highlight the role of the loading phase with ranibizumab treatment . this is consistent with results observed in interventional studies with ranibizumab and bevacizumab.1921 however , the reasons for this are not fully understood , and may be related to a preconditioning effect of the loading phase , resulting in modification of subsequent disease course . overall , our results demonstrate that good outcomes are achievable in real - life settings , particularly if high quality control by payers ( such as the situation in the uk ) means that physicians adhere to protocols for monthly monitoring ( including injections being around or above seven in the first year ) ; however , this is associated with high resource utilization and increased burden for patients and health care providers , and may depend on reimbursement . in countries where monitoring and treatment were less frequent , and audits and funding were limited , visual outcomes were poorer and did not reflect the changes seen in clinical studies . further research into optimal treatment regimens and alternative therapeutic options may help to further improve patient outcomes in real - life settings . approval from the country - specific independent ethics committees or institutional review boards was received based on the requirements of local law and/or regulations , as follows : uk : nres committee north west greater manchester west ; france : the authors advise that in france only a data privacy committee is involved , as no institutional review board or ethics committee is involved for observational studies ; canada : the st . michael s hospital research ethics board , institutional review board services ( irb services ) , university health network research ethics board , the university of western ontario research ethics board for health sciences research involving human subjects ( hsreb ) ; germany : ethik kommission , medizinische fakultt bonn , biomedizinisches zentrum , ( first approval in germany ) ; netherlands : ethical committee of catharina hospital in eindhoven ; venezuela : centro nacional de biotica ( cenabi ) , iec del hospital militar , iec del centro mdico de ojos ; italy : comitato etico per la sperimentazione , ospedale luigi sacco , azienda ospedaliera polo universitario ; ireland : research ethics committee , health service executive , south east area ( this was the bigger site ) , mater misericordiae university hospital and mater private hospital research ethics committee . timeline for study design . abbreviations : fpfv , first patient , first visit ; lpfv , last patient , first visit ; lplv , last patient , last visit ; vegf , vascular endothelial growth factor .

purposeaura was an international , retrospective , observational study that monitored the real - life use and effectiveness of ranibizumab injections in patients with neovascular age - related macular degeneration ( namd ) . this paper reports the findings from the uk.methodspatients who started treatment with ranibizumab between january 1 , 2009 , and august 31 , 2009 , and had documented follow - up to the end of their treatment and/or monitoring or until august 31 , 2011 , were retrospectively monitored ; the diagnosis and subsequent decision to treat was made by the patient s own physician . assessments included the change in visual acuity ( standardized letter count ) during the first and second years after start of ranibizumab therapy and resource utilization.resultsfour hundred and ten patients from 13 uk centers were analyzed . the mean ( standard deviation [ sd ] ) letter score at baseline was 55.0 ( 17.8 ) . the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 and + 4.1 ( 16.9 ) at year 2 . most of the patients ( 86.6% ) completed a 3-month loading phase ; the visual improvements were numerically higher in these patients . over 2 years , the mean ( sd ) number of clinic visits and injections was 18.4 ( 5.0 ) and 9.0 ( 4.7 ) , respectively . resource use and visual acuity gains were greater than those observed in the global population , which included other countries enrolled in aura ( canada , france , germany , ireland , italy , the netherlands , and venezuela ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was also higher for the uk than for the global population across all subgroups.conclusionmonitoring and treatment rates were high in the uk , resulting in better visual acuity outcomes compared with other included countries . this suggests that translation of clinical study outcomes into real - life settings is achievable , but at the expense of higher resource utilization than is currently the norm in most developed countries .

Introduction Materials and methods Study design and participants Assessments Statistical analysis Results Participants Change in visual acuity over time Resource utilization Discontinuations (UK cohort) Discussion Ethical approval Supplementary material

the current standard of care for neovascular age - related macular degeneration ( namd ) is treatment with agents ( such as ranibizumab ) that inhibit vascular endothelial growth factor ( vegf ) , a potent angiogenic mediator associated with age - dependent choroidal neovascularization.1 ranibizumab was approved for use in namd largely due to the findings from two pivotal clinical studies.2,3 in these studies , monthly ranibizumab ( 0.3 or 0.5 mg ) was shown to be superior to sham or photodynamic therapy for the 12-month primary efficacy outcome , and visual and anatomical benefits were sustained over a 24-month follow - up.24 based on these studies , monthly dosing with ranibizumab was recommended.5 however , this approach can be unsustainable in many clinical practices . less frequent dosing is now the norm , and the label for all the currently available anti - vegf agents recommends an initial loading phase followed by as - needed ( prn ) dosing with frequent monitoring.5,6 however , it is acknowledged that prn or quarterly dosing may be less effective compared with monthly dosing , and optimal regimens need to be established.711 some studies show that the visual benefit declines during the maintenance phase despite an initial marked improvement in acuity at the completion of the loading phase.7,8,10,11 in contrast , the sustain study found that ranibizumab prn was comparable with monthly dosing.12 the aura study was undertaken to monitor ranibizumab use and treatment outcomes in a real - life setting . aura was a large , retrospective study conducted in eight countries ( canada , france , germany , ireland , italy , the netherlands , the uk , and venezuela ) that evaluated the real - life clinical use of ranibizumab treatment for patients with namd who were diagnosed and treated by their own physicians.13 in the global population ( all eight included countries ) , there was a mean increase in visual acuity of + 2.4 letters during year 1 , which decreased to + 0.6 letters during year 2 . the aim of the present report is to describe the outcomes of the uk cohort of aura and to compare with the global aura cohort ( canada , france , germany , ireland , italy , the netherlands , and venezuela ) . the study design has been reported elsewhere.13 in brief , patients who were diagnosed with namd and given ranibizumab injections during january 1 , 2009 to august 31 , 2009 were eligible ; the diagnosis and subsequent decision to treat was made by the patient s own physician . for inclusion , patients were required to have received one or more ranibizumab injections with documented follow - up to the end of their treatment and/or monitoring , or until august 31 , 2011 , whichever came first ( figure s1 ) . this paper reports the findings from the uk cohort only , but makes explicit comparisons with the global results . the primary assessment was the mean change in visual acuity over the 2-year period ( with key assessments at year 1 [ day 360 ] and year 2 [ day 720 ] ) after the start of ranibizumab treatment . the conversion chart used is provided in the online supplementary material for the primary manuscript.13 secondary assessments included resource utilization during the 2-year period ( ie , the number of clinic visits , use of optical coherence tomography [ oct ] and visual acuity tests , and the number of injections ) . the mean change in visual acuity over the 2-year period was calculated for patients who received one or more doses of ranibizumab treatment and had one or more post - baseline assessments of visual acuity for the treated eye . the study design has been reported elsewhere.13 in brief , patients who were diagnosed with namd and given ranibizumab injections during january 1 , 2009 to august 31 , 2009 were eligible ; the diagnosis and subsequent decision to treat was made by the patient s own physician . for inclusion , patients were required to have received one or more ranibizumab injections with documented follow - up to the end of their treatment and/or monitoring , or until august 31 , 2011 , whichever came first ( figure s1 ) . this paper reports the findings from the uk cohort only , but makes explicit comparisons with the global results . the primary assessment was the mean change in visual acuity over the 2-year period ( with key assessments at year 1 [ day 360 ] and year 2 [ day 720 ] ) after the start of ranibizumab treatment . the conversion chart used is provided in the online supplementary material for the primary manuscript.13 secondary assessments included resource utilization during the 2-year period ( ie , the number of clinic visits , use of optical coherence tomography [ oct ] and visual acuity tests , and the number of injections ) . a total of 313 patients were excluded for the following reasons : participation in an investigational study ( n=40 ) , no diagnosis of namd ( n=3 ) , no informed consent form signed ( n=257 ) , and start of ranibizumab therapy outside inclusion dates ( n=13 ) . visual acuity ( letter count ) improved from baseline following initiation of ranibizumab treatment , peaking at day 270 with a mean ( standard deviation [ sd ] ) gain in visual acuity of + 6.5 ( 14.4 ) letters in the overall uk cohort . the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 , but there was a small decline in visual acuity over time , and the mean ( sd ) change was + 4.1 ( 16.9 ) at year 2 in the uk cohort ( figure 1a ) . the mean change in visual acuity was greater in these patients compared with patients who did not receive a loading phase ; however , a small decline in visual acuity was observed during the follow - up in both groups ( figure 1a ) . the mean change in visual acuity from baseline was + 6.8 ( 15.5 ) letters in year 1 and + 4.8 ( 17.1 ) letters in year 2 in patients receiving a loading phase , and + 0.5 ( 13.7 ) and 0.2 ( 14.3 ) in patients who did not receive a loading phase . the mean improvement in visual acuity was also greater in the uk compared with the global population ( figure 1b ) ; there was a decline in both the uk and global populations , but this occurred earlier in the global population ( from approximately day 120 onward ) and was more marked . compared with the other countries enrolled in aura , the uk demonstrated the greatest visual acuity improvements over time , followed by the netherlands ( figure 1c ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was higher for the uk than for the global population ( figure 2 ) . in the uk , mean ( sd ) improvement in visual acuity was also higher in patients with a baseline score of < 35 letters ( + 15.1 [ 16.9 ] at year 1 and + 11.6 [ 16.9 ] at year 2 ) ( figure 2 ) . overall , 43.4% of patients in the uk cohort received ranibizumab treatment within 2 weeks of receiving their namd diagnosis ( figure 3 ) , and the mean ( sd ) treatment duration was 634.7 ( 292.4 ) days . in the uk cohort , the mean ( sd ) number of visits to the ophthalmologist was 18.4 ( 5.0 ) over the full 2-year observation period ( figure 4a ) . there was a higher mean ( sd ) number of visits in the first year ( 10.4 [ 2.2 ] ) than in the second year ( 8.0 [ 3.3 ] ) . similar findings were observed for the mean ( sd ) number of visual acuity tests ( 10.0 [ 2.4 ] in year 1 and 7.8 [ 3.3 ] in year 2 ) and oct tests ( 9.2 [ 2.4 ] in year 1 and 7.4 [ 3.0 ] in year 2 ) ( figure 4a ) . patients from the uk received a mean ( sd ) of 9.0 ( 4.7 ) injections during the full 2-year period ( 5.8 [ 2.4 ] in year 1 and 3.2 [ 2.8 ] in year 2 ) ( figure 4a ) . there were more visits , tests , and injections in the uk compared with the global population during year 1 and year 2 ( figure 4b and c ) . the mean number of injections in the uk compared with the global population ( excluding the uk ) was 5.8 versus 4.8 in year 1 and 3.2 versus 2.0 in year 2 . in the global cohort , 80.2% ( n=1,786/2,227 ) completed a 3-month loading phase , and 19.9% ( 355/1,786 ) of these patients were from the uk . the mean ( sd ) time from the end of the loading phase to the next injection ( maintenance phase ) was 102.8 ( 121.4 ) days in the uk and 134.0 ( 141.2 ) days in the global cohort ( excluding the uk ) . the mean ( sd ) duration between visits in the maintenance phase was 42.1 ( 11.7 ) days in the uk compared with 59.3 ( 51.0 ) in the global cohort ( excluding the uk ) . the mean ( sd ) duration between injections in the maintenance phase was 100.0 ( 80.3 ) in the uk cohort versus 104.1 ( 98.7 ) days in the global cohort ( excluding the uk ) . a total of 313 patients were excluded for the following reasons : participation in an investigational study ( n=40 ) , no diagnosis of namd ( n=3 ) , no informed consent form signed ( n=257 ) , and start of ranibizumab therapy outside inclusion dates ( n=13 ) . visual acuity ( letter count ) improved from baseline following initiation of ranibizumab treatment , peaking at day 270 with a mean ( standard deviation [ sd ] ) gain in visual acuity of + 6.5 ( 14.4 ) letters in the overall uk cohort . the mean ( sd ) change in visual acuity from baseline was + 6.0 ( 15.4 ) letters at year 1 , but there was a small decline in visual acuity over time , and the mean ( sd ) change was + 4.1 ( 16.9 ) at year 2 in the uk cohort ( figure 1a ) . the mean change in visual acuity was greater in these patients compared with patients who did not receive a loading phase ; however , a small decline in visual acuity was observed during the follow - up in both groups ( figure 1a ) . the mean change in visual acuity from baseline was + 6.8 ( 15.5 ) letters in year 1 and + 4.8 ( 17.1 ) letters in year 2 in patients receiving a loading phase , and + 0.5 ( 13.7 ) and 0.2 ( 14.3 ) in patients who did not receive a loading phase . the mean improvement in visual acuity was also greater in the uk compared with the global population ( figure 1b ) ; there was a decline in both the uk and global populations , but this occurred earlier in the global population ( from approximately day 120 onward ) and was more marked . compared with the other countries enrolled in aura , the uk demonstrated the greatest visual acuity improvements over time , followed by the netherlands ( figure 1c ) . when patients were stratified according to severity of namd ( based on letter count at baseline ) , the mean change in visual acuity score at years 1 and 2 was higher for the uk than for the global population ( figure 2 ) . in the uk , mean ( sd ) improvement in visual acuity was also higher in patients with a baseline score of < 35 letters ( + 15.1 [ 16.9 ] at year 1 and + 11.6 [ 16.9 ] at year 2 ) ( figure 2 ) . overall , 43.4% of patients in the uk cohort received ranibizumab treatment within 2 weeks of receiving their namd diagnosis ( figure 3 ) , and the mean ( sd ) treatment duration was 634.7 ( 292.4 ) days . in the uk cohort , the mean ( sd ) number of visits to the ophthalmologist was 18.4 ( 5.0 ) over the full 2-year observation period ( figure 4a ) . there was a higher mean ( sd ) number of visits in the first year ( 10.4 [ 2.2 ] ) than in the second year ( 8.0 [ 3.3 ] ) . similar findings were observed for the mean ( sd ) number of visual acuity tests ( 10.0 [ 2.4 ] in year 1 and 7.8 [ 3.3 ] in year 2 ) and oct tests ( 9.2 [ 2.4 ] in year 1 and 7.4 [ 3.0 ] in year 2 ) ( figure 4a ) . patients from the uk received a mean ( sd ) of 9.0 ( 4.7 ) injections during the full 2-year period ( 5.8 [ 2.4 ] in year 1 and 3.2 [ 2.8 ] in year 2 ) ( figure 4a ) . there were more visits , tests , and injections in the uk compared with the global population during year 1 and year 2 ( figure 4b and c ) . the mean number of injections in the uk compared with the global population ( excluding the uk ) was 5.8 versus 4.8 in year 1 and 3.2 versus 2.0 in year 2 . in the global cohort , 80.2% ( n=1,786/2,227 ) completed a 3-month loading phase , and 19.9% ( 355/1,786 ) of these patients were from the uk . the mean ( sd ) time from the end of the loading phase to the next injection ( maintenance phase ) was 102.8 ( 121.4 ) days in the uk and 134.0 ( 141.2 ) days in the global cohort ( excluding the uk ) . the mean ( sd ) duration between visits in the maintenance phase was 42.1 ( 11.7 ) days in the uk compared with 59.3 ( 51.0 ) in the global cohort ( excluding the uk ) . the mean ( sd ) duration between injections in the maintenance phase was 100.0 ( 80.3 ) in the uk cohort versus 104.1 ( 98.7 ) days in the global cohort ( excluding the uk ) . also , most patients in the uk ( 86.6% ) received a loading phase ( ie , three injections within 90 days ) , which is more than was observed in the global population ( 80.2% ) . the improvement in visual acuity was higher in patients who received a loading phase in both populations , but there was still decline over time , which was more marked in the global population . the mean number of ophthalmology visits in years 1 and 2 was 10.4 and 8.0 in the uk compared with 8.6 and 4.9 in the global population , respectively . the mean number of injections was also higher in the uk ( 5.8 in year 1 and 3.2 in year 2 ) compared with the global population ( 5.0 in year 1 and 2.2 in year 2 ) . although less frequent ranibizumab dosing is not as effective as monthly dosing , four to five injections in the 9 months following a 3-month loading phase is still expected to maintain visual acuity based on clinical study outcomes . the catt study showed that the mean change in visual acuity was + 8.8 letters with monthly ranibizumab ( mean number of injections was 22.4 ) and + 6.7 letters with prn ranibizumab ( mean number of injections was 12.6 ) over a 2-year period.14 in the anchor study , ranibizumab 0.5 mg monthly was associated with a mean change in visual acuity of + 11.3 letters in year 1 and + 10.7 letters in year 2.2,15 the corresponding values in the marina study were + 7.2 letters in year 1 and + 6.6 in year 2.3 in comparison , the current observational study showed that the mean change in visual acuity was + 6.8 letters in year 1 and + 4.8 letters in year 2 ( with a loading phase ) , with an average of nine injections over a 2-year period . the emr study , a national namd database study of ranibizumab in the uk that included information from 11,135 patients ( 12,951 eyes ) and more than 300,000 clinic visits showed that mean visual acuity only improved by + 2 letters at the end of year 1 and by + 1 letter at the end of year 2 ( the mean baseline visual acuity was 55 letters).16 however , the median number of visits and injections were similar to those observed in the current study ( 9.2 and five in year 1 and 8.2 and four in year 2 , respectively ) . consequently , monthly monitoring and relatively frequent treatment are likely to be more prevalent in the uk , resulting in better functional outcomes compared with other countries . in the uk cohort , visual acuity changed from 55.0 letters ( n=408 ) at baseline to 62.1 letters ( + 7.1 ) at year 1 ( n=263 ) and to 60.8 letters ( + 5.8 letters ) at year 2 ( n=209 ) using observational data ( data not shown ) . the relatively good results seen with ranibizumab in the uk cohort does not exclude the possibility that results with ranibizumab over the whole of the uk may be closer to those seen in larger real - world studies such as the emr study , which found that real world outcomes did not match the results in randomized studies.16 the ongoing luminous study ( nct01318941 ) , which includes patients from a larger number of uk centers , may also provide additional information from a real - world perspective . this study also showed that anti - vegf agents are effective in patients with poor baseline visual acuity , which is similar to what was reported in another uk study.18 the findings in the uk also highlight the role of the loading phase with ranibizumab treatment . this is consistent with results observed in interventional studies with ranibizumab and bevacizumab.1921 however , the reasons for this are not fully understood , and may be related to a preconditioning effect of the loading phase , resulting in modification of subsequent disease course . overall , our results demonstrate that good outcomes are achievable in real - life settings , particularly if high quality control by payers ( such as the situation in the uk ) means that physicians adhere to protocols for monthly monitoring ( including injections being around or above seven in the first year ) ; however , this is associated with high resource utilization and increased burden for patients and health care providers , and may depend on reimbursement .

atherosclerosis is the major cause of death in western countries ; atherosclerosis leads to cardiovascular diseases such as peripheral artery disease , acute coronary syndromes , and aneurysms . the pathology of atherosclerosis develops in discrete stages : normal vessel wall , fatty streaks , atherosclerotic plaques , and ruptured plaques with thrombosis . the cellular and molecular events that lead to these pathological changes are well studied and include endothelial dysfunction , monocyte adherence and entry into the vessel wall , monocyte development into foam cells , smooth muscle cell migration and proliferation , and platelet adhesion and aggregation [ 2 , 3 ] . healthy endothelial cells ( ecs ) control vascular tone , limit vascular smooth muscle cells ( vsmcs ) proliferation , inhibit leukocyte adherence , and block thrombosis . ecs release a set of factors that promote vascular homeostasis , including nitric oxide and prostacyclin . however , a variety of vascular injuries destroy the ability of the endothelium to protect the vessel wall . furthermore , injured ecs express proinflammatory soluble and membrane bound mediators , including chemokines and p - selectin and vascular cell adhesion molecule-1 ( vcam-1 ) , which increase leukocyte trafficking , as well as von willebrand factor ( vwf ) which promotes thrombosis . for example , oxidized ldl can activate the nuclear factor b ( nf-b ) pathway , inducing the expression of a set of inflammatory genes . also , angiotensin ii ( angii ) activates ets-1 , a key endothelial transcription factor , leading to expression of vcam-1 by several stimuli . recent work by several investigators has revealed that micrornas ( mirnas ) can also control vascular inflammation . this paper summarizes the role of mirnas in vascular inflammation and highlights recent evidence that circulating mirnas are not only biomarkers for disease but also serve as cell - to - cell messengers . micrornas ( mirnas ) are small noncoding rnas of 1822 nucleotides in length , which regulate gene expression posttranscriptionally [ 1719 ] . mirnas regulate diverse biological functions , including cell proliferation , apoptosis , senescence , differentiation , metabolism , tumorigenesis , and developments . mature mirnas are generated from primary mirnas ( pri - mirnas ) by two rnase iii enzymes drosha and dicer . the drosha complex processes pri - mirnas into hairpin mirna precursors ( pre - mirnas ) in the nucleus ; then dicer cleaves these pre - mirnas into mirna duplexes in the cytoplasm . recently it has shown that some mirna precursors are generated by a drosha - independent pathway . one strand of this mirna duplex is incorporated into the rna - induced silencing complex ( risc ) and acts to guide the risc complex to its targets . in mammals , mature mirnas can bind to the 3 untranslated region ( 3-utr ) of target genes by partial complementarity . the interaction of the 5 end of mirnas ( the seed sequence ) with the target mrna is sufficient to stop translation of target genes . mirnas limit gene expression by ( 1 ) degradation of mrna or ( 2 ) inhibition of translation initiation . computer algorithms predict that most mirnas have multiple potential target genes , based on potential interactions between the 3utr of mrna and the mirna seed sequences . in fact , it is predicted that mirnas can manage the regulation of at least 60% of protein - coding genes in humans [ 18 , 22 ] . dysregulation of mirnas cause a variety of diseases , including cancer [ 23 , 24 ] , neuropsychiatric disease [ 25 , 26 ] , diabetes , and renal failure . a series of mirnas control inflammation and oxidative stress in vascular cells including ecs , vsmcs , and inflammatory cells [ 20 , 30 , 31 ] . elimination of most endothelial mirnas by knockdown of dicer in ecs inhibits proliferation and tube formation in vitro . moreover , ec - specific dicer knockout mice have impaired blood vessel development [ 35 , 36 ] . these findings suggest that mirnas in ecs are indispensable for the maintenance of vascular homeostasis . which mirnas are important in ecs and why ? mirna profiling data suggest that mir-126 is expressed mainly in ecs and platelets [ 34 , 37 , 38 ] . interestingly , mir-126 is located in the intron of epithelial growth factor like domain containing protein 7 ( egfl7 ) , an endothelial - specific protein involved in development of the vasculature . during splicing of egfl7 pre - mrna , knockout of mir-126 in mice and zebrafish decreases vascular integrity and impairs proliferation , migration , and angiogenic activity of ecs [ 40 , 41 ] . mir-126 knockout in mice is partially embryonic lethal , and surviving mir-126 knockout mice have defective cardiac neovascularization after myocardial infarction . mir-126 enhances vegf signaling by inhibiting sprouty - related protein ( spred1 ) and phosphoinositol-3 kinase regulatory subunit 2 ( pik3r2/p85-beta ) to maintain vascular integrity [ 4042 ] . thus mir-126 acts as a proangiogenic mirna by increasing pi3k and map kinase signaling . during vascular inflammation , mir-126 senescent ecs have increased apoptosis , induce inflammation , and have decreased nitric oxide production by endothelial nitric oxide synthase ( enos ) , causing endothelial dysfunction , followed by progression of atherosclerosis [ 48 , 49 ] . in cultured ecs , both replicative senescence and stress - induced premature senescence release proinflammatory mediators and decrease expression of anti - inflammatory proteins such as enos [ 50 , 51 ] . several mirnas are identified as senescent associated mirnas in many cancers and fibroblasts [ 5254 ] . the profiling of mirnas in senescent human primary ecs shows that a set of mirnas , such as mir-17 - 5p , mir-21 , mir-216 , mir-217 , mir-31b , and mir-181a / b , are highly expressed by aging cells . mir-146a regulates nox4 , which is one of nadph oxidase isoforms and contributes to generation of reactive oxidative stress ( ros ) . since ros promotes ecs senescence , mir-146a suppresses senescence by inhibiting nox4 , suggesting that the decrease level of mir-146a in senescent ecs may promote more aging by enhancing nox4 expression . mir-217 is minimally expressed in normal ecs , but mir-217 expression increases in senescent cells . sirt1 promotes longevity and prevents stress - induced senescence in ecs [ 57 , 58 ] . sirt1 controls a variety of transcription factors such as p53 , foxo ( forkhead box o ) , and pgc-1a ( peroxisome proliferators activated receptor gamma coactivator-1a ) . overexpression of mir-217 decreases sirt1 expression , which increases acetylation of foxo1 in young ecs . since ectopic expression of foxo1 inhibits ecs migration and tube formation , mir-217 blocks angiogenic property in ecs by inhibiting sirt1-foxo1 function . menghini et al . also demonstrated that mir-217 is negatively correlated with sirt1 expression in human atherosclerotic plaques . these results suggest that mir-217 has an important role in the pathogenesis of atherosclerosis in vitro and in vivo . demonstrated that the expression of mir-34a in heart and spleen are higher in aged mice than in young mice . since sirt1 has been shown to be a direct target of mir-34a , mir-34a promotes aging of ecs through sirt1 inhibition . mir-34a epcs are involved in new blood vessel formation to maintain ecs homeostasis and the number of epcs is reduced in atherosclerotic patients , indicating that mir-34a may be implicated in the progression of atherosclerosis ; however , the relationship between mir-34a and atherogenesis is not defined yet . several mirnas including mir-21 and mir-214 are downregulated in senescent human aortic endothelial cells ( haec ) compared with young haec . mir-21 regulates cell proliferation by suppressing phosphatase and tensin homolog deleted on chromosome 10 ( pten ) , a potent negative regulator of pi3k / akt signaling pathway . pten suppresses akt signaling , which decreases enos activity and pten also inhibits vcam-1 expression in tnf--stimulated ecs , suggesting that mir-21 promotes inflammation in ecs . shear stress induces mir-21 expression and high mir-21 level is observed in vessels during pulmonary hypertension [ 65 , 66 ] . blockage of mir-21 suppresses tgf--induced endmt by inhibiting phosphatase and tensin homolog ( pten ) in ecs . pressure overload of left ventricular in mice increases mir-21 expression and fibroblast markers in cardiac ecs ; mir-21 antagomir blocks this effect . angiogenesis plays an important role in the development of atherosclerosis [ 68 , 69 ] . the mir-17 - 92 cluster is a polycistronic mirna gene ( c13orf25 transcript ) , containing six tandem stem - loop hairpin structure that produce six mature mirnas : mir-17 , mir-18a , mir-19a , mir-19b-1 , mir-20a , and mir-92 [ 70 , 71 ] . moreover , two mir-17 - 92 cluster paralogs exist , mir-106a-363 and mir-106b-25 . this mirna polycistron is functionally categorized into four families : ( 1 ) mir-17 family , ( 2 ) mir-18 family , ( 3 ) mir-19 family , and ( 4 ) mir-92 family . the c13orf25 transcript containing mir-17 - 92 precursor is often elevated in many cancers [ 7275 ] . in ecs , impaired angiogenic activity by knockdown of dicer in ecs is rescued by adding individual mirnas in the mir-17 - 92 cluster . overexpression of mir-92a targets integrin 5 ( itga5 ) and inhibits angiogenic activity in ecs . administration of antagomir-92a blocks neovascularization in mouse hindlimb ischemia model and limits tissue injury in myocardial infarction . overexpression of mir-17 , mir-18a , mir-19a , and mir-20a inhibits endothelial sprouting in vitro . in vivo , inhibition of mir-17 and mir-20a increase the number of lectin - perfused vessels in matrigel plugs , but knockdown of mir-18a and mir-19a does not . these findings indicate that individual mirnas in this cluster function as negative regulators of angiogenesis . there are two highly conserved clusters : an intergenic mir-23a-27a-24 - 2 cluster and an intronic mir-24b-27b-24 - 1 cluster . mir-23a is upregulated during hypertrophy by pressure overload or isoproterenol treatment [ 81 , 82 ] . mir-27b targets thrombospondin-1 ( tsp-1 ) , an endogenous angiogenesis inhibitor [ 34 , 79 ] . inhibition of mir-27b reduced in vitro sprout formation . tsp-1 deficiency accelerates atherosclerotic plaque maturation in apoe knockout mice and dysregulates vsmcs activation in the arterial wall [ 84 , 85 ] . another study identified semaphorin 6a ( sema6a ) as a target of mir-27a / b . mir-27a / b negatively regulates ecs sprout formation and knockdown of mir-27a / b blocks embryonic vessel formation in zebrafish . zhou et al . demonstrated that knockdown of mir-23 and mir-27 impairs sprouting of aorta ring cells , migration , and tube formation of ecs in vitro . mir-23 and mir-27 inhibit expression of sprouty2 , semaphorin 6a , and semaphorin 6d , which inhibit angiogenesis . , cells express prolyl hydroxylase domain protein 2 ( phd2 or egln ) , which hydroxylates prolyl residues on hypoxia - inducible factor-1 alpha ( hif-1 ) . prolyl hydroxylated hif-1 is immediately degraded by binding to von hippel - lindau ( vhl ) . however , hypoxia suppresses phd2 activity , stabilizing hif-1 , which then forms heterodimers with its partner hypoxia - inducible factor-1 beta ( hif-1 ) . this complex is translocated into the nucleus and promotes expression of hundreds of hypoxia regulated gene , such as vascular endothelial growth factor ( vegf ) . hypoxia induces mir-210 expression in ecs as well as in cancer cells . in cancer , the expression level of mir-210 is correlated with poor survival in cancer patients [ 90 , 91 ] . interesting target genes of mir-210 include glycerol-3-phosphate dehydrogenase ( gpd1l ) and mitochondrial components ( nadh dehydrogenase ( ubiquinone ) 1 alpha subcomplex 4 ( ndufa4 ) and succinate dehydrogenase complex , subunit d ( sdhd ) ) . moreover , mir-210 expression is associated with expression of vegf signaling molecules in clinical breast cancer . in ecs , mir-210 controls the receptor tyrosine kinase ephrin - a3 ( efna3 ) that is involved in vascular remodeling . overexpression of mir-210 increases ecs migration ; inhibition of mir-210 decreases ecs tube formation under hypoxia . hif-1 directly binds to hypoxia responsive element ( hre ) on the promoter of mir-210 , following production of mir-210 transcripts . mir-424 and mir-503 are derived from a polycistronic precursor mir-424 - 503 . these mirnas are induced during monocyte differentiation and myogenesis . mir-503 expression in ecs is upregulated by high glucose or the absence of growth factors . cdc25a is a protein phosphatase that drives cell cycle by activating cyclin - dependent protein kinases ( cdks ) and ccne1 functions as a regulator of cdks . mir-503 expression is increased in ischemic adductor muscles of hindlimb ischemia model in streptozotocin - induced diabetic mice and administration of mir-503 decoy to inhibit mir-503 recovers postischemic angiogenesis . an ubiquitin ligase scaffold protein cullin-2 ( clu2 ) destabilizes hif-1 to assemble an e3 ubiquitin ligase complex . hypoxia - induced mir-424 decreases clu2 protein expression , which in turn stabilizes hif-1 and promotes hypoxia regulated gene expression , which increases proliferation and migration of ecs and angiogenesis in mice . c / ebp bound with runx-1 activates the pu.1 promoter and increased pu.1 then induces the expression of mir-424 . vegf and fibroblast growth factor 2 ( fgf2 ) increase mir-424 and mir-16 , and these mirnas target vegf receptor 2 ( vegfr2 ) and fgf receptor 1 ( fgfr1 ) . mir-16 and mir-424 are located in different gene locations but have the same seed sequence , so it is not surprising that mir-16 and mir-424 share the same target genes . in this case , mir-424 overexpression reduces proliferation and migration in ecs . interestingly vegf and fgf2 increase mature mir-424 , but not pri - mir-424 in ecs , suggesting that increase of mir-424 expression by vegf and bfgf stimulation are not because the induction of transcription , but due to a positive regulation of mirna processing from the preexisting primary transcript . vascular inflammation is an early step in atherogenesis , and many mirnas are induced in inflamed ecs . proinflammatory cytokines ( tnf- , il-1 ) and lps increase a set of adhesion molecules in ecs , which recruit inflammatory cells to the site of inflammation . this induction of adhesion molecules is mainly mediated by the nf-b pathway . among many different homodimers and heterodimers in the nf-b / rel family , the p50/p65 heterodimer is predominant in ecs . in resting ecs , nf-b binds to ib protein , an inhibitor protein of nf-b , and localized in the cytoplasm . once ecs are activated , ib kinase ( ikk ) complex is phosphorylated , which rapidly degrades ib by the 26s proteasome . this leads to translocate nf-b heterodimers into nucleus immediately , following the induction of the number of inflammatory response genes . overexpression of mir-181b blocks the induction of adhesion molecules , such as vcam-1 , in vitro and in vivo . systematic administration of mir-181b mimics reduces leukocyte accumulation and ecs activation in lps - induced lung injury . sun et al . demonstrated that mir-181b targets importin-3 , which is required for nuclear translocation of nf-b , suggesting that the inhibitory effects of mir-181b on tnf- induced expression of adhesion molecules are mediated by repression of nf-b nuclear translocation . fang et al . demonstrated that mir-10a / b expression is lower at athero - susceptible arterial sites compared with athero - protected sites in dorsal thoracic aorta from swine . fang et al . then showed that mir-10a directly inhibits two key molecules of ib degradation , mitogen - activated kinase kinase kinase 7 ( map3k7 or tak1 ) and beta - transducin repeat - containing gene ( betatrc ) . knockdown of mir-10a decreases the expression of map3k7 and betatrc , which upregulates phosphorylation of ib , causing more nuclear transport of nf-b p65 and upregulation of the inflammatory cytokines such as mcp-1 , il-6 , il-8 , vcam1 , and e - selectin ( sele ) . this suggests that mir-10a contributes to the regulation of inflammatory response through nf-b pathway in ecs . mir-31 and mir-17 are induced by tnf- in human umbilical cord endothelial cells ( huvec ) and mir-31 regulates sele and mir-17 - 3p targets intercellular adhesion molecule 1 ( icam1 ) in ecs . both mirnas control neutrophil adhesion to ecs in vitro , suggesting that mir-31 and mir-17 - 3p limit vascular inflammation by regulating the expression of adhesion molecule . tnf- treatment of ecs induces other mirnas such as mir-155 , mir-221 , and mir-222 . these mirnas are enriched in ecs and target ets-1 , a key endothelial transcription factor . stimulation with angiotensin ii increases downstream genes of ets-1 , including vcam1 , monocyte chemotactic protein 1 ( mcp1 ) , and fms - related tyrosine kinase 1 ( flt1 ) ; overexpression of mir-155 partially restores this effect , suggesting that mir-155 regulates adhesion of t cells to activated ecs . interestingly , the human at1r contains a + 1166 a / c polymorphism , which enhances at1r activity . since this + 1166 a / c mutation destroys mir-155 binding element ( the seed sequence ) , this mutation often maintains high at1r activity . dicer knockdown enhances enos expression in huvec , and mir-221 and mir-222 overexpression rescues the enhanced enos suppression [ 20 , 35 ] . of note , the 3utr of enos has no target sequence for mir-221/222 , suggesting that this regulation is indirect . liu et al . have shown that mir-221/222 targets p27 ( kip1 ) to suppress endothelial proliferation and growth of vsmcs is promoted by inhibiting c - kit . mir-221/222 increases neointimal growth but decrease reendothelialization in balloon - injury rat carotid artery model . krppel - like family of transcription factor , the zinc finger family of dna - binding transcription factor , is regulated by several stimuli such as laminar flow and statins in ecs . kruppel - like factor 2 ( klf2 ) and klf4 are implicated in protection of atherogenesis through anti - inflammatory and anticoagulant pathways [ 116 , 117 ] . especially klf2 plays a pivotal role in endothelial biology . klf2 inhibits cytokine - mediated induction of vcam-1 and sele expression , resulting in decreasing inflammation in ecs . klf2 induces thrombomodulin ( tm ) , a cell surface factor involved in antithrombotic function on the surface of ecs . mir-92a negatively regulates klf4 and klf2 expression in arterial endothelium [ 120 , 121 ] . mir-92a , a member of the mir-17 - 92 cluster , has been identified as an endogenous repressor of angiogenesis ( see section 3.3.1 ) . overexpression of mir-92a inhibits the expression of enos and tm , downstream molecules of klf2 , and administration of mir-92a into mice decreases the expression of klf2 and enos in the arteries . fang and davies also demonstrated that atheroprone flow increases the interaction between mir-92a and klf2 mrna with ago proteins , one of the major rna induced silencing complex , indicating direct evidence that mir-92a regulates klf2 expression . tnf- increases the expression of monocyte chemotactic protein 1 ( mcp-1 ) , vcam-1 , and sele in human aortic endothelial cells ( haec ) . fang and davies demonstrated that knockdown of mir-92a partially suppresses these tnf--induced endothelial inflammatory mediators through klf4 and mir-92 knockdown inhibits tnf--induced leukocyte adhesion to ecs in vitro . these findings suggest that mir-92a in ecs acts as an atheroprotective mirna by regulating klf2 and klf4 . human studies have revealed a set of mirna in blood , joint fluid , and other extracellular locations [ 122 , 123 ] . extracellular mirnas have been used as biomarkers to classify diseases and progression of diseases [ 124 , 125 ] . recent studies have revealed that mirnas also serve as messengers between cells [ 126128 ] . the other mode is by active secretion from living cells within microvesicles ( mvs ) or in rna - lipid / protein complexes . cytokines or shear stress induce ecs - derived mvs release . in response to these stresses , ecs release three types of mvs : exosomes , microparticles , and apoptotic bodies . exosomes , lipid bound particles about 30100 nm in size , are generated through the endosomal pathway from multivesicular bodies ( mvb ) , and then secreted by the fusion of endosome and plasma membrane . microparticles are released by budding from the outer layers of plasma membranes , and their size is larger than exosomes ( 100 nm1 m ) . the much larger size of apoptotic bodies , about 13 m in size , contains mirnas , dna , and histones . apoptotic bodies are released by ecs in atherosclerotic lesions and can fuse to other vascular cells , delivering their contents . some mirnas are incorporated into rna - binding proteins such as argonaute 2 or nucleophosmin 1 ( npm1 ) and high - density lipoprotein ( hdl ) and exist as mvs - free conditions [ 32 , 33 , 136 ] . however , neutral sphingomyelinase 2 ( nsmase2 ) controls ceramide biosynthesis and inhibition of nsmase2 by gw4869 or a silencing rna decreases secretion of mirna , suggesting that ceramide pathway is involved in mvs secretion . mvs are released into microenvironments near their origin and can be detected in plasma , urine , bile , ascites , cerebrospinal fluid , and breast milk . circulating mirnas are also detected in body fluids , such as serum , plasma , urine , and saliva [ 124 , 139141 ] . mvs derived from ecs and platelet are elevated in hypertensive patients , suggesting that pressure induced activation of ecs and platelets increase mvs productions . human atherosclerotic plaques contain a lot of microparticles , which comes from other origins such as platelets , ecs , and monocytes [ 143 , 144 ] . moreover , mvs affect the progression and development of human atherosclerotic lesions by transferring adhesion molecules and cytokines . interestingly , chronic treatment with antioxidants decreases ecs - derived mvs in patients with heart failure . these reports suggest that molecules including mirnas inside mvs can regulate functions of recipient cells . a variety cells secrete mirnas , including t cells , monocytes , endothelial cells , adipocytes , and cancer cells [ 126128 , 148150 ] . since many cell types express a set of mirnas , circulating mirnas released from the cells represent their original source . therefore circulating mirnas have diagnostic and prognostic potential . for example , high levels of mir-208b , mir-499 , and mir-133a are detected in plasma of myocardial infarction patients [ 151 , 152 ] . especially , high levels of mir-133a and mir-208b are significantly associated with the risk of death in acute coronary syndrome . since these mirnas are expressed well in the heart , most of these mirnas might be released from cardiomyocytes or fibroblasts during myocardial injury or infarction . in plasma of type 2 vascular mirnas , such as mir-126 , mir17 , mir-92a , and mir-155 are significantly lower levels in serum of patients with coronary artery disease compared with healthy subjects . possible reasons for this reduction of mirna levels are ( 1 ) lack of mirnas storage and production in vascular cells after dramatic release and activation of vasculature , ( 2 ) increased uptake of mirnas into blood cells or into atherosclerotic lesions , or ( 3 ) decrease of nsmase activity in blood vessels . mirnas released by ecs can regulate the biology of vascular cells , including vsmcs , leukocytes and other ecs . demonstrated that apoptotic bodies from ecs trigger cxcl12 production in other cells in the vascular wall in a paracrine manner . mir-126 is packaged in ecs derived apoptotic bodies , and directly suppressed a set of genes , including regulator of g protein signaling 16 ( rgs16 ) , which is known to negatively regulate cxcr4 , the cxcl12 receptor . this upregulation of cxcr4 by mir-126 uptake promotes cxcl12 production through an autoregulatory feedback loop . transfer of mir-126 enriched apoptotic bodies or even mir-126 itself into the apoe knockout mice reduces the size of lesions , suggesting that the antiatherosclerotic effect of ecs derived apoptotic bodies is at least partially performed by mir-126 . various stimuli , including lps , oxidative stress , and advanced glycosylated end - product ( age ) , trigger mir-150 release from monocytes in vitro . mir-150 is packaged into 20400 nm sized mvs and these mvs deliver mir-150 into human cultural ecs and inhibit c - myb expression . in vivo , communication via mirnas between vascular cells might play a role in inflammatory events leading to atherosclerosis . kruppel - like factor 2 ( klf2 ) is a key molecule induced by atheroprotective shear stress , and it regulates a set of genes expressed in ecs described above ( see section 3.6 ) . in vitro , klf2 expression is upregulated by laminar flow or statin . discovered that physiological shear stress and statin treatment activate expression of the mir-143/145 cluster through klf2 in ecs . mir-143 and mir-145 are intergenic mirnas , which control the vsmcs phenotypic switch , tumorigenesis , and adipocyte differentiation [ 156158 ] . interestingly mir-143/145 synthesized in ecs are secreted into extracellular vesicles and transported into vsmcs . mir-143 and mir-145 in contrast , incomplete differentiation of vsmcs is observed in aortas from mir-143/145 knockout mice . hergenreider et al . demonstrated that injection of ecs derived mvs containing mir-143/145 reduces the formation of atherosclerotic lesion in apoe knockout mice . these studies demonstrated that atheroprotective flow increases mir-143/145 expression and secretion , and mir-143/145 can be transferred into vsmcs , preventing dedifferentiation . although the in vitro evidence is compelling , definitive in vivo evidence for intercellular communication through mirna is still lacking . mirnas function as fine tuners of various biological processes to maintain homeostasis and play a key role in atherogenesis . mirnas within ecs and vsmcs and monocytes regulate their proliferation , migration , and inflammatory profile . mirnas can be released by cells and taken up by vascular cells , modulating their cellular biology . mirna profiles in the blood of humans provide diagnostic and prognostic information during acute vascular events . the rapid development of rna chemistry has led to the invention of novel modifications of rna bases and the synthesis of artificial antisense mirna or antagomir , which may be used as novel therapeutic tools in the future to manipulate mirna and control vascular inflammatory diseases .

vascular inflammation is an important component of the pathophysiology of cardiovascular diseases , such as hypertension , atherosclerosis , and aneurysms . all vascular cells , including endothelial cells ( ecs ) and vascular smooth muscle cells ( vsmcs ) , and infiltrating cells , such as macrophages , orchestrate a series of pathological events . despite dramatic improvements in the treatment of atherosclerosis , the molecular basis of vascular inflammation is not well understood . in the last decade , micrornas ( mirnas ) have been revealed as novel regulators of vascular inflammation . each mirnas suppresses a set of genes , forming complex regulatory network . this paper provides an overview of current advances that have been made in revealing the roles of mirnas during vascular inflammation . recent studies show that mirnas not only exist inside cells but also circulate in blood . these circulating mirnas are useful biomarkers for diagnosis of cardiovascular diseases . furthermore , recent studies demonstrate that circulating mirnas are delivered into certain recipient cells and act as messengers . these studies suggest that mirnas provide new therapeutic opportunities .

1. Introduction 2. Biogenesis of miRNAs (See 3. Endothelial miRNAs (See 4. The Communication of miRNAs between Cells 5. Summary

atherosclerosis is the major cause of death in western countries ; atherosclerosis leads to cardiovascular diseases such as peripheral artery disease , acute coronary syndromes , and aneurysms . the pathology of atherosclerosis develops in discrete stages : normal vessel wall , fatty streaks , atherosclerotic plaques , and ruptured plaques with thrombosis . the cellular and molecular events that lead to these pathological changes are well studied and include endothelial dysfunction , monocyte adherence and entry into the vessel wall , monocyte development into foam cells , smooth muscle cell migration and proliferation , and platelet adhesion and aggregation [ 2 , 3 ] . healthy endothelial cells ( ecs ) control vascular tone , limit vascular smooth muscle cells ( vsmcs ) proliferation , inhibit leukocyte adherence , and block thrombosis . ecs release a set of factors that promote vascular homeostasis , including nitric oxide and prostacyclin . however , a variety of vascular injuries destroy the ability of the endothelium to protect the vessel wall . furthermore , injured ecs express proinflammatory soluble and membrane bound mediators , including chemokines and p - selectin and vascular cell adhesion molecule-1 ( vcam-1 ) , which increase leukocyte trafficking , as well as von willebrand factor ( vwf ) which promotes thrombosis . for example , oxidized ldl can activate the nuclear factor b ( nf-b ) pathway , inducing the expression of a set of inflammatory genes . recent work by several investigators has revealed that micrornas ( mirnas ) can also control vascular inflammation . this paper summarizes the role of mirnas in vascular inflammation and highlights recent evidence that circulating mirnas are not only biomarkers for disease but also serve as cell - to - cell messengers . micrornas ( mirnas ) are small noncoding rnas of 1822 nucleotides in length , which regulate gene expression posttranscriptionally [ 1719 ] . mirnas regulate diverse biological functions , including cell proliferation , apoptosis , senescence , differentiation , metabolism , tumorigenesis , and developments . mature mirnas are generated from primary mirnas ( pri - mirnas ) by two rnase iii enzymes drosha and dicer . the drosha complex processes pri - mirnas into hairpin mirna precursors ( pre - mirnas ) in the nucleus ; then dicer cleaves these pre - mirnas into mirna duplexes in the cytoplasm . one strand of this mirna duplex is incorporated into the rna - induced silencing complex ( risc ) and acts to guide the risc complex to its targets . the interaction of the 5 end of mirnas ( the seed sequence ) with the target mrna is sufficient to stop translation of target genes . computer algorithms predict that most mirnas have multiple potential target genes , based on potential interactions between the 3utr of mrna and the mirna seed sequences . in fact , it is predicted that mirnas can manage the regulation of at least 60% of protein - coding genes in humans [ 18 , 22 ] . dysregulation of mirnas cause a variety of diseases , including cancer [ 23 , 24 ] , neuropsychiatric disease [ 25 , 26 ] , diabetes , and renal failure . a series of mirnas control inflammation and oxidative stress in vascular cells including ecs , vsmcs , and inflammatory cells [ 20 , 30 , 31 ] . these findings suggest that mirnas in ecs are indispensable for the maintenance of vascular homeostasis . which mirnas are important in ecs and why ? mirna profiling data suggest that mir-126 is expressed mainly in ecs and platelets [ 34 , 37 , 38 ] . interestingly , mir-126 is located in the intron of epithelial growth factor like domain containing protein 7 ( egfl7 ) , an endothelial - specific protein involved in development of the vasculature . during splicing of egfl7 pre - mrna , knockout of mir-126 in mice and zebrafish decreases vascular integrity and impairs proliferation , migration , and angiogenic activity of ecs [ 40 , 41 ] . mir-126 knockout in mice is partially embryonic lethal , and surviving mir-126 knockout mice have defective cardiac neovascularization after myocardial infarction . mir-126 enhances vegf signaling by inhibiting sprouty - related protein ( spred1 ) and phosphoinositol-3 kinase regulatory subunit 2 ( pik3r2/p85-beta ) to maintain vascular integrity [ 4042 ] . during vascular inflammation , mir-126 senescent ecs have increased apoptosis , induce inflammation , and have decreased nitric oxide production by endothelial nitric oxide synthase ( enos ) , causing endothelial dysfunction , followed by progression of atherosclerosis [ 48 , 49 ] . in cultured ecs , both replicative senescence and stress - induced premature senescence release proinflammatory mediators and decrease expression of anti - inflammatory proteins such as enos [ 50 , 51 ] . several mirnas are identified as senescent associated mirnas in many cancers and fibroblasts [ 5254 ] . the profiling of mirnas in senescent human primary ecs shows that a set of mirnas , such as mir-17 - 5p , mir-21 , mir-216 , mir-217 , mir-31b , and mir-181a / b , are highly expressed by aging cells . sirt1 controls a variety of transcription factors such as p53 , foxo ( forkhead box o ) , and pgc-1a ( peroxisome proliferators activated receptor gamma coactivator-1a ) . these results suggest that mir-217 has an important role in the pathogenesis of atherosclerosis in vitro and in vivo . mir-34a epcs are involved in new blood vessel formation to maintain ecs homeostasis and the number of epcs is reduced in atherosclerotic patients , indicating that mir-34a may be implicated in the progression of atherosclerosis ; however , the relationship between mir-34a and atherogenesis is not defined yet . several mirnas including mir-21 and mir-214 are downregulated in senescent human aortic endothelial cells ( haec ) compared with young haec . mir-21 regulates cell proliferation by suppressing phosphatase and tensin homolog deleted on chromosome 10 ( pten ) , a potent negative regulator of pi3k / akt signaling pathway . angiogenesis plays an important role in the development of atherosclerosis [ 68 , 69 ] . the mir-17 - 92 cluster is a polycistronic mirna gene ( c13orf25 transcript ) , containing six tandem stem - loop hairpin structure that produce six mature mirnas : mir-17 , mir-18a , mir-19a , mir-19b-1 , mir-20a , and mir-92 [ 70 , 71 ] . this mirna polycistron is functionally categorized into four families : ( 1 ) mir-17 family , ( 2 ) mir-18 family , ( 3 ) mir-19 family , and ( 4 ) mir-92 family . in ecs , impaired angiogenic activity by knockdown of dicer in ecs is rescued by adding individual mirnas in the mir-17 - 92 cluster . overexpression of mir-92a targets integrin 5 ( itga5 ) and inhibits angiogenic activity in ecs . overexpression of mir-17 , mir-18a , mir-19a , and mir-20a inhibits endothelial sprouting in vitro . mir-27b targets thrombospondin-1 ( tsp-1 ) , an endogenous angiogenesis inhibitor [ 34 , 79 ] . tsp-1 deficiency accelerates atherosclerotic plaque maturation in apoe knockout mice and dysregulates vsmcs activation in the arterial wall [ 84 , 85 ] . demonstrated that knockdown of mir-23 and mir-27 impairs sprouting of aorta ring cells , migration , and tube formation of ecs in vitro . mir-23 and mir-27 inhibit expression of sprouty2 , semaphorin 6a , and semaphorin 6d , which inhibit angiogenesis . , cells express prolyl hydroxylase domain protein 2 ( phd2 or egln ) , which hydroxylates prolyl residues on hypoxia - inducible factor-1 alpha ( hif-1 ) . this complex is translocated into the nucleus and promotes expression of hundreds of hypoxia regulated gene , such as vascular endothelial growth factor ( vegf ) . in cancer , the expression level of mir-210 is correlated with poor survival in cancer patients [ 90 , 91 ] . interesting target genes of mir-210 include glycerol-3-phosphate dehydrogenase ( gpd1l ) and mitochondrial components ( nadh dehydrogenase ( ubiquinone ) 1 alpha subcomplex 4 ( ndufa4 ) and succinate dehydrogenase complex , subunit d ( sdhd ) ) . these mirnas are induced during monocyte differentiation and myogenesis . cdc25a is a protein phosphatase that drives cell cycle by activating cyclin - dependent protein kinases ( cdks ) and ccne1 functions as a regulator of cdks . vegf and fibroblast growth factor 2 ( fgf2 ) increase mir-424 and mir-16 , and these mirnas target vegf receptor 2 ( vegfr2 ) and fgf receptor 1 ( fgfr1 ) . mir-16 and mir-424 are located in different gene locations but have the same seed sequence , so it is not surprising that mir-16 and mir-424 share the same target genes . vascular inflammation is an early step in atherogenesis , and many mirnas are induced in inflamed ecs . proinflammatory cytokines ( tnf- , il-1 ) and lps increase a set of adhesion molecules in ecs , which recruit inflammatory cells to the site of inflammation . among many different homodimers and heterodimers in the nf-b / rel family , the p50/p65 heterodimer is predominant in ecs . in resting ecs , nf-b binds to ib protein , an inhibitor protein of nf-b , and localized in the cytoplasm . this leads to translocate nf-b heterodimers into nucleus immediately , following the induction of the number of inflammatory response genes . overexpression of mir-181b blocks the induction of adhesion molecules , such as vcam-1 , in vitro and in vivo . then showed that mir-10a directly inhibits two key molecules of ib degradation , mitogen - activated kinase kinase kinase 7 ( map3k7 or tak1 ) and beta - transducin repeat - containing gene ( betatrc ) . knockdown of mir-10a decreases the expression of map3k7 and betatrc , which upregulates phosphorylation of ib , causing more nuclear transport of nf-b p65 and upregulation of the inflammatory cytokines such as mcp-1 , il-6 , il-8 , vcam1 , and e - selectin ( sele ) . mir-31 and mir-17 are induced by tnf- in human umbilical cord endothelial cells ( huvec ) and mir-31 regulates sele and mir-17 - 3p targets intercellular adhesion molecule 1 ( icam1 ) in ecs . both mirnas control neutrophil adhesion to ecs in vitro , suggesting that mir-31 and mir-17 - 3p limit vascular inflammation by regulating the expression of adhesion molecule . tnf- treatment of ecs induces other mirnas such as mir-155 , mir-221 , and mir-222 . stimulation with angiotensin ii increases downstream genes of ets-1 , including vcam1 , monocyte chemotactic protein 1 ( mcp1 ) , and fms - related tyrosine kinase 1 ( flt1 ) ; overexpression of mir-155 partially restores this effect , suggesting that mir-155 regulates adhesion of t cells to activated ecs . interestingly , the human at1r contains a + 1166 a / c polymorphism , which enhances at1r activity . since this + 1166 a / c mutation destroys mir-155 binding element ( the seed sequence ) , this mutation often maintains high at1r activity . dicer knockdown enhances enos expression in huvec , and mir-221 and mir-222 overexpression rescues the enhanced enos suppression [ 20 , 35 ] . of note , the 3utr of enos has no target sequence for mir-221/222 , suggesting that this regulation is indirect . krppel - like family of transcription factor , the zinc finger family of dna - binding transcription factor , is regulated by several stimuli such as laminar flow and statins in ecs . kruppel - like factor 2 ( klf2 ) and klf4 are implicated in protection of atherogenesis through anti - inflammatory and anticoagulant pathways [ 116 , 117 ] . klf2 induces thrombomodulin ( tm ) , a cell surface factor involved in antithrombotic function on the surface of ecs . mir-92a , a member of the mir-17 - 92 cluster , has been identified as an endogenous repressor of angiogenesis ( see section 3.3.1 ) . overexpression of mir-92a inhibits the expression of enos and tm , downstream molecules of klf2 , and administration of mir-92a into mice decreases the expression of klf2 and enos in the arteries . fang and davies also demonstrated that atheroprone flow increases the interaction between mir-92a and klf2 mrna with ago proteins , one of the major rna induced silencing complex , indicating direct evidence that mir-92a regulates klf2 expression . tnf- increases the expression of monocyte chemotactic protein 1 ( mcp-1 ) , vcam-1 , and sele in human aortic endothelial cells ( haec ) . these findings suggest that mir-92a in ecs acts as an atheroprotective mirna by regulating klf2 and klf4 . human studies have revealed a set of mirna in blood , joint fluid , and other extracellular locations [ 122 , 123 ] . extracellular mirnas have been used as biomarkers to classify diseases and progression of diseases [ 124 , 125 ] . recent studies have revealed that mirnas also serve as messengers between cells [ 126128 ] . exosomes , lipid bound particles about 30100 nm in size , are generated through the endosomal pathway from multivesicular bodies ( mvb ) , and then secreted by the fusion of endosome and plasma membrane . microparticles are released by budding from the outer layers of plasma membranes , and their size is larger than exosomes ( 100 nm1 m ) . the much larger size of apoptotic bodies , about 13 m in size , contains mirnas , dna , and histones . apoptotic bodies are released by ecs in atherosclerotic lesions and can fuse to other vascular cells , delivering their contents . some mirnas are incorporated into rna - binding proteins such as argonaute 2 or nucleophosmin 1 ( npm1 ) and high - density lipoprotein ( hdl ) and exist as mvs - free conditions [ 32 , 33 , 136 ] . mvs are released into microenvironments near their origin and can be detected in plasma , urine , bile , ascites , cerebrospinal fluid , and breast milk . circulating mirnas are also detected in body fluids , such as serum , plasma , urine , and saliva [ 124 , 139141 ] . human atherosclerotic plaques contain a lot of microparticles , which comes from other origins such as platelets , ecs , and monocytes [ 143 , 144 ] . these reports suggest that molecules including mirnas inside mvs can regulate functions of recipient cells . a variety cells secrete mirnas , including t cells , monocytes , endothelial cells , adipocytes , and cancer cells [ 126128 , 148150 ] . since many cell types express a set of mirnas , circulating mirnas released from the cells represent their original source . therefore circulating mirnas have diagnostic and prognostic potential . for example , high levels of mir-208b , mir-499 , and mir-133a are detected in plasma of myocardial infarction patients [ 151 , 152 ] . since these mirnas are expressed well in the heart , most of these mirnas might be released from cardiomyocytes or fibroblasts during myocardial injury or infarction . in plasma of type 2 vascular mirnas , such as mir-126 , mir17 , mir-92a , and mir-155 are significantly lower levels in serum of patients with coronary artery disease compared with healthy subjects . possible reasons for this reduction of mirna levels are ( 1 ) lack of mirnas storage and production in vascular cells after dramatic release and activation of vasculature , ( 2 ) increased uptake of mirnas into blood cells or into atherosclerotic lesions , or ( 3 ) decrease of nsmase activity in blood vessels . mirnas released by ecs can regulate the biology of vascular cells , including vsmcs , leukocytes and other ecs . demonstrated that apoptotic bodies from ecs trigger cxcl12 production in other cells in the vascular wall in a paracrine manner . mir-126 is packaged in ecs derived apoptotic bodies , and directly suppressed a set of genes , including regulator of g protein signaling 16 ( rgs16 ) , which is known to negatively regulate cxcr4 , the cxcl12 receptor . various stimuli , including lps , oxidative stress , and advanced glycosylated end - product ( age ) , trigger mir-150 release from monocytes in vitro . in vivo , communication via mirnas between vascular cells might play a role in inflammatory events leading to atherosclerosis . kruppel - like factor 2 ( klf2 ) is a key molecule induced by atheroprotective shear stress , and it regulates a set of genes expressed in ecs described above ( see section 3.6 ) . discovered that physiological shear stress and statin treatment activate expression of the mir-143/145 cluster through klf2 in ecs . mir-143 and mir-145 are intergenic mirnas , which control the vsmcs phenotypic switch , tumorigenesis , and adipocyte differentiation [ 156158 ] . these studies demonstrated that atheroprotective flow increases mir-143/145 expression and secretion , and mir-143/145 can be transferred into vsmcs , preventing dedifferentiation . mirnas within ecs and vsmcs and monocytes regulate their proliferation , migration , and inflammatory profile . mirnas can be released by cells and taken up by vascular cells , modulating their cellular biology . the rapid development of rna chemistry has led to the invention of novel modifications of rna bases and the synthesis of artificial antisense mirna or antagomir , which may be used as novel therapeutic tools in the future to manipulate mirna and control vascular inflammatory diseases .

histamine , a low - molecular - weight amine synthesized from l - histidine in the reaction catalyzed by l - histidine decarboxylase , is known to activate the four g protein - coupled receptors : h1 , h2 , h3 and h4 , through which it is involved in the regulation of several physiological processes . although it is contained in almost all tissues in human organism , it is most frequently found in mast cells and basophilic leukocytes . this biogenic amine is released by endothelial cells , aggregating platelets , lymphocytes , and monocytes / macrophages [ 2 , 3 ] . mast cells and histamine also have cranium - caudal distribution in the heart which means that the largest quantity of this amine is in the right atrium and the least in left ventricle tissue . increased number of mast cells was found in coronary blood vessels adventitia in the patients suffering from ischemic heart diseases , especially in the narrow parts of blood vessels , or at the site of plaque rupture [ 4 , 5 ] . obvious connection was also determined between coronary blood vessel inflammation and mast cells activation in the process of atherogenesis [ 6 , 7 ] . for example , histamine enhances the expression of adhesion molecules in vascular endothelial cells , thereby augmenting leukocyte - endothelial cell interactions , an important onset event in atherogenesis . moreover , this biogenic amine suppresses hepatic ldl receptor expression and reduces plasma hdl cholesterol in rats . this suggests that histamine may play an important role in lipoprotein metabolism , which may be related to its role in the development of atherosclerosis . histamine has also been shown to increase smooth muscle cell proliferation and migration and implicated in intimal thickening and atherogenesis . histamine 's effects on coronary arteries are the result of multiple actions of this molecule on both smooth muscle and endothelial cells . these effects are species specific and depend on dose of histamine , diameter and initial vessel tone , and relative location within the coronary circulation . when atherosclerotic coronary artery is concerned histamine is a vasoconstrictor . on the other hand histamine is a proven endothelium - dependent vasodilator which bound with h1 receptor of endothelial cells activates endothelial nitric oxide synthase [ 1214 ] . although this enos upregulation may be protective under normal circumstances , as it increases no production , it may become harmful under conditions of oxidative stress when enos produces reactive oxygen species ( ros ) at the expense of no [ 1518 ] . also , it is shown that histamine produces coronary dilation via h2 receptor , which is not endothelium dependent [ 11 , 12 ] , as well as through h2- and h3-dependent mechanism involving the generation of nitric oxide . as said above , histamine is also released from the aggregating platelets , and these observations suggested a possible role of this amine in the pathogenesis of acute coronary syndrome ( acs ) . furthermore , histamine via h1 receptors induces expression of tissue factor ( tf ) , a key enzyme in the activation of coagulation . the elevated levels of quantity and activity of tf antigen have been detected in plasma and atherectomy specimens of patients with unstable angina and acute myocardial infarction [ 5 , 6 ] . recent research clearly indicates a great role of histamine in inflammation processes through its influence on cyclooxygenases and increased production of pgi2 and pge2 . considering the fact that inflammation is the key factor for atherosclerosis , finally , it is essential to point out the fact that blood histamine alone may be a good prognostic indicator for ischemic event ( acs ) , better than crp , even when it is combined with lipid indexes . up to now research showed that the patients suffering from ischemic heart diseases ( stable coronary artery disease ( scad ) and acute coronary syndrome ( acs ) ) have statistically significantly higher total blood histamine than the population without these diseases . however , these data about histamine blood levels in acs patients have been obtained only within the first 24 hours of acute event , which is why the aim of this study was to examine histamine blood concentration in subjects suffering from ischemic heart diseases during the period of eight days . furthermore , we intended to analyze whether histamine blood concentrations in patients depend on different types of ischemic heart diseases such as stable coronary artery disease , acute coronary syndrome , nonocclusive type , or acute coronary syndrome , occlusive type . all researches were performed according to the ethical standards of the local ethics committee and helsinki declaration . group i : control group included age , sex , and risk factors matched volunteers without coronary disease . all volunteers underwent an initial clinical assessment that included a clinical history taking , a physical examination , electrocardiography , standard blood measurements , and stress echocardiography . furthermore , none of the volunteers had shown any allergy manifestations ( n = 32 ) . group ii : subjects suffering from stable coronary artery disease ( scad patients ) determined by symptoms during physical activity , stress echocardiography , coronarography , and so forth ( n = 28 ) . patients suffering from this disease manifest symptoms ( like chest pain , fatigue , etc . ) and changes in ecg ( such as negative t - wave or st depression ) under exertion while the values of troponin i and ckmb remain normal . group iii : subjects with acute coronary syndrome , nonocclusive type ( unstable angina or acs - ua patients ) determined by symptoms during resting period , acute electrocardiographic changes , and coronarography ( n = 26 ) . apart from chest pain these subjects also have changes in ecg ( such as negative t - wave or the positivity of previously negative t - wave ) , while the values of troponin i and ckmb are within physiological range . group iv : subjects with acute coronary syndrome , occlusive type ( acs - stemi patients ) determined by typical accelerating symptoms , acute electrocardiographic changes and levels of serum enzymes ( troponin i , ckmb ) , and coronarography ( n = 37 ) . patients suffering from this form of acute coronary syndrome in addition to prolonged chest pain also have changes in ecg ( such as elevation of st segment ) , and the values of troponin i and ckmb are increased . patients in groups ii , iii , and iv were hospitalized in the medical centre ( kragujevac , serbia ) . first blood samples were collected within either ( 1 ) the first 24 h of their acute events ( acs - ua and acs - stemi patients ) after initial medical stabilization or ( 2 ) the first 24 h of their hospitalization in medical center of kragujevac ( scad patients ) . data points included sex , age , illness , work activities , pain , history of exercise , smoking habits , and other observations ( such as anger or nervousness , etc . ) , as well . the estimation of whole blood histamine was performed immediately after the collection from the study subjects using modified shore 's method for the fluorometric assay of histamine in the tissue . this method is precise , sensitive , and specific for histamine and involves extraction of histamine into n - butanol ( polskie odczynniki chemiczne s.a . , poland ) from alkalinized perchloric acid ( mallinckrodt baker b.v . , holland ) blood sample , return of the histamine to an aqueous solution and condensation with o - phthalaldehyde ( opt ) ( sigma , germany ) to yield a product with strong and stable fluorescence , which is measured in a spectrofluorometer ( perkin - elmer ls-5b ) . ( standard error of the mean ) and analyzed after determination of normality of distribution with student 's t - test and anova test , where p value of < .05 was considered statistically significant . this study examined , as described in section 2 , patients with stable coronary artery disease ( scad ) , acute coronary syndrome , nonocclusive type ( acs - ua ) , and acute coronary syndrome , occlusive type ( acs - stemi ) , as well as control group . the analyzed groups were uniform in terms of age , sex , smoking habits , or physical activity . hypertension , heredity , and diabetes mellitus type ii , as well as total cholesterol , low - density lipoproteins ( ldls ) , and triglycerides were more frequent in patients with ischemic heart disease than in those of control group ( see table 1 ) . in terms of medication usage the groups differed as could be expected . beta - blockers use was the highest in scad patients ( 80% ) and the least in the control group ( 10% ) while it ranged from 60% in acs - ua patients to 25% in acs - stemi patients . the usage of calcium channel blockers was the highest in scad and acs - ua patients ( 40% ) , intermediate in acs - stemi patients ( 25% ) , and the least in control group ( 5% ) . ace inhibitor usage was similar in all patients ( about 50% ) , but much lower in control group ( 10% ) . the usage of nitrates was almost equal in all patients ( about 80% ) versus none in control . antiplatelets drugs , such as clopidogrel , were taken by acs patients ( ua and stemi ) . all the patients in heart disease groups were taking acetylsalicylic acid in comparison to 40% of the control group . it is important to underline the fact that there were no statistically significant differences in histamine blood concentrations among patients in the same group with different therapy ( p > .05 ) . histamine blood concentrations in the control group , as well as in patients with coronary diseases ( scad , acs - ua and acs - stemi ) , during the eight - day period are presented in figure 1 . histamine blood concentrations in patients with different types of coronary diseases ( scad , acs - ua and acs - stemi ) remained increased in comparison with the control group during the whole testing period . whole blood histamine concentration in the control group was 44.87 1.09 ng ml . the highest increase of histamine was in acs - stemi patients , who had 2- to 3-fold more of this biogenic amine in blood than those in control group ( figure 1 ) . furthermore , histamine blood concentration in acs - stemi patients achieved one peak value on the second day of their acute events ( 127 6.34 ng ml ) , after which point histamine blood level steadily decreased throughout the testing period remaining significantly higher when compared with the corresponding histamine blood levels in the other analyzed groups . intermediate increase of histamine blood concentration was recorded in acs - ua patients , where values remained significantly lower than histamine blood concentrations in acs - stemi patients during the entire testing period . the highest increase of histamine blood concentrations in acs - ua patients occurred on the second day ( 90.85 6.34 ng ml ) , the same as in acs - stemi patients ( figure 1 ) . during the first five days of histamine blood analysis concentrations in acs - ua patients were significantly higher than in scad patients , whereas there were no significant differences in this respect between these two groups in the last three days . when scad patients are concerned , the histamine blood concentration was significantly increased in comparison with the control group on one hand and significantly lower than that in acs - stemi patients on the other hand throughout the testing . during the eight - day observation , concentrations of histamine in patients with stable angina did not vary considerably on daily basis . the lowest value of 74.8 4.1 ng ml was measured on the last day , while the highest , measured on the third day , was 79.7 3.1 ng ml . troponin i blood concentration ( ng ml ) in stemi , acs - ua , and acs - stemi patients and subjects of control group during the eight - day period is shown in figure 2 . troponin i blood values were within normal physiological range ( less than 0.01 ng ml ) during the whole testing period in control , scad , and acs - ua groups . on the other hand , troponin i blood concentrations in acs - stemi patients were significantly increased . troponin i blood level in acs - stemi patients achieved peak value in the first 24 hours , which is much higher ( 3.2 0.23 ng ml ) than physiological ranges for this cardiac specific enzyme ( < 0.01 ng ml ) after which point it steadily decreased till the end of the period remaining significantly higher . ckmb blood concentrations ( u l ) in control group and scad , acs - ua , and acs - stemi patients during the eight - day period are shown in figure 3 . ckmb blood values remained within normal physiological range ( between 0 and 25 u l ) in control group as well as in scad and acs - ua patients . on the other hand , ckmb blood concentrations in acs - stemi patients were significantly increased on the first and second day ( see figure 3 ) . ckmb level in acs - stemi patients achieved a peak value in the first 24 hours of their acute events ( 4 times higher than physiological value for this cardiac specific enzyme ) , and after that point its level decreased and returned to its normal physiological range after the third day of analysis . for more than two decades histamine has been considered to be one of the vasoactive metabolites which are released in coronary circulation during myocardial ischemia . clinically , increased blood histamine levels have been described in patients with ischemic heart diseases by kounis and zavras . furthermore , experimental studies have documented that coronary histamine mostly originates from cardiac mast cells and activated platelets [ 25 , 26 ] . up to now the histamine blood levels in the patients suffering from ischemic heart diseases have been measured only in the first 24 hours of acute event . however , there is a deficiency of data regarding histamine blood concentration in patients suffering from ischemic heart diseases for longer periods , which is why this study examines histamine blood concentration during the eight - day period in subjects suffering from different types of ischemic heart diseases . in this study ( table 1 ) , observed groups were uniform in terms of age , sex , smoking habits , and physical activity . hypertension , heredity , and diabetes mellitus type ii , as well as lipids in the blood were more frequent in patients with ischemic heart disease than in the control group . these data are in accordance with previous reports [ 2731 ] , reflecting relationship between mentioned risk factors and coronary endothelial dysfunction . statistically speaking , significantly higher levels of blood histamine are found in scad , acs - ua , and acs - stemi patients compared with control group during the whole period ( figure 1 ) . scad patients showed significantly increased histamine blood concentration ( up to 79.5 3.6 ng ml ) compared with the control group ( 44.87 1.09 ng ml ) . obtained values were lower than those in acs - stemi patients during the whole eight - day period , in comparison to acs - ua patients where they were lower only during the first five days . the process of atherosclerosis leads to lipids accumulation in the blood vessel wall , activating cardiac mast cells , which , in turn , cause an increase in coronary histamine concentration . furthermore , atherosclerosis leads to narrowing of epicardial coronary arteries , thus compromising the flow of coronary vascular bed , resulting in inadequate blood supply to the heart muscle ( ischemia ) . myocardial ischemia and consequent hypoxia also promote the degranulation of cardiac mast cells , leading to the release of histamine [ 24 , 32 ] . this can be the cause of significantly higher and sustained histamine blood concentration in scad patients compared with the control group . our results demonstrate that the increased concentration of histamine in patients with stable form of coronary artery disease was not significantly different depending on the time of blood sampling during the eight - day monitoring and remained at about 80 ng ml . figure 1 shows that acs - ua patients had higher histamine blood concentration compared with the control group during the entire monitoring , and for the first five days these values were significantly higher than those in scad patients as well . on the sixth day the measured value was 81.1 1.2 ng ml , while on the seventh and eighth days the measured concentrations of histamine in acs - ua patients were below 80 ng ml . these results suggest that acs - ua patients have significantly higher blood histamine levels on the 1st admission day ( 84.8 1.2 ng ml ) as well as within the next four days when compared to scad patients . interestingly , the peak of histamine increase in the acs - ua patients ( 90.85 2.4 ng ml ) occurs on the second day indicating additional histamine release within the first 48 hours . in the last three days , none of the measured concentrations differed significantly from the blood histamine concentration of scad patients for the same period this indicates stabilization and transition of unstable form of disease to a stable one . pathophysiological basis for unstable angina pectoris is unstable atherosclerosis plaque complicated with endothelial damage , activation of platelets and spasm ( reversible ischemia ) . the role of histamine in the erosion or rupture of coronary plaque is not defined , but there are investigations that show that chronic activation of mast cells in the atherosclerotic lesions predisposes the subject to this event . further , the proportion of activated and degranulated adventitial mast cells was found to be the highest in the segments with ruptured plaques leading to higher histamine release . the damaged endothelium leads to activation and secretion of platelet which , in addition to adp , coagulation factors , growth factors , and serotonin , secretes histamine too . furthermore , via h1 receptors histamine induces expression of tissue factor ( tf ) , a key enzyme in the activation of coagulation . the process of coagulation with the formation of a thrombus results in the narrowing of blood vessels , ischemia , and hypoxia which leads to further activation of mast cells and platelets , which then release new amounts of histamine [ 26 , 35 , 36 ] . all this indicates the reasons of significantly increased histamine concentration in our patients with unstable angina . also , unstable angina is characterized by dynamic narrowing due to spasm of the coronary arteries in addition to the mechanical narrowing . spasm , together with the process of coagulation , significantly increases ischemia leading to the histamine release from activated mast cells . further , sakata et al . observed that the coronary histamine is elevated shortly before coronary spasm , as discussed elsewhere , and kounis and zavras suggest that chest pain in patients with unstable angina triggered by coronary spasm can be provoked by histamine . thus a vicious circle is created : ( 1 ) erosion or rupture of unstable atherosclerotic plaque leads to mechanical constriction that causes the release of histamine , ( 2 ) released histamine increases coronary artery spasm , and ( 3 ) spasm exacerbates ischemia and hypoxia releasing new amounts of histamine . perhaps the reason for several days of increased concentrations of histamine in our acs - ua patients is the given mechanism . however , unstable angina is characterized by reversible ischemia , which was confirmed in our study by the negative values of cardiac enzymes . so the stabilization of plaque leads to better blood circulation and consequently less hypoxia , which could be the reason for significantly lower concentrations of histamine in our patients on the sixth , seventh , and eighth days of testing . the blood levels of troponin i ( figure 2 ) as well as ckmb ( figure 3 ) were in normal physiological range in acs - ua patients during the testing . this suggests that in our study histamine appears to be a more sensitive parameter for myocardial ischemia than the cardiac specific enzymes . of all the four groups , the highest increase of histamine blood concentration was measured in acs - stemi patients ( figure 1 ) . our results show that histamine blood concentration in acs - stemi patients achieved a peak value ( 121.7 6.34 ng ml ) on the second day of their acute events , after which point histamine blood level decreased to the end of the observed period steadily remaining significantly higher when compared with the corresponding histamine blood levels in the other groups . these data are in accordance with previous data that histamine blood concentration increases as the disease becomes more severe . acs - stemi is the most serious form of unstable coronary disease characterized by irreversible ischemia and necrosis of myocardial tissue , caused by occlusion of blood vessel . cardiac troponins are the biomarkers of choice for detection of myocardial necrosis , and the determination of these enzymes is very useful for the measurement of the extent of necrosis [ 3942 ] . also , it is noted that histamine blood level has been dependent on the infarct size . the increased number of adventitial mast cells was found to correlate with the progression of atherosclerosis , and their presence is particularly high in areas of arterial thrombosis . importantly , the highest numbers of intimal mast cells have been found at sites of rupture and/or erosion in infarct - related coronary arteries . we must not forget that in these patients thrombin and collagen stimulate platelets to release histamine as well [ 25 , 43 ] . these data are in accordance with our results that acs - stemi patients have the highest histamine blood concentration compared with the other groups and its level remained above 80 ng ml during the whole eight - day period ( figure 1 ) . acute coronary event with occlusion of coronary blood vessel causes severe injury to neighboring cells ( mast cell necrosis ) and leads to massive histamine release as illustrated by high histamine values in our patients . furthermore , troponin i blood concentration as well as ckmb concentration in acs - stemi patients was significantly increased , and their values peaked in the first 24 hours ( figures 2 and 3 ) . these data are in accordance with other reports , proving the correlation between myocardial cell injury and release of cardiac specific enzymes . also , as in acs - ua patients , in acs - stemi patients , additional histamine release has been detected within 48 hours after admission . this peak of histamine concentration on the second day was even more pronounced when compared with acs - ua patients . the additional histamine release on the second day in acs - ua and acs - stemi patients was not detected in scad patients which can be explained as the consequence of the reperfusion after previous ischemia . the fact that histamine is released in ischemia - reperfusion conditions is well known [ 45 , 46 ] . previous studies have shown that half - life of plasma histamine and plasma troponin i measures in minutes and days , respectively [ 47 , 48 ] . because of troponin long half - life , it is not useful for detection of early reinfarction , which is why the rise of histamine blood concentration may be a parameter for detection of such events . finally , it is well known that smoking habit is also a risk factor for atherosclerosis , and nicotine present in smoke extract has been shown to activate human mast cells [ 6 , 49 ] and histamine release . however , in our study , the groups did not differ in smoking habits ( table 1 ) . it is important to underline the fact that there were no statistically significant differences in histamine blood concentration between patients in the same group according to smoking habits ( p > .05 ) . that means that mast cell activation provoked by smoking did not significantly influence histamine release in our study . furthermore , in our study there were no statistically significant differences in histamine blood concentration among subjects in control group according to the incidence of hypertension , type ii diabetes , or dyslipidemias , and there are no known data published indicating that such diseases may have impact on histamine blood concentration . in this study we investigated histamine blood concentration in subjects suffering from different types of ischemic heart diseases ( stable coronary artery disease , acute coronary syndrome , nonocclusive type , and acute coronary syndrome , occlusive type ) during the eight - day testing period . we found evidence that the histamine blood level is associated with different types of ischemic heart disease . the histamine level in blood in all research subjects was significantly higher when compared to control subjects , indicating the increase of histamine release in the patients suffering from coronary diseases . in the patients suffering from acs - ua and acs - stemi , the second day peak of histamine level furthermore , our data suggest that histamine can be additional parameter of myocardial ischemia along with cardiac specific enzymes such as troponin i and ckmb and may prove to be an excellent single prognostic marker for multitude of ischemic heart diseases .

the aim of this study was to investigate histamine blood concentration in subjects suffering from different types of ischemic heart diseases during the period of eight days . our results showed that the histamine blood level was associated with different types of ischemic heart diseases . the blood histamine level in all investigated patients was significantly higher when compared to control subjects ( 44.87 1.09 ng ml1 ) , indicating the increase of histamine release in patients suffering from coronary diseases . in patients suffering from acs - ua and acs - stemi , the second day peak of histamine level occurs ( 90.85 6.34 ng ml1 and 121.7 6.34 ng ml1 , resp . ) probably as the reperfusion event . furthermore , our data suggest that histamine can be additional parameter of myocardial ischemia along with cardiac specific enzymes and may prove to be an excellent single prognostic marker for multitude of ischemic heart diseases .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

mast cells and histamine also have cranium - caudal distribution in the heart which means that the largest quantity of this amine is in the right atrium and the least in left ventricle tissue . increased number of mast cells was found in coronary blood vessels adventitia in the patients suffering from ischemic heart diseases , especially in the narrow parts of blood vessels , or at the site of plaque rupture [ 4 , 5 ] . histamine 's effects on coronary arteries are the result of multiple actions of this molecule on both smooth muscle and endothelial cells . these effects are species specific and depend on dose of histamine , diameter and initial vessel tone , and relative location within the coronary circulation . furthermore , histamine via h1 receptors induces expression of tissue factor ( tf ) , a key enzyme in the activation of coagulation . recent research clearly indicates a great role of histamine in inflammation processes through its influence on cyclooxygenases and increased production of pgi2 and pge2 . considering the fact that inflammation is the key factor for atherosclerosis , finally , it is essential to point out the fact that blood histamine alone may be a good prognostic indicator for ischemic event ( acs ) , better than crp , even when it is combined with lipid indexes . up to now research showed that the patients suffering from ischemic heart diseases ( stable coronary artery disease ( scad ) and acute coronary syndrome ( acs ) ) have statistically significantly higher total blood histamine than the population without these diseases . however , these data about histamine blood levels in acs patients have been obtained only within the first 24 hours of acute event , which is why the aim of this study was to examine histamine blood concentration in subjects suffering from ischemic heart diseases during the period of eight days . furthermore , we intended to analyze whether histamine blood concentrations in patients depend on different types of ischemic heart diseases such as stable coronary artery disease , acute coronary syndrome , nonocclusive type , or acute coronary syndrome , occlusive type . furthermore , none of the volunteers had shown any allergy manifestations ( n = 32 ) . group ii : subjects suffering from stable coronary artery disease ( scad patients ) determined by symptoms during physical activity , stress echocardiography , coronarography , and so forth ( n = 28 ) . patients suffering from this disease manifest symptoms ( like chest pain , fatigue , etc . ) group iii : subjects with acute coronary syndrome , nonocclusive type ( unstable angina or acs - ua patients ) determined by symptoms during resting period , acute electrocardiographic changes , and coronarography ( n = 26 ) . apart from chest pain these subjects also have changes in ecg ( such as negative t - wave or the positivity of previously negative t - wave ) , while the values of troponin i and ckmb are within physiological range . group iv : subjects with acute coronary syndrome , occlusive type ( acs - stemi patients ) determined by typical accelerating symptoms , acute electrocardiographic changes and levels of serum enzymes ( troponin i , ckmb ) , and coronarography ( n = 37 ) . patients suffering from this form of acute coronary syndrome in addition to prolonged chest pain also have changes in ecg ( such as elevation of st segment ) , and the values of troponin i and ckmb are increased . first blood samples were collected within either ( 1 ) the first 24 h of their acute events ( acs - ua and acs - stemi patients ) after initial medical stabilization or ( 2 ) the first 24 h of their hospitalization in medical center of kragujevac ( scad patients ) . the estimation of whole blood histamine was performed immediately after the collection from the study subjects using modified shore 's method for the fluorometric assay of histamine in the tissue . this method is precise , sensitive , and specific for histamine and involves extraction of histamine into n - butanol ( polskie odczynniki chemiczne s.a . , holland ) blood sample , return of the histamine to an aqueous solution and condensation with o - phthalaldehyde ( opt ) ( sigma , germany ) to yield a product with strong and stable fluorescence , which is measured in a spectrofluorometer ( perkin - elmer ls-5b ) . this study examined , as described in section 2 , patients with stable coronary artery disease ( scad ) , acute coronary syndrome , nonocclusive type ( acs - ua ) , and acute coronary syndrome , occlusive type ( acs - stemi ) , as well as control group . hypertension , heredity , and diabetes mellitus type ii , as well as total cholesterol , low - density lipoproteins ( ldls ) , and triglycerides were more frequent in patients with ischemic heart disease than in those of control group ( see table 1 ) . beta - blockers use was the highest in scad patients ( 80% ) and the least in the control group ( 10% ) while it ranged from 60% in acs - ua patients to 25% in acs - stemi patients . the usage of calcium channel blockers was the highest in scad and acs - ua patients ( 40% ) , intermediate in acs - stemi patients ( 25% ) , and the least in control group ( 5% ) . ace inhibitor usage was similar in all patients ( about 50% ) , but much lower in control group ( 10% ) . the usage of nitrates was almost equal in all patients ( about 80% ) versus none in control . it is important to underline the fact that there were no statistically significant differences in histamine blood concentrations among patients in the same group with different therapy ( p > .05 ) . histamine blood concentrations in the control group , as well as in patients with coronary diseases ( scad , acs - ua and acs - stemi ) , during the eight - day period are presented in figure 1 . histamine blood concentrations in patients with different types of coronary diseases ( scad , acs - ua and acs - stemi ) remained increased in comparison with the control group during the whole testing period . whole blood histamine concentration in the control group was 44.87 1.09 ng ml . the highest increase of histamine was in acs - stemi patients , who had 2- to 3-fold more of this biogenic amine in blood than those in control group ( figure 1 ) . furthermore , histamine blood concentration in acs - stemi patients achieved one peak value on the second day of their acute events ( 127 6.34 ng ml ) , after which point histamine blood level steadily decreased throughout the testing period remaining significantly higher when compared with the corresponding histamine blood levels in the other analyzed groups . intermediate increase of histamine blood concentration was recorded in acs - ua patients , where values remained significantly lower than histamine blood concentrations in acs - stemi patients during the entire testing period . the highest increase of histamine blood concentrations in acs - ua patients occurred on the second day ( 90.85 6.34 ng ml ) , the same as in acs - stemi patients ( figure 1 ) . during the first five days of histamine blood analysis concentrations in acs - ua patients were significantly higher than in scad patients , whereas there were no significant differences in this respect between these two groups in the last three days . when scad patients are concerned , the histamine blood concentration was significantly increased in comparison with the control group on one hand and significantly lower than that in acs - stemi patients on the other hand throughout the testing . during the eight - day observation , concentrations of histamine in patients with stable angina did not vary considerably on daily basis . troponin i blood concentration ( ng ml ) in stemi , acs - ua , and acs - stemi patients and subjects of control group during the eight - day period is shown in figure 2 . troponin i blood values were within normal physiological range ( less than 0.01 ng ml ) during the whole testing period in control , scad , and acs - ua groups . on the other hand , troponin i blood concentrations in acs - stemi patients were significantly increased . troponin i blood level in acs - stemi patients achieved peak value in the first 24 hours , which is much higher ( 3.2 0.23 ng ml ) than physiological ranges for this cardiac specific enzyme ( < 0.01 ng ml ) after which point it steadily decreased till the end of the period remaining significantly higher . ckmb blood concentrations ( u l ) in control group and scad , acs - ua , and acs - stemi patients during the eight - day period are shown in figure 3 . ckmb blood values remained within normal physiological range ( between 0 and 25 u l ) in control group as well as in scad and acs - ua patients . on the other hand , ckmb blood concentrations in acs - stemi patients were significantly increased on the first and second day ( see figure 3 ) . ckmb level in acs - stemi patients achieved a peak value in the first 24 hours of their acute events ( 4 times higher than physiological value for this cardiac specific enzyme ) , and after that point its level decreased and returned to its normal physiological range after the third day of analysis . for more than two decades histamine has been considered to be one of the vasoactive metabolites which are released in coronary circulation during myocardial ischemia . clinically , increased blood histamine levels have been described in patients with ischemic heart diseases by kounis and zavras . furthermore , experimental studies have documented that coronary histamine mostly originates from cardiac mast cells and activated platelets [ 25 , 26 ] . up to now the histamine blood levels in the patients suffering from ischemic heart diseases have been measured only in the first 24 hours of acute event . however , there is a deficiency of data regarding histamine blood concentration in patients suffering from ischemic heart diseases for longer periods , which is why this study examines histamine blood concentration during the eight - day period in subjects suffering from different types of ischemic heart diseases . in this study ( table 1 ) , observed groups were uniform in terms of age , sex , smoking habits , and physical activity . hypertension , heredity , and diabetes mellitus type ii , as well as lipids in the blood were more frequent in patients with ischemic heart disease than in the control group . statistically speaking , significantly higher levels of blood histamine are found in scad , acs - ua , and acs - stemi patients compared with control group during the whole period ( figure 1 ) . scad patients showed significantly increased histamine blood concentration ( up to 79.5 3.6 ng ml ) compared with the control group ( 44.87 1.09 ng ml ) . obtained values were lower than those in acs - stemi patients during the whole eight - day period , in comparison to acs - ua patients where they were lower only during the first five days . the process of atherosclerosis leads to lipids accumulation in the blood vessel wall , activating cardiac mast cells , which , in turn , cause an increase in coronary histamine concentration . myocardial ischemia and consequent hypoxia also promote the degranulation of cardiac mast cells , leading to the release of histamine [ 24 , 32 ] . this can be the cause of significantly higher and sustained histamine blood concentration in scad patients compared with the control group . our results demonstrate that the increased concentration of histamine in patients with stable form of coronary artery disease was not significantly different depending on the time of blood sampling during the eight - day monitoring and remained at about 80 ng ml . figure 1 shows that acs - ua patients had higher histamine blood concentration compared with the control group during the entire monitoring , and for the first five days these values were significantly higher than those in scad patients as well . on the sixth day the measured value was 81.1 1.2 ng ml , while on the seventh and eighth days the measured concentrations of histamine in acs - ua patients were below 80 ng ml . these results suggest that acs - ua patients have significantly higher blood histamine levels on the 1st admission day ( 84.8 1.2 ng ml ) as well as within the next four days when compared to scad patients . interestingly , the peak of histamine increase in the acs - ua patients ( 90.85 2.4 ng ml ) occurs on the second day indicating additional histamine release within the first 48 hours . in the last three days , none of the measured concentrations differed significantly from the blood histamine concentration of scad patients for the same period this indicates stabilization and transition of unstable form of disease to a stable one . further , the proportion of activated and degranulated adventitial mast cells was found to be the highest in the segments with ruptured plaques leading to higher histamine release . furthermore , via h1 receptors histamine induces expression of tissue factor ( tf ) , a key enzyme in the activation of coagulation . the process of coagulation with the formation of a thrombus results in the narrowing of blood vessels , ischemia , and hypoxia which leads to further activation of mast cells and platelets , which then release new amounts of histamine [ 26 , 35 , 36 ] . all this indicates the reasons of significantly increased histamine concentration in our patients with unstable angina . spasm , together with the process of coagulation , significantly increases ischemia leading to the histamine release from activated mast cells . observed that the coronary histamine is elevated shortly before coronary spasm , as discussed elsewhere , and kounis and zavras suggest that chest pain in patients with unstable angina triggered by coronary spasm can be provoked by histamine . thus a vicious circle is created : ( 1 ) erosion or rupture of unstable atherosclerotic plaque leads to mechanical constriction that causes the release of histamine , ( 2 ) released histamine increases coronary artery spasm , and ( 3 ) spasm exacerbates ischemia and hypoxia releasing new amounts of histamine . perhaps the reason for several days of increased concentrations of histamine in our acs - ua patients is the given mechanism . so the stabilization of plaque leads to better blood circulation and consequently less hypoxia , which could be the reason for significantly lower concentrations of histamine in our patients on the sixth , seventh , and eighth days of testing . the blood levels of troponin i ( figure 2 ) as well as ckmb ( figure 3 ) were in normal physiological range in acs - ua patients during the testing . this suggests that in our study histamine appears to be a more sensitive parameter for myocardial ischemia than the cardiac specific enzymes . of all the four groups , the highest increase of histamine blood concentration was measured in acs - stemi patients ( figure 1 ) . our results show that histamine blood concentration in acs - stemi patients achieved a peak value ( 121.7 6.34 ng ml ) on the second day of their acute events , after which point histamine blood level decreased to the end of the observed period steadily remaining significantly higher when compared with the corresponding histamine blood levels in the other groups . these data are in accordance with previous data that histamine blood concentration increases as the disease becomes more severe . acs - stemi is the most serious form of unstable coronary disease characterized by irreversible ischemia and necrosis of myocardial tissue , caused by occlusion of blood vessel . cardiac troponins are the biomarkers of choice for detection of myocardial necrosis , and the determination of these enzymes is very useful for the measurement of the extent of necrosis [ 3942 ] . also , it is noted that histamine blood level has been dependent on the infarct size . importantly , the highest numbers of intimal mast cells have been found at sites of rupture and/or erosion in infarct - related coronary arteries . these data are in accordance with our results that acs - stemi patients have the highest histamine blood concentration compared with the other groups and its level remained above 80 ng ml during the whole eight - day period ( figure 1 ) . acute coronary event with occlusion of coronary blood vessel causes severe injury to neighboring cells ( mast cell necrosis ) and leads to massive histamine release as illustrated by high histamine values in our patients . furthermore , troponin i blood concentration as well as ckmb concentration in acs - stemi patients was significantly increased , and their values peaked in the first 24 hours ( figures 2 and 3 ) . these data are in accordance with other reports , proving the correlation between myocardial cell injury and release of cardiac specific enzymes . also , as in acs - ua patients , in acs - stemi patients , additional histamine release has been detected within 48 hours after admission . this peak of histamine concentration on the second day was even more pronounced when compared with acs - ua patients . the additional histamine release on the second day in acs - ua and acs - stemi patients was not detected in scad patients which can be explained as the consequence of the reperfusion after previous ischemia . the fact that histamine is released in ischemia - reperfusion conditions is well known [ 45 , 46 ] . because of troponin long half - life , it is not useful for detection of early reinfarction , which is why the rise of histamine blood concentration may be a parameter for detection of such events . finally , it is well known that smoking habit is also a risk factor for atherosclerosis , and nicotine present in smoke extract has been shown to activate human mast cells [ 6 , 49 ] and histamine release . it is important to underline the fact that there were no statistically significant differences in histamine blood concentration between patients in the same group according to smoking habits ( p > .05 ) . that means that mast cell activation provoked by smoking did not significantly influence histamine release in our study . furthermore , in our study there were no statistically significant differences in histamine blood concentration among subjects in control group according to the incidence of hypertension , type ii diabetes , or dyslipidemias , and there are no known data published indicating that such diseases may have impact on histamine blood concentration . in this study we investigated histamine blood concentration in subjects suffering from different types of ischemic heart diseases ( stable coronary artery disease , acute coronary syndrome , nonocclusive type , and acute coronary syndrome , occlusive type ) during the eight - day testing period . we found evidence that the histamine blood level is associated with different types of ischemic heart disease . the histamine level in blood in all research subjects was significantly higher when compared to control subjects , indicating the increase of histamine release in the patients suffering from coronary diseases . in the patients suffering from acs - ua and acs - stemi , the second day peak of histamine level furthermore , our data suggest that histamine can be additional parameter of myocardial ischemia along with cardiac specific enzymes such as troponin i and ckmb and may prove to be an excellent single prognostic marker for multitude of ischemic heart diseases .

prenatal alcohol exposure may lead to a range of physical and cognitive abnormalities in children that persist into adulthood ( mattson et al . , 2011 ; riley et al . , the range of deficiencies observed in affected individuals is collectively described as fetal alcohol spectrum disorders ( fasd ) ( sampson et al . , 1997 ) , with fetal alcohol syndrome ( fas ) representing the most severe form . the diagnosis for fasd is based on three major domains of deficiencies : brain growth , cognitive dysmorphology , and facial dysmorphology ( jones and smith , 1973 ; hoyme et al . , 2005 ) . smaller parietal and frontal white matter volumes and smaller corpus callosum size and/or area have been specifically reported in this population ( archibald et al . , 2001 ; wozniak and muetzel , 2011 ) and may occur in tandem with abnormal gray matter thickness observed in the parietal and frontal cortices ( sowell et al . change in white matter development over time , however , remains under - investigated in the fasd literature . in typically developing children , post - natal development of white matter is more protracted than gray matter , and continues during childhood and adolescence and into midlife ( riddle et al . , 2010 ; welker and patton , 2012 ) . specifically , during childhood and adolescence , there are significant age - related increases in white matter volume , with peak volumes occurring between 12 and 14 years in the frontal and temporal lobes to around 2024 years for the parietal lobes ( giedd et al . , 1999 ; ge et al . , 2002 ; gogtay et al . , 2004 ; tamnes et al . , 2010 ; increases in white matter volume are attributed to increases in myelination and potentially to changes in the size , density , and number of white matter fibers over time ( paus et al . , 1999 ; welker and patton , 2012 ) . past studies have shown that those with fasd demonstrate very little improvement of cognitive function over time , and differences in cognitive ability between exposed and typically developing subjects are even more apparent during adolescence and adulthood ( streissguth et al . , 1991 ) . cross - sectional relationships between gray and white matter and cognitive function have been assessed previously in this population . for instance , earlier studies from our group have shown significant positive correlations between verbal recall and frontal gray matter thickness only in those with fasd ( sowell et al . , 2008a ) . similarly , white matter integrity estimated with diffusion tensor imaging ( dti ) and measures of fractional anisotropy ( fa ) in the external capsule ( significant differences near the fronto - temporal region ) have been found to be correlated with performance of visuomotor tasks in individuals with fasd but not in controls ( sowell et al . others have also found lower fa ( li et al . , 2009 ) and moderate correlations between fa and math performance in individuals with fasd ( lebel et al . , 2010 however , relationships between longitudinal change in white matter and other executive functions over time in those with fasd have not been previously assessed . while changes in white matter volume and executive function in typically developing children remain under investigated , cross - sectional studies of typically developing cohorts using dti show that higher fa near frontal and parietal areas is positively associated with better reading ability , lexical decision making ( nagy et al . , 2004 ; gold et al . , 2007 ) and iq ( schmithorst et al . , 2005 ) . positive relationships have also been reported between lower reaction times and greater white matter volumes in a lifespan sample of healthy volunteers ( walhovd and fjell , 2007 ) . however , in typically developing children , such relationships are not consistently found , and null results have also been reported when compared with those with fasd ( lebel et al . , 2010 ; treit et al . , 2013 ) . in the current study , we investigated age - related changes in white matter volume over time within individuals with fasd , and how these changes relate to executive function change over time . behavior relationships with an executive function battery because of the following : i ) these functions are known to be subserved by these cortical association regions ( smith and jonides , 1997 ; smith and jonides , 1999 ; cabeza , 2008 ) ; ii ) due to the prominent attention and working memory deficits reported in fasd , these structures were hypothesized to be the most likely to be related to the deficits in function . the corpus callosum was additionally chosen as a roi as numerous studies have previously documented abnormalities in its structure in fasd ( riley et al . , 1995 ; sowell et al . , 2001 ; astley et al . , 2009 ) , and we hypothesized that this structure would show differential volume changes in children with fasd compared to controls . hence , we first investigated whether the rate of change in white matter volume in frontal and parietal regions differed between children and adolescents who were typically developing from those with fasd . secondly , we investigated whether regional white matter volume changes predict the rate of change in executive functions over time and differ in children with fasd and typically developing children . we expected children with fasd to show smaller white matter volumes even after adjusting for overall brain size . specifically , we hypothesized that volumes of frontal , parietal and total white matter would be smaller in participants with fasd than in controls independent of brain size and age . given that previous studies have consistently found positive relationships between measures of white matter macro- and microstructure and cognitive function in fasd , we expected positive brain behavior relationships in children with fasd , but not in controls . all participant data were obtained as part of the longitudinal collaborative initiative on fetal alcohol spectrum disorders ( cifasd ) and included subjects from los angeles , california . participants were recruited via advertisements and word - of - mouth , and alcohol exposed participants were also recruited through the fetal alcohol and related disorders clinic at the university of california , los angeles . prenatally exposed participants were identified as those who were exposed to more than 4 drinks per occasion at least once per week or more than 13 drinks per week during pregnancy . control subjects had none or less than 2 drinks ( per week or on any one occasion ) throughout pregnancy . participants with incomplete exposure documentation were classified as alcohol exposed if they displayed the physical characteristics of fetal alcohol syndrome ( fas ) as documented by an expert dysmorphologist . in the case of adoptive children , information about maternal alcohol use during pregnancy was gathered through sources other than the biological parent during the developmental history interview ( stratton , 1996 ; hoyme et al . , 2005 ) . out of the 49 fasd children in the study , only 7 children were living with their biological parents , and the rest were adopted . detailed explanations of fasd diagnosis for cifasd have been published previously ( jones et al . , 2006 ; mattson et al . , 2010 a total of 103 participants were included in the study , out of which 49 were classified as having fasd and 54 as controls . of these , data were available for 41 participants ( 25 with fasd , 16 controls ) for two time points . for the remaining 38 controls and 24 participants with fasd , data were available for one time point . mean ages , scan intervals , and other demographic variables are presented in table 1 . there were no significant differences in age , sex , or severity of diagnosis between participants who did and did not return for a follow - up visit . scans were acquired on 1.5 t siemens sonata , tr = 1900 ms , te = 4.38 ms , flip angle = 15 , matrix size = 256 256 160 ; fov , 256 256 mm ; total acquisition time = 8 min 8 s , voxel size = 1 1 1 mm . freesurfer allows for semi - automatic reconstruction of the cortical surface using t1-weighted mri images . major steps during image analyses include motion correction , removal of non - brain tissue , automated talairach transformation , subcortical and cortical matter segmentation , intensity correction and delineation of gray / white / pial boundaries ( dale et al . , 1999 ; after the formation of cortical models , deformable procedures are applied including cortical inflation , registration to a spherical atlas and parcellation of the cerebral cortex into gyral and sulcal units ( desikan et al . , 2006 ) . for the participants with data at two time points , the structural scans were additionally processed through the longitudinal stream ( reuter et al . , 2010 ) . after initial processing by freesurfer , all mri scans were visually checked slice - by - slice to ensure there was no misclassification of gray and white matter voxels ; scans were reprocessed if errors were detected and rechecked visually a second time . using the inbuilt atlas ( fischl et al . , 2004 ) , the volumes of the superior frontal , middle frontal , superior frontal , supramarginal , superior parietal , inferior parietal , and corpus callosum white matter , as well as total white matter volume was obtained from freesurfer and imported to spss 19.0 for preliminary analyses . left and right hemisphere volumes for each region in the frontal and parietal cortices were added to give bilateral estimates to reduce numbers of hypothesis tested . . performance on verbal working memory was measured through digit - backward span ( db ) , while attention was measured through digit - forward span ( df ) , both subtests of the wechsler intelligence scale for children , fourth edition ( wisc - iv ) ( wechsler , 2003 ) . attention and processing speed were measured through the trail making test a ( tmt - a ) and mental flexibility was measured with the trail making test b ( tmt - b ) ( reitan and wolfson , 1985 ) . finally , free recall was measured through california verbal learning test c ( cvlt - c ) for delayed total free - recall ( delis et al . , 1987 ) . for db , total completion times in seconds were utilized for tmt - a and -b , hence higher scores represent poorer performance . the cognitive tests utilized in the study are widely used in neuropsychological examinations and their test retest reliabilities have been previously reported by other studies ( for cvlt - c see paolo et al . , 1997 ; for db see iverson , 2001 ; and for tmt - a and -b see cangoz et al . , 2009 ) . descriptive statistics were analyzed using spss ( v 19.0 . ) and are presented in table 1 . this allowed us to confidently assess white matter volumes in the age ranges between 6 and 17 . to minimize the effects of attrition , we adopted the linear mixed modeling ( lesaffre and verbeke , 1998 ; rabe - hesketh et al . , 2001 ) and maximum likelihood approaches , which are able to estimate the most likely parameters based on available time points and are robust to missing data . changes in white matter volume , as well as change in cognitive function over time were modeled taking sex , age , and icv into account . first , the effects of age and status ( prenatal alcohol exposure status : controls , fasd group ) on white matter volumes were investigated for total white matter and each region of interest ( roi ) separately . in the model , volumes were dependent variables while age , status , sex , icv , and the interaction term of age - by - status were fixed variables . a significant effect for group status indicated significantly different volumes between the groups , while a significant effect for age indicated a significantly different volume with age . a significant age - by - status interaction would indicate a significant volume difference between groups in relation to age . for the second set of analyses , the effects of change in cognitive variables were modeled for each white matter roi , and additionally included two - way interactions for roi - by - age , roi - by - status , age - by - status , and finally the three - way interaction of roi - by - age - by - status . age , sex , icv , and status were again included as fixed variables , and subject intercept was included as a random variable . for significant three - way interactions , roi - by - age interactions were also separately investigated for each group ( controls / fasd group ) to determine which group was driving the relationship among the variables . similar to the first set of analyses , these analyses allowed us to determine if status ( i.e. , being alcohol exposed or not ) , age , and age - related change in white matter volume were differentially related to change in cognitive function . mean age for controls was 11.83 ( 2.93 ) years and for the fasd group was 11.15 ( 2.81 ) years . there was no statistically significant difference in age between groups ( p= 0.54 ) . mean interval between scans was 2.36 ( 3.05 ) years for controls and 2.46 ( 2.98 ) years for the fasd group . independent sample t - tests revealed that cognitive function was significantly poorer in fasd participants compared to typically developing controls in tmt - b , df , db , and cvlt - c . mean times for tmt - a were higher in those with fasd but were not significantly different from controls ( fig . 2 , table 1 ) . as predicted , for both groups , age was significantly related to white matter volume change over time in each of the frontal and parietal regions , including total white matter and corpus callosum , except in the supramarginal region ( table 2 ) . also as predicted , participants with fasd had significantly smaller regional volumes than controls , and this difference remained significant even after adjusting for intracranial volumes , for the corpus callosum ( t = 2.18 , p= 0.031 ) , middle frontal ( t= 2.24 , p= 0.027 ) , supramarginal ( t= 2.58 , p= 0.037 ) , and inferior parietal regions ( t= 2.11 , p = 0.011 ) . both groups showed similar rates of increase in regional white matter volumes and there were no significant age - by - status interactions for any of the regions investigated . both groups improved with age significantly on all cognitive tests ( table 3 ) . after controlling for age , controls were also significantly better than those with fasd over time on all cognitive tests except tmt - a ( table 4 ) . differential relationships between increased white matter volume and improved cognitive function were observed between the two groups ( table 4 ) . such roi - by - status interactions were significant for i ) df for increases in volumes of corpus callosum ( t = 3.08 , p= 0.002 ) , inferior parietal ( t= 2.27 , p = 0.03 ) , middle frontal ( t= 2.28 , p = 0.03 ) , superior parietal ( t = 2.23 , p= 0.02 ) , inferior frontal ( t= 2.34 , p= 0.02 ) , and total white matter ( t= 2.35 , p = 0.02 ) ; ii ) tmt - b times for increases in volumes of corpus callosum ( t = 2.20 , p= 0.02 ) , inferior parietal ( t= 2.37 , p = 0.02 ) , supra marginal ( t= 2.30 , p = 0.03 ) , middle frontal ( t= 2.26 , p = 0.03 ) , and superior frontal ( t= 2.03 , p= 0.04 ) regions ; and iii ) db for corpus callosum volume increase ( t = 2.04 , p = 0.04 ) . these findings showed that there were significant differences in change in cognitive function and change in volumes between those with fasd and controls . follow - up analyses confirmed the direction of the findings , where larger white matter volumes were related to better performance in those with fasd , but not in controls . in addition , only fasd participants showed significant relationships between white matter volume increase and better cognitive function with increasing age ( table 4 ) . such three - way interactions ( roi - by - status - by - age ) were significant for i ) df for increases in volumes of corpus callosum ( t = 3.65 , p < 0.001 ) , inferior parietal ( t= 2.08 , p= 0.04 ) , middle frontal ( t = 2.08 , p= 0.04 ) , superior frontal ( t= 2.26 , p= 0.03 ) , inferior frontal ( t= 2.26 , p= 0.03 ) , and total white matter ( t= 2.63 , p < 0.01 ) ; ii ) cvlt - c long delay free recall for corpus callosum ( t = 2.11 , p = 0.04 ) . such significant interactions show that the relationship between regional white matter volume , age , and cognition is mediated differently in the fasd and control groups . analyses of t - values from tables 3 and 4 reveal that individuals with fasd have consistently lower cognition and volumes compared to controls . finally , follow - up analyses conducted for each group separately revealed significant roi - by - age interactions for all of the regions with significant three - way interactions in the fasd group , but not in the control group . in contrast , while controls showed a significant positive effect of age on cognitive function , none of the interactions with age were significant for any of the cognitive variables . a representative plot for significant three way interactions for each group has been presented in fig . as highlighted in the plot , the relationship between white matter volume and cognitive function increases with age in the fasd group ( i.e. , increases in volume with age are related to better improvements in cognitive scores ) , but such relationships with age are not significant in controls . we show for the first time that age - related regional increase in white matter volume is differentially related to cognitive change between children with fasd and typically developing children . specifically , age - related increases in callosal , frontal , and parietal white matter volume showed significant associations with cognitive improvements in children with fasd , but this relationship was not observed in those that were typically developing . since both groups showed improvement of cognitive performance and increases in white matter volume during the periods examined , results support the fact that age - related plasticity in white matter structure impacts the development of cognitive function in fasd children and adolescents . these findings suggest that fasd - related disturbances or disorganization of white matter structure contribute to cognitive impairments over time in individuals with fasd and/or that variations in other structural characteristics not measured here ( such as gray matter volume or thickness ) play a more central role in cognitive development in typically developing controls . abnormalities in white matter macro- and micro - structure are widely documented in fasd ( riley et al . , 1995 ; sowell et al . , we have extended these findings to show that while developmental trajectories for white matter volume continues at the same rate as typical children , these trajectories have a strong positive effect on cognitive function in those with fasd . these findings may have implications for continuing effective social and behavioral support for better health , and socioeconomic status shown to relate to better brain outcomes in those with fasd that may in turn have long - lasting positive effects on executive function . results revealed that both typically developing children and those with fasd showed significant improvement of cognitive function over time , and age and time were significantly related to cognitive function for both groups . significant group differences still remained for all the cognitive variables after taking age into account , and fasd children performed more poorly than typically developing children for all tasks examined with the exception of the tmt - a , which showed a trend for faster completion times in controls . these results suggest that while those with fasd do not catch up to typically developing children in standardized tests , they nonetheless make significant improvements in cognitive function over time . since results show that those with fasd can potentially improve in executive functioning with age , it appears to be of particular importance to enhance opportunities to support the social and behavioral development of those with fasd . environmental support is especially relevant , as previous studies have shown that the biological parents of those with fasd have poorer health behaviors and education and are of lower socioeconomic status ( ses ) ( streissguth et al . , 2004 ; furthermore , other studies of early life adversity have been related to smaller white matter volume ( teicher et al . , 2004 ) while adequate care early in life in typically developing children is associated with greater white matter volume later on ( als et al . , these results suggest that providing an environment with health and educational opportunities at home and in the community for those with fasd might continue to enhance overall cognitive development . given that most of the children with fasd in the current study were not living with biological parents , but instead with adopted families , it is possible that participants in this study were exposed to improved home and school environments , which might have led to better brain and cognitive outcomes . similarly , although individuals with fasd had significantly smaller white matter volumes at both time points , results revealed that the rate of increase in white matter volume with age is similar in the two groups : there were no significant exposure status - by - age interactions for change in white matter volume . these findings suggest that even though those with fasd begin with smaller white matter volumes , this does not affect the subsequent increase / development of white matter over time in this population . furthermore , results also showed that despite having smaller volumes , these differences only remained significant for the middle and superior frontal and the supramarginal regions for individuals with fasd after taking brain sizes into account . our results of similar rate of white matter volume increase in those with fasd are in contrast with studies of gray matter development , where rates of change of gray matter have been found to be different between fasd and control children . ( 2012 ) found that while control children showed an inverted - u shaped development of the gray matter , those with fasd only showed linear decreases implying different timing of gray matter maturation between these two populations . hence , while gray matter development seems to show ongoing difference in developmental trajectory between groups , our results suggest that differences in white matter are static with pronounced but stable group differences . these results could possibly be explained through the different developmental timelines for gray and white matter . though some fiber connections develop early , the majority of white matter development ( myelination and changes in the size , density and organization of axons ) continues to develop after birth ( welker and patton , 2012 ) and increases in volume are reported at least until the first four decades of life ( thompson et al . , 2005 ; riddle et al . , 2010 ) . hence , postnatal development of white matter occurs after the direct teratogenic effect of alcohol in utero . , 2012 ; rubia , 2013 ) , hence the initial brain neurons may be directly affected by alcohol for a longer period . gray matter volume also has a narrower developmental trajectory , as volumes peak in early adolescence ( giedd et al . while no longitudinal studies addressing white matter volume development over time in fasd children exist , follow - up on preterm children have shown that after controlling for brain size , gray matter volumes are more affected than white matter at adolescence ( cheong et al . , 2013 ) . hence , it is possible that , due to its relatively prolonged maturation period , white matter development could be more resistant to the long - lasting effects of prenatal alcohol exposure and/or be more amenable to plasticity relating to environmental factors . future investigations should test our findings across a wider age range to verify the long - term effects of prenatal alcohol exposure beyond adolescence . further , as mentioned above , increase in white matter volume showed different relationships with cognitive function depending on exposure status . specifically , performance in df improved with age and larger white matter regional volumes in those with fasd . however , for typically developing children who also improved over time in cognitive function , this relationship was not shown as mediated by white matter volume increases . significant interactions were also observed for db and cvlt - c where increased callosal volume with age was related to better cognitive function in the exposed group . these results were less robust than those observed for tests of attention capacity and correlations were significant predominantly for the corpus callosum . overall , our findings suggest that in typically developing children , cognitive function may rely more heavily on other neurodevelopmental changes in brain macro- or microstructure . for instance , previous research has also shown that relationships between brain measures and cognitive function are stronger in study populations with neurological disorders , whereas such brain structure relationships during normal development are less consistently found ( van petten , 2004 ; treit et al . , 2013 ) . thus , in a typically developing population , it is possible that the contribution of other neurodevelopmental processes might mask white matter contributions , making these relationships harder to detect . in contrast , children with fasd have documented abnormalities of gray matter structure as well as function . for instance , abnormal activation of the parietal and frontal cortices ( ohare et al . , 2009 ; meintjes et al . , 2010 ) as well as atypical morphology of cortical regions ( riley et al . , 1995 ; mattson et al . , 1996 ) have been previously reported . for children with fasd , typical relationships between gray and white matter structure and their connectivity across the brain since children with fasd have smaller overall white matter volumes compared to age - matched typically developing peers , increase of white matter might significantly contribute to better communication among brain regions and consequently lead to improved cognitive abilities . since increases in white matter volume are at least partly attributable to age - related increases in myelination ( welker and patton , 2012 ) , improvements in conduction velocity of axonal fibers may facilitate communication among brain regions . not surprisingly , the time course of increased myelination is related to language function in typically developing young children ( pujol et al . , 2006 ) and may provide a possible explanation for the improvement of executive function with age in the current sample . several factors may contribute to our observations for more pronounced group differences in attention than in other functional domains such as mental flexibility as measured by tmt - b . first , since tasks like working memory and recall require more complex functional networks for successful task completion , it is possible that gross measures of white matter volume were not sensitive to variations associated with these specific cognitive functions . second , only a small proportion of the participants in the fasd group had the full fas diagnosis ( n= 5 ) . less severe neuropsychological deficits have been previously documented for children in the spectrum compared to those with full fas , and children with fetal alcohol effects but without the facial dysmorphology or growth abnormalities to warrant an fas diagnosis can show normal iq and display only subtle behavioral or cognitive deficits . hence , the results could be more representative of those with less severe deficits who show fewer differences from control children in executive tasks . although we did not find differences in the developmental trajectory of white matter volume between diagnostic groups , it is possible that there might be changes occurring at the microstructural level . for instance , a recent study has found different rates of white matter maturation using dti measures between controls and children with fasd ( treit et al . , 2013 ) . although this study did not examine executive functioning , they reported positive correlations between change in mean diffusivity ( the overall magnitude of water diffusion in a voxel ) and reading scores in the fasd group . future longitudinal research will need to clarify relationships between diffusion imaging indices of white matter integrity and structural connectivity and executive functions . in addition , while we included participants with data on both first and follow - up visits in order to increase power in detecting significant relationships , this is a limitation of our study and our findings need to be understood in this context . although there were no significant age , sex , or diagnosis severity differences in participants who did and did not complete the follow - up study , there might be other unknown factors that determined attrition . finally , additional time points of 3 or more , would also have allowed us to assess more detailed changes in developmental trajectories . since development is a time of rapid change , it is possible that increasing the sample size and sampling at more time points would have allowed us to detect other subtler group differences . this study showed that while the rate of development of white matter volume is similar with age in both typically developing and exposed children , there are differential relationships between cognitive function and white matter change over time . specifically , for those with fasd , age - related increases in volumes were related to better cognitive function , while for controls , this positive relationship was not detected . findings suggest that increasing white matter volume with age allows those with fasd to improve in their cognitive abilities over time , possibly by facilitating information processing among connected brain areas . findings also suggest that better cognitive outcomes could be possible for fasd subjects through interventions targeting better cognitive and behavioral outcomes through education and/or other environmental factors .

prenatal alcohol exposure can cause a wide range of deficits in executive function that persist throughout life , but little is known about how changes in brain structure relate to cognition in affected individuals . in the current study , we predicted that the rate of white matter volumetric development would be atypical in children with fetal alcohol spectrum disorders ( fasd ) when compared to typically developing children , and that the rate of change in cognitive function would relate to differential white matter development between groups . data were available for 103 subjects [ 49 with fasd , 54 controls , age range 617 , mean age = 11.83 ] with 153 total observations . groups were age - matched . participants underwent structural magnetic resonance imaging ( mri ) and an executive function ( ef ) battery . using white matter volumes measured bilaterally for frontal and parietal regions and the corpus callosum , change was predicted by modeling the effects of age , intracranial volume , sex , and interactions with exposure status and ef measures . while both groups showed regional increases in white matter volumes and improvement in cognitive performance over time , there were significant effects of exposure status on age - related relationships between white matter increases and ef measures . specifically , individuals with fasd consistently showed a positive relationship between improved cognitive function and increased white matter volume over time , while no such relationships were seen in controls . these novel results relating improved cognitive function with increased white matter volume in fasd suggest that better cognitive outcomes could be possible for fasd subjects through interventions that enhance white matter plasticity .

Introduction Materials and methods Results Discussion Conclusions

prenatal alcohol exposure may lead to a range of physical and cognitive abnormalities in children that persist into adulthood ( mattson et al . , the range of deficiencies observed in affected individuals is collectively described as fetal alcohol spectrum disorders ( fasd ) ( sampson et al . smaller parietal and frontal white matter volumes and smaller corpus callosum size and/or area have been specifically reported in this population ( archibald et al . change in white matter development over time , however , remains under - investigated in the fasd literature . in typically developing children , post - natal development of white matter is more protracted than gray matter , and continues during childhood and adolescence and into midlife ( riddle et al . specifically , during childhood and adolescence , there are significant age - related increases in white matter volume , with peak volumes occurring between 12 and 14 years in the frontal and temporal lobes to around 2024 years for the parietal lobes ( giedd et al . , 2010 ; increases in white matter volume are attributed to increases in myelination and potentially to changes in the size , density , and number of white matter fibers over time ( paus et al . past studies have shown that those with fasd demonstrate very little improvement of cognitive function over time , and differences in cognitive ability between exposed and typically developing subjects are even more apparent during adolescence and adulthood ( streissguth et al . cross - sectional relationships between gray and white matter and cognitive function have been assessed previously in this population . similarly , white matter integrity estimated with diffusion tensor imaging ( dti ) and measures of fractional anisotropy ( fa ) in the external capsule ( significant differences near the fronto - temporal region ) have been found to be correlated with performance of visuomotor tasks in individuals with fasd but not in controls ( sowell et al . , 2009 ) and moderate correlations between fa and math performance in individuals with fasd ( lebel et al . , 2010 however , relationships between longitudinal change in white matter and other executive functions over time in those with fasd have not been previously assessed . while changes in white matter volume and executive function in typically developing children remain under investigated , cross - sectional studies of typically developing cohorts using dti show that higher fa near frontal and parietal areas is positively associated with better reading ability , lexical decision making ( nagy et al . however , in typically developing children , such relationships are not consistently found , and null results have also been reported when compared with those with fasd ( lebel et al . in the current study , we investigated age - related changes in white matter volume over time within individuals with fasd , and how these changes relate to executive function change over time . behavior relationships with an executive function battery because of the following : i ) these functions are known to be subserved by these cortical association regions ( smith and jonides , 1997 ; smith and jonides , 1999 ; cabeza , 2008 ) ; ii ) due to the prominent attention and working memory deficits reported in fasd , these structures were hypothesized to be the most likely to be related to the deficits in function . the corpus callosum was additionally chosen as a roi as numerous studies have previously documented abnormalities in its structure in fasd ( riley et al . , 2009 ) , and we hypothesized that this structure would show differential volume changes in children with fasd compared to controls . hence , we first investigated whether the rate of change in white matter volume in frontal and parietal regions differed between children and adolescents who were typically developing from those with fasd . secondly , we investigated whether regional white matter volume changes predict the rate of change in executive functions over time and differ in children with fasd and typically developing children . we expected children with fasd to show smaller white matter volumes even after adjusting for overall brain size . specifically , we hypothesized that volumes of frontal , parietal and total white matter would be smaller in participants with fasd than in controls independent of brain size and age . given that previous studies have consistently found positive relationships between measures of white matter macro- and microstructure and cognitive function in fasd , we expected positive brain behavior relationships in children with fasd , but not in controls . all participant data were obtained as part of the longitudinal collaborative initiative on fetal alcohol spectrum disorders ( cifasd ) and included subjects from los angeles , california . out of the 49 fasd children in the study , only 7 children were living with their biological parents , and the rest were adopted . of these , data were available for 41 participants ( 25 with fasd , 16 controls ) for two time points . for the remaining 38 controls and 24 participants with fasd , data were available for one time point . there were no significant differences in age , sex , or severity of diagnosis between participants who did and did not return for a follow - up visit . , 2004 ) , the volumes of the superior frontal , middle frontal , superior frontal , supramarginal , superior parietal , inferior parietal , and corpus callosum white matter , as well as total white matter volume was obtained from freesurfer and imported to spss 19.0 for preliminary analyses . this allowed us to confidently assess white matter volumes in the age ranges between 6 and 17 . to minimize the effects of attrition , we adopted the linear mixed modeling ( lesaffre and verbeke , 1998 ; rabe - hesketh et al . changes in white matter volume , as well as change in cognitive function over time were modeled taking sex , age , and icv into account . first , the effects of age and status ( prenatal alcohol exposure status : controls , fasd group ) on white matter volumes were investigated for total white matter and each region of interest ( roi ) separately . in the model , volumes were dependent variables while age , status , sex , icv , and the interaction term of age - by - status were fixed variables . for the second set of analyses , the effects of change in cognitive variables were modeled for each white matter roi , and additionally included two - way interactions for roi - by - age , roi - by - status , age - by - status , and finally the three - way interaction of roi - by - age - by - status . age , sex , icv , and status were again included as fixed variables , and subject intercept was included as a random variable . , being alcohol exposed or not ) , age , and age - related change in white matter volume were differentially related to change in cognitive function . independent sample t - tests revealed that cognitive function was significantly poorer in fasd participants compared to typically developing controls in tmt - b , df , db , and cvlt - c . as predicted , for both groups , age was significantly related to white matter volume change over time in each of the frontal and parietal regions , including total white matter and corpus callosum , except in the supramarginal region ( table 2 ) . also as predicted , participants with fasd had significantly smaller regional volumes than controls , and this difference remained significant even after adjusting for intracranial volumes , for the corpus callosum ( t = 2.18 , p= 0.031 ) , middle frontal ( t= 2.24 , p= 0.027 ) , supramarginal ( t= 2.58 , p= 0.037 ) , and inferior parietal regions ( t= 2.11 , p = 0.011 ) . both groups showed similar rates of increase in regional white matter volumes and there were no significant age - by - status interactions for any of the regions investigated . after controlling for age , controls were also significantly better than those with fasd over time on all cognitive tests except tmt - a ( table 4 ) . differential relationships between increased white matter volume and improved cognitive function were observed between the two groups ( table 4 ) . such roi - by - status interactions were significant for i ) df for increases in volumes of corpus callosum ( t = 3.08 , p= 0.002 ) , inferior parietal ( t= 2.27 , p = 0.03 ) , middle frontal ( t= 2.28 , p = 0.03 ) , superior parietal ( t = 2.23 , p= 0.02 ) , inferior frontal ( t= 2.34 , p= 0.02 ) , and total white matter ( t= 2.35 , p = 0.02 ) ; ii ) tmt - b times for increases in volumes of corpus callosum ( t = 2.20 , p= 0.02 ) , inferior parietal ( t= 2.37 , p = 0.02 ) , supra marginal ( t= 2.30 , p = 0.03 ) , middle frontal ( t= 2.26 , p = 0.03 ) , and superior frontal ( t= 2.03 , p= 0.04 ) regions ; and iii ) db for corpus callosum volume increase ( t = 2.04 , p = 0.04 ) . these findings showed that there were significant differences in change in cognitive function and change in volumes between those with fasd and controls . follow - up analyses confirmed the direction of the findings , where larger white matter volumes were related to better performance in those with fasd , but not in controls . in addition , only fasd participants showed significant relationships between white matter volume increase and better cognitive function with increasing age ( table 4 ) . such three - way interactions ( roi - by - status - by - age ) were significant for i ) df for increases in volumes of corpus callosum ( t = 3.65 , p < 0.001 ) , inferior parietal ( t= 2.08 , p= 0.04 ) , middle frontal ( t = 2.08 , p= 0.04 ) , superior frontal ( t= 2.26 , p= 0.03 ) , inferior frontal ( t= 2.26 , p= 0.03 ) , and total white matter ( t= 2.63 , p < 0.01 ) ; ii ) cvlt - c long delay free recall for corpus callosum ( t = 2.11 , p = 0.04 ) . such significant interactions show that the relationship between regional white matter volume , age , and cognition is mediated differently in the fasd and control groups . analyses of t - values from tables 3 and 4 reveal that individuals with fasd have consistently lower cognition and volumes compared to controls . in contrast , while controls showed a significant positive effect of age on cognitive function , none of the interactions with age were significant for any of the cognitive variables . as highlighted in the plot , the relationship between white matter volume and cognitive function increases with age in the fasd group ( i.e. , increases in volume with age are related to better improvements in cognitive scores ) , but such relationships with age are not significant in controls . we show for the first time that age - related regional increase in white matter volume is differentially related to cognitive change between children with fasd and typically developing children . specifically , age - related increases in callosal , frontal , and parietal white matter volume showed significant associations with cognitive improvements in children with fasd , but this relationship was not observed in those that were typically developing . since both groups showed improvement of cognitive performance and increases in white matter volume during the periods examined , results support the fact that age - related plasticity in white matter structure impacts the development of cognitive function in fasd children and adolescents . these findings suggest that fasd - related disturbances or disorganization of white matter structure contribute to cognitive impairments over time in individuals with fasd and/or that variations in other structural characteristics not measured here ( such as gray matter volume or thickness ) play a more central role in cognitive development in typically developing controls . abnormalities in white matter macro- and micro - structure are widely documented in fasd ( riley et al . , we have extended these findings to show that while developmental trajectories for white matter volume continues at the same rate as typical children , these trajectories have a strong positive effect on cognitive function in those with fasd . these findings may have implications for continuing effective social and behavioral support for better health , and socioeconomic status shown to relate to better brain outcomes in those with fasd that may in turn have long - lasting positive effects on executive function . results revealed that both typically developing children and those with fasd showed significant improvement of cognitive function over time , and age and time were significantly related to cognitive function for both groups . significant group differences still remained for all the cognitive variables after taking age into account , and fasd children performed more poorly than typically developing children for all tasks examined with the exception of the tmt - a , which showed a trend for faster completion times in controls . these results suggest that while those with fasd do not catch up to typically developing children in standardized tests , they nonetheless make significant improvements in cognitive function over time . since results show that those with fasd can potentially improve in executive functioning with age , it appears to be of particular importance to enhance opportunities to support the social and behavioral development of those with fasd . , 2004 ) while adequate care early in life in typically developing children is associated with greater white matter volume later on ( als et al . , these results suggest that providing an environment with health and educational opportunities at home and in the community for those with fasd might continue to enhance overall cognitive development . given that most of the children with fasd in the current study were not living with biological parents , but instead with adopted families , it is possible that participants in this study were exposed to improved home and school environments , which might have led to better brain and cognitive outcomes . similarly , although individuals with fasd had significantly smaller white matter volumes at both time points , results revealed that the rate of increase in white matter volume with age is similar in the two groups : there were no significant exposure status - by - age interactions for change in white matter volume . these findings suggest that even though those with fasd begin with smaller white matter volumes , this does not affect the subsequent increase / development of white matter over time in this population . furthermore , results also showed that despite having smaller volumes , these differences only remained significant for the middle and superior frontal and the supramarginal regions for individuals with fasd after taking brain sizes into account . our results of similar rate of white matter volume increase in those with fasd are in contrast with studies of gray matter development , where rates of change of gray matter have been found to be different between fasd and control children . hence , while gray matter development seems to show ongoing difference in developmental trajectory between groups , our results suggest that differences in white matter are static with pronounced but stable group differences . though some fiber connections develop early , the majority of white matter development ( myelination and changes in the size , density and organization of axons ) continues to develop after birth ( welker and patton , 2012 ) and increases in volume are reported at least until the first four decades of life ( thompson et al . while no longitudinal studies addressing white matter volume development over time in fasd children exist , follow - up on preterm children have shown that after controlling for brain size , gray matter volumes are more affected than white matter at adolescence ( cheong et al . hence , it is possible that , due to its relatively prolonged maturation period , white matter development could be more resistant to the long - lasting effects of prenatal alcohol exposure and/or be more amenable to plasticity relating to environmental factors . future investigations should test our findings across a wider age range to verify the long - term effects of prenatal alcohol exposure beyond adolescence . further , as mentioned above , increase in white matter volume showed different relationships with cognitive function depending on exposure status . specifically , performance in df improved with age and larger white matter regional volumes in those with fasd . however , for typically developing children who also improved over time in cognitive function , this relationship was not shown as mediated by white matter volume increases . significant interactions were also observed for db and cvlt - c where increased callosal volume with age was related to better cognitive function in the exposed group . these results were less robust than those observed for tests of attention capacity and correlations were significant predominantly for the corpus callosum . overall , our findings suggest that in typically developing children , cognitive function may rely more heavily on other neurodevelopmental changes in brain macro- or microstructure . for instance , previous research has also shown that relationships between brain measures and cognitive function are stronger in study populations with neurological disorders , whereas such brain structure relationships during normal development are less consistently found ( van petten , 2004 ; treit et al . thus , in a typically developing population , it is possible that the contribution of other neurodevelopmental processes might mask white matter contributions , making these relationships harder to detect . for children with fasd , typical relationships between gray and white matter structure and their connectivity across the brain since children with fasd have smaller overall white matter volumes compared to age - matched typically developing peers , increase of white matter might significantly contribute to better communication among brain regions and consequently lead to improved cognitive abilities . since increases in white matter volume are at least partly attributable to age - related increases in myelination ( welker and patton , 2012 ) , improvements in conduction velocity of axonal fibers may facilitate communication among brain regions . , 2006 ) and may provide a possible explanation for the improvement of executive function with age in the current sample . less severe neuropsychological deficits have been previously documented for children in the spectrum compared to those with full fas , and children with fetal alcohol effects but without the facial dysmorphology or growth abnormalities to warrant an fas diagnosis can show normal iq and display only subtle behavioral or cognitive deficits . although we did not find differences in the developmental trajectory of white matter volume between diagnostic groups , it is possible that there might be changes occurring at the microstructural level . for instance , a recent study has found different rates of white matter maturation using dti measures between controls and children with fasd ( treit et al . although this study did not examine executive functioning , they reported positive correlations between change in mean diffusivity ( the overall magnitude of water diffusion in a voxel ) and reading scores in the fasd group . future longitudinal research will need to clarify relationships between diffusion imaging indices of white matter integrity and structural connectivity and executive functions . although there were no significant age , sex , or diagnosis severity differences in participants who did and did not complete the follow - up study , there might be other unknown factors that determined attrition . this study showed that while the rate of development of white matter volume is similar with age in both typically developing and exposed children , there are differential relationships between cognitive function and white matter change over time . specifically , for those with fasd , age - related increases in volumes were related to better cognitive function , while for controls , this positive relationship was not detected . findings suggest that increasing white matter volume with age allows those with fasd to improve in their cognitive abilities over time , possibly by facilitating information processing among connected brain areas . findings also suggest that better cognitive outcomes could be possible for fasd subjects through interventions targeting better cognitive and behavioral outcomes through education and/or other environmental factors .

patients were identified from registries from four urban community - based medical groups and four diabetes education settings . after physician permission was obtained , patients received a letter , a screening phone call , and then a personal visit from a project staff member to introduce them to the study and collect informed consent . inclusion criteria were type 2 diabetes , age 2175 years , ability to read and speak english or spanish fluently , no severe diabetes complications ( undergoing dialysis or legally blind ) , and no diagnosis of dementia or psychosis . at the initial assessment ( t1 ) patients participated in a 1.5-h visit that included questionnaires , physical measurements and interviews , a 150-item mail - back questionnaire , and forms for a visit to a local laboratory for collection of blood and urine specimens . patients were contacted again 9 ( t2 ) and 18 ( t3 ) months later , at which time the same assessments were repeated . patients who met the criteria for affective or anxiety disorder and who were not being treated were referred to their physician . approval was received by the institutional review board at the university of california , san francisco and at each participating facility . patient demographics included sex , self - identified ethnicity ( white or nonwhite ) , age , education ( years ) , and time since diagnosis ( years ) . also collected were use of insulin ( yes or no ) , bmi , number of complications , and number of comorbidities . diet and exercise were measured by the summary of diabetes self - care activities , which has demonstrated reliability and sensitivity to change over time ( 16 ) . patients reported the number of days in the past week they followed their diet or exercise plans . life stress unrelated to diabetes was assessed by the negative life events scale ( 17 ) , based on a list of 22 potential stressful events , such as death of a friend or being a crime victim . life context stressors have been shown to affect glucose levels ( 18 ) and self - management behavior ( 19 ) , thus potentially affecting glycemic control over time . mdd was assessed by the cidi ( 11 ) , a frequently used , reliable , structured diagnostic interview based on dsm - iv criteria . the time frame for mdd at t1 was occurrence during the past year and since we saw you last was used for t2 and t3 . depressive symptoms were assessed by the ces - d ( 13 ) , a frequently used , reliable , continuous scale that assesses the number of days during the last week that each of 20 free - standing depressive symptoms occurred . diabetes distress was assessed by the dds ( 15 ) , a continuous scale ( = 0.93 ) that assesses each of 17 items across six levels of severity of emotional , regimen - related , social , and medical care distress related to diabetes and its management . we used multilevel modeling ( mlm ) ( 20 ) to assess the independent relationship among demographics , diabetes status , stress , self - management , the three affective variables , and baseline level and change in a1c across three assessments , covering 18 months . mlm accounts for correlations among nested responses in repeated - measures designs and maximizes efficiency by including all available information for each respondent , even if an assessment is missed . a distinctive feature of mlm is that it allows for an estimation of between - person and within - person models to test whether the effect of a predictor on an outcome varies by patient subgroup . finally , it accommodates tests of time - varying covariates , reflecting how subgroups of variables change together over time . an unconditional means model partitioned the total variance of a1c across people and waves into two pieces : the between - person and the within - person variance . an unconditional growth model further partitioned the within - person variance into two pieces : the estimated variance of the slope of change in a1c over time and other changes in a1c not related to time . first , we evaluated how baseline predictors were related to baseline levels of a1c ( cross - sectional analyses ) and second , how these predictors were related to linear change in a1c over time ( prospective analyses ) . third , we also explored a set of time - varying covariates : how changes in a predictor over time were related to changes in a1c over time . time was centered at t1 and coded in years ( 0 , 0.75 , and 1.5 ) . baseline predictor variables were centered at their grand means so that the estimates of the intercept and time were interpretable . estimates were obtained with proc mixed ( sas version 9.2 ) using full maximum likelihood and robust ses ( 20 ) . we also examined nonlinearities among the continuous variables and assessed a series of interactions among the three affective variables , as well as between each with age , sex , time with diabetes , and insulin use : all were nonsignificant . we also explored the impact of use of psychotropic medication in all models : again , all were nonsignificant . at each stage , residuals were examined for normality and heterogeneity . we used multilevel modeling ( mlm ) ( 20 ) to assess the independent relationship among demographics , diabetes status , stress , self - management , the three affective variables , and baseline level and change in a1c across three assessments , covering 18 months . mlm accounts for correlations among nested responses in repeated - measures designs and maximizes efficiency by including all available information for each respondent , even if an assessment is missed . a distinctive feature of mlm is that it allows for an estimation of between - person and within - person models to test whether the effect of a predictor on an outcome varies by patient subgroup . finally , it accommodates tests of time - varying covariates , reflecting how subgroups of variables change together over time . an unconditional means model partitioned the total variance of a1c across people and waves into two pieces : the between - person and the within - person variance . an unconditional growth model further partitioned the within - person variance into two pieces : the estimated variance of the slope of change in a1c over time and other changes in a1c not related to time . first , we evaluated how baseline predictors were related to baseline levels of a1c ( cross - sectional analyses ) and second , how these predictors were related to linear change in a1c over time ( prospective analyses ) . third , we also explored a set of time - varying covariates : how changes in a predictor over time were related to changes in a1c over time . time was centered at t1 and coded in years ( 0 , 0.75 , and 1.5 ) . baseline predictor variables were centered at their grand means so that the estimates of the intercept and time were interpretable . estimates were obtained with proc mixed ( sas version 9.2 ) using full maximum likelihood and robust ses ( 20 ) . we also examined nonlinearities among the continuous variables and assessed a series of interactions among the three affective variables , as well as between each with age , sex , time with diabetes , and insulin use : all were nonsignificant . we also explored the impact of use of psychotropic medication in all models : again , all were nonsignificant . at each stage , residuals were examined for normality and heterogeneity . telephone screening identified 640 eligible individuals , and 506 of these completed the t1 assessment ( 79.0% ) ( table 1 ) . no significant differences were recorded between those who refused initially and those who participated on all major study variables . sample description ( n = 506 ) data are means sd or n ( % ) . approximately 81% of patients completed all three study waves , 21 ( 4.2% ) missed t2 only , 40 ( 7.9% ) missed t3 only , and 34 ( 6.7% ) missed both t2 and t3 . patients who completed all three waves those who missed a wave more often spoke spanish than english ( r = 0.09 , p = 0.04 ) and had diabetes longer . those with mdd , high depressive affect , or diabetes distress did not miss a wave or drop out more often than those without these conditions . all three affective variables were significantly intercorrelated at t1 , although the relationship between ces - d and dds ( r = 0.48 , p < 0.001 ) was notably higher than the relationship of these two variables with mdd ( mdd with ces - d , r = 0.29 , p < both ces - d ( r = 0.14 ; p = 0.002 ) and dds ( r = 0.17 ; p = 0.001 ) were significantly correlated with a1c , whereas mdd was not ( r = 0.05 ) . of the 12 other variables in the multivariate model ( table 2 ) , 8 displayed significant zero - order correlations with a1c : race / ethnicity ( r = 0.19 , p < 0.001 ) , age ( r = 0.08 , p < 0.05 ) , education ( r = 0.16 , p < 0.001 ) , time with diabetes ( r = 0.27 , p < 0.001 ) , insulin use ( r = 0.29 , p ( r = 0.17 , p < 0.001 ) , life events ( r = 0.13 , p < 0.01 ) , and diet ( r = 0.09 , p < 0.05 ) . cross - sectional and prospective models predicting glycemic control ( a1c ) data are unstandardized regression coefficients . the cross - sectional model uses data from t1 ; the prospective model uses t1 predictors of change in a1c over time . table 2 shows the cross - sectional relationships between each variable in the model and a1c at t1 , with controls for all other variables . patients who were nonwhite , had more comorbidities , had diabetes longer , and were receiving insulin had higher a1c at t1 than those who were white , had few comorbidities , had diabetes a shorter time , and were not receiving insulin . of the three affective variables , however , a significant positive relationship with a1c was found only for dds but not for mdd or ces - d . not shown are models in which each of the three affective variables was entered into separate equations individually . only dds reached significance ( p < 0.004 ) ; mdd and ces - d did not . although the average change in a1c over time was not significantly different from zero for the sample as a whole , there was significant within - person variation in a1c change over time : slopes for 95% of the sample ranged from 0.122 to 0.128 , with some decreasing and some increasing systematically over time . table 2 shows that three t1 variables independently predicted change in a1c over time : older patients and those having diabetes longer displayed significantly greater decreases in a1c over time than younger patients and those with a more recent diagnosis of diabetes . also , those with more comorbidities at t1 displayed greater increases in a1c over time than those with fewer comorbidities . also , none of the three reached significance when each was entered individually into separate regression models . these analyses added sets of time - varying covariates to the previous model to examine whether change in each characteristic was independently related to change in a1c over time . we included patient sex , ethnicity , age , education , and time with diabetes as covariates in these analyses but did not explore their time - varying relationships with a1c because these variables were viewed as being relatively fixed over time . these analyses provided statistical information only about the degree of time - concordant association between changes in a characteristic and changes in a1c over time ; they did not provide information about the causal linkages between the two . the first columns of table 3 show the coefficients for the time - varying associations among the three affective variables and a1c , with all other variables entered as controls . only dds , but not mdd or ces - d , displayed a significant time concordant association with a1c ( b = 0.024 , p = 0.001 ) . the right - hand columns of table 3 show the independent time - varying relationships for all variables in the model . again , only dds displayed a significant time concordant relationship with a1c ( b = 0.023 , p = 0.001 ) . when each of the affective variables was entered individually into separate models , only dds reached significance ( p = 0.001 ) . time - covarying models predicting change in glycemic control over time ( a1c ) data are unstandardized regression coefficients . patient sex , race , age , education , and time since diagnosis also were included in the model . telephone screening identified 640 eligible individuals , and 506 of these completed the t1 assessment ( 79.0% ) ( table 1 ) . no significant differences were recorded between those who refused initially and those who participated on all major study variables . sample description ( n = 506 ) data are means sd or n ( % ) . approximately 81% of patients completed all three study waves , 21 ( 4.2% ) missed t2 only , 40 ( 7.9% ) missed t3 only , and 34 ( 6.7% ) missed both t2 and t3 . patients who completed all three waves those who missed a wave more often spoke spanish than english ( r = 0.09 , p = 0.04 ) and had diabetes longer . those with mdd , high depressive affect , or diabetes distress did not miss a wave or drop out more often than those without these conditions . all three affective variables were significantly intercorrelated at t1 , although the relationship between ces - d and dds ( r = 0.48 , p < 0.001 ) was notably higher than the relationship of these two variables with mdd ( mdd with ces - d , r = 0.29 , p < 0.001 ; mdd with dds , r = 0.15 , p < 0.001 ) . both ces - d ( r = 0.14 ; p = 0.002 ) and dds ( r = 0.17 ; p = 0.001 ) were significantly correlated with a1c , whereas mdd was not ( r = 0.05 ) . of the 12 other variables in the multivariate model ( table 2 ) , 8 displayed significant zero - order correlations with a1c : race / ethnicity ( r = 0.19 , p < 0.001 ) , age ( r = 0.08 , p < 0.05 ) , education ( r = 0.16 , p < 0.001 ) , time with diabetes ( r = 0.27 , p < 0.001 ) , insulin use ( r = 0.29 , p < 0.001 ) , complications ( r = 0.17 , p < 0.001 ) , life events ( r = 0.13 , p < 0.01 ) , and diet ( r = 0.09 , p < 0.05 ) . cross - sectional and prospective models predicting glycemic control ( a1c ) data are unstandardized regression coefficients . the cross - sectional model uses data from t1 ; the prospective model uses t1 predictors of change in a1c over time . table 2 shows the cross - sectional relationships between each variable in the model and a1c at t1 , with controls for all other variables . patients who were nonwhite , had more comorbidities , had diabetes longer , and were receiving insulin had higher a1c at t1 than those who were white , had few comorbidities , had diabetes a shorter time , and were not receiving insulin . of the three affective variables , however , a significant positive relationship with a1c was found only for dds but not for mdd or ces - d . not shown are models in which each of the three affective variables was entered into separate equations individually . only dds reached significance ( p < 0.004 ) ; mdd and ces - d did not . although the average change in a1c over time was not significantly different from zero for the sample as a whole , there was significant within - person variation in a1c change over time : slopes for 95% of the sample ranged from 0.122 to 0.128 , with some decreasing and some increasing systematically over time . table 2 shows that three t1 variables independently predicted change in a1c over time : older patients and those having diabetes longer displayed significantly greater decreases in a1c over time than younger patients and those with a more recent diagnosis of diabetes . also , those with more comorbidities at t1 displayed greater increases in a1c over time than those with fewer comorbidities . also , none of the three reached significance when each was entered individually into separate regression models . these analyses added sets of time - varying covariates to the previous model to examine whether change in each characteristic was independently related to change in a1c over time . we included patient sex , ethnicity , age , education , and time with diabetes as covariates in these analyses but did not explore their time - varying relationships with a1c because these variables were viewed as being relatively fixed over time . these analyses provided statistical information only about the degree of time - concordant association between changes in a characteristic and changes in a1c over time ; they did not provide information about the causal linkages between the two . the first columns of table 3 show the coefficients for the time - varying associations among the three affective variables and a1c , with all other variables entered as controls . only dds , but not mdd or ces - d , displayed a significant time concordant association with a1c ( b = 0.024 , p = 0.001 ) . the right - hand columns of table 3 show the independent time - varying relationships for all variables in the model . again , only dds displayed a significant time concordant relationship with a1c ( b = 0.023 , p = 0.001 ) . when each of the affective variables was entered individually into separate models , only dds reached significance ( p = 0.001 ) . time - covarying models predicting change in glycemic control over time ( a1c ) data are unstandardized regression coefficients . patient sex , race , age , education , and time since diagnosis also were included in the model . ours is one of the few observational , noninterventional studies that explored both the cross - sectional and longitudinal relationships of mdd , depressive symptoms , and diabetes distress with glycemic control using well - established scales that specifically addressed each of the three affective constructs . with a comprehensive battery of controls in the models , we found no statistically significant cross - sectional , prospective , or time - concordant relationship between mdd and a1c or between depressive symptoms and a1c . only distress specifically linked to diabetes displays both cross - sectional and time - varying longitudinal relationships with a1c . congruent with prior research , we find no evidence of a statistically significant relationship between mdd and glycemic control or between depressive symptoms and glycemic control ( 8,21 ) . a similar finding is provided by a recent study with both type 1 and type 2 diabetic patients that showed that improvements in depressive symptoms after cognitive behavior therapy were not associated with changes in a1c ( 7 ) . thus , in both prospective studies , in which changes in mdd or in depressive symptoms occur after behavioral or pharmacological intervention , and in noninterventional studies , in which changes in symptoms or mdd are recorded over time , we see little or no concomitant changes in glycemic control . we conclude from these studies that the association between mdd and depressive symptoms with glycemic control is most likely modest at best and may be an artifact of the complex pattern of frequently uncontrolled interrelationships often found among a host of mood , diabetes status , treatment , behavioral , and life context variables ( 22 ) . if mdd and glycemic control are linked , it may be that depressive states have to be of sufficient intensity and duration to demonstrate the effect , or it may be that there are multiple pathways between mdd and depressive symptoms with glycemic control and that they operate differently for different patients under different life contexts . if a causal link does exist , most likely there is no single , easily identified common pathway . in contrast , we find that emotional distress specifically tied to diabetes and its management displays both cross - sectional and time - concordant relationships with a1c . these results do not necessarily imply a causative relationship between the two , especially because no significant prospective linkages between dds and a1c were found . we suspect that each most likely influences the other over time , suggesting a bidirectional relationship ( 5 ) within the context of other co - occurring diabetes and life context variables ( 22 ) . for example , for some patients , high disease distress can influence self - management and medication adherence with subsequent effects on glycemic control , and for other patients , poor control can lead to distress , which can influence disease management ( 23 ) . this formulation of the relationship between diabetes distress and glycemic control does not assume the direct involvement of any physiological process but instead emphasizes the ongoing negative subjective experience of emotional distress around the management of a significant chronic condition that has implications for ongoing disease - related behavior , motivation , self - efficacy , and problem solving . it is also likely that some depressive symptoms partly reflect the negative emotional experience that surrounds disease - specific distress ( 26 ) . this factor may explain the significant association between ces - d and dds ( r = 0.48 ) , coupled with the finding that only dds , but not ces - d , displays both independent cross - sectional and time concordant relationships with a1c . thus , symptom inventories , such as ces - d , may tap into the negative emotional component of diabetes - specific distress . in an effort to clarify and be more precise about what has been called depression in diabetes , it may be helpful clinically to consider and assess two relatively common conditions : mdd and diabetes - specific distress . for example , it has been shown that most individuals who are distressed about their chronic disease are not clinically depressed ( 27 ) , that distress can be conceptually and empirically differentiated from depression and depressive symptoms , and that distress has stronger linkages with common psychological , behavioral , and social factors than clinical depression or depressive symptoms ( 26,28 ) . diabetes distress is about twice as prevalent as mdd in this population , is more persistent over time than mdd and high depressive symptoms , and is significantly and independently associated with a host of diabetes - related variables , e.g. , bmi , complications , comorbidities , and self - management behaviors ( 26 ) . both mdd and diabetes distress are serious , treatable , and worthy of clinical concern . first , our use of a diverse community sample led to somewhat small subsamples of patients with defined affective conditions . although we had sufficient statistical power to address the research questions posed , larger stratified samples might permit more comprehensive subgroup analyses . studies with more frequent assessments that continue for a longer duration may yield additional findings . third , the failure to observe a relationship between mdd and glycemic control may be partially due to a statistical issue : mdd is a dichotomous variable , whereas dds , ces - d , and a1c are continuous variables , and correlations between continuous variables generally will be higher than correlations between a continuous variable and a binary variable . this is a problem inherent in a diagnostic approach and may argue for the use of more dimensional measures , which are generally more powerful . fourth , some of the findings from the time - covarying analyses may have been influenced by patient knowledge of their a1c level . in contrast , the strengths of the study include a diverse community - based sample with high rates of participation and retention , and the use of sophisticated data analytic procedures that permit maximum flexibility and power in analyzing both cross - sectional and longitudinal data . in summary , we found no cross - sectional , prospective , or time - concordant associations between mdd and depressive symptoms with glycemic control , whereas significant cross - sectional and time - concordant relationships were found between diabetes distress and glycemic control . given the linkages between diabetes distress and a host of diabetes management variables , we emphasize the importance of exploring further with empirical studies the interactive relationship between diabetes distress and glycemic control , screening for both mdd and disease - related distress in the clinical setting , and development of interventions for nondepressed but distressed patients with diabetes .

objectiveto determine the concurrent , prospective , and time - concordant relationships among major depressive disorder ( mdd ) , depressive symptoms , and diabetes distress with glycemic control.research design and methodsin a noninterventional study , we assessed 506 type 2 diabetic patients for mdd ( composite international diagnostic interview ) , for depressive symptoms ( center for epidemiological studies - depression ) , and for diabetes distress ( diabetes distress scale ) , along with self - management , stress , demographics , and diabetes status , at baseline and 9 and 18 months later . using multilevel modeling ( mlm ) , we explored the cross - sectional relationships of the three affective variables with a1c , the prospective relationships of baseline variables with change in a1c over time , and the time - concordant relationships with a1c.resultsall three affective variables were moderately intercorrelated , although the relationship between depressive symptoms and diabetes distress was greater than the relationship of either with mdd . in the cross - sectional mlm , only diabetes distress but not mdd or depressive symptoms was significantly associated with a1c . none of the three affective variables were linked with a1c in prospective analyses . only diabetes distress displayed significant time - concordant relationships with a1c.conclusionswe found no concurrent or longitudinal association between mdd or depressive symptoms with a1c , whereas both concurrent and time - concordant relationships were found between diabetes distress and a1c . what has been called depression among type 2 diabetic patients may really be two conditions , mdd and diabetes distress , with only the latter displaying significant associations with a1c . ongoing evaluation of both diabetes distress and mdd may be helpful in clinical settings .

RESEARCH DESIGN AND METHODS Data analysis RESULTS Preliminary analyses Concurrent relationships Prospective analyses Time-varying relationships CONCLUSIONS

after physician permission was obtained , patients received a letter , a screening phone call , and then a personal visit from a project staff member to introduce them to the study and collect informed consent . inclusion criteria were type 2 diabetes , age 2175 years , ability to read and speak english or spanish fluently , no severe diabetes complications ( undergoing dialysis or legally blind ) , and no diagnosis of dementia or psychosis . patients were contacted again 9 ( t2 ) and 18 ( t3 ) months later , at which time the same assessments were repeated . patient demographics included sex , self - identified ethnicity ( white or nonwhite ) , age , education ( years ) , and time since diagnosis ( years ) . also collected were use of insulin ( yes or no ) , bmi , number of complications , and number of comorbidities . diet and exercise were measured by the summary of diabetes self - care activities , which has demonstrated reliability and sensitivity to change over time ( 16 ) . patients reported the number of days in the past week they followed their diet or exercise plans . life stress unrelated to diabetes was assessed by the negative life events scale ( 17 ) , based on a list of 22 potential stressful events , such as death of a friend or being a crime victim . life context stressors have been shown to affect glucose levels ( 18 ) and self - management behavior ( 19 ) , thus potentially affecting glycemic control over time . mdd was assessed by the cidi ( 11 ) , a frequently used , reliable , structured diagnostic interview based on dsm - iv criteria . the time frame for mdd at t1 was occurrence during the past year and since we saw you last was used for t2 and t3 . depressive symptoms were assessed by the ces - d ( 13 ) , a frequently used , reliable , continuous scale that assesses the number of days during the last week that each of 20 free - standing depressive symptoms occurred . diabetes distress was assessed by the dds ( 15 ) , a continuous scale ( = 0.93 ) that assesses each of 17 items across six levels of severity of emotional , regimen - related , social , and medical care distress related to diabetes and its management . we used multilevel modeling ( mlm ) ( 20 ) to assess the independent relationship among demographics , diabetes status , stress , self - management , the three affective variables , and baseline level and change in a1c across three assessments , covering 18 months . finally , it accommodates tests of time - varying covariates , reflecting how subgroups of variables change together over time . an unconditional growth model further partitioned the within - person variance into two pieces : the estimated variance of the slope of change in a1c over time and other changes in a1c not related to time . first , we evaluated how baseline predictors were related to baseline levels of a1c ( cross - sectional analyses ) and second , how these predictors were related to linear change in a1c over time ( prospective analyses ) . third , we also explored a set of time - varying covariates : how changes in a predictor over time were related to changes in a1c over time . baseline predictor variables were centered at their grand means so that the estimates of the intercept and time were interpretable . we also examined nonlinearities among the continuous variables and assessed a series of interactions among the three affective variables , as well as between each with age , sex , time with diabetes , and insulin use : all were nonsignificant . we used multilevel modeling ( mlm ) ( 20 ) to assess the independent relationship among demographics , diabetes status , stress , self - management , the three affective variables , and baseline level and change in a1c across three assessments , covering 18 months . finally , it accommodates tests of time - varying covariates , reflecting how subgroups of variables change together over time . an unconditional growth model further partitioned the within - person variance into two pieces : the estimated variance of the slope of change in a1c over time and other changes in a1c not related to time . first , we evaluated how baseline predictors were related to baseline levels of a1c ( cross - sectional analyses ) and second , how these predictors were related to linear change in a1c over time ( prospective analyses ) . third , we also explored a set of time - varying covariates : how changes in a predictor over time were related to changes in a1c over time . baseline predictor variables were centered at their grand means so that the estimates of the intercept and time were interpretable . we also examined nonlinearities among the continuous variables and assessed a series of interactions among the three affective variables , as well as between each with age , sex , time with diabetes , and insulin use : all were nonsignificant . those with mdd , high depressive affect , or diabetes distress did not miss a wave or drop out more often than those without these conditions . all three affective variables were significantly intercorrelated at t1 , although the relationship between ces - d and dds ( r = 0.48 , p < 0.001 ) was notably higher than the relationship of these two variables with mdd ( mdd with ces - d , r = 0.29 , p < both ces - d ( r = 0.14 ; p = 0.002 ) and dds ( r = 0.17 ; p = 0.001 ) were significantly correlated with a1c , whereas mdd was not ( r = 0.05 ) . of the 12 other variables in the multivariate model ( table 2 ) , 8 displayed significant zero - order correlations with a1c : race / ethnicity ( r = 0.19 , p < 0.001 ) , age ( r = 0.08 , p < 0.05 ) , education ( r = 0.16 , p < 0.001 ) , time with diabetes ( r = 0.27 , p < 0.001 ) , insulin use ( r = 0.29 , p ( r = 0.17 , p < 0.001 ) , life events ( r = 0.13 , p < 0.01 ) , and diet ( r = 0.09 , p < 0.05 ) . cross - sectional and prospective models predicting glycemic control ( a1c ) data are unstandardized regression coefficients . the cross - sectional model uses data from t1 ; the prospective model uses t1 predictors of change in a1c over time . table 2 shows the cross - sectional relationships between each variable in the model and a1c at t1 , with controls for all other variables . patients who were nonwhite , had more comorbidities , had diabetes longer , and were receiving insulin had higher a1c at t1 than those who were white , had few comorbidities , had diabetes a shorter time , and were not receiving insulin . of the three affective variables , however , a significant positive relationship with a1c was found only for dds but not for mdd or ces - d . not shown are models in which each of the three affective variables was entered into separate equations individually . although the average change in a1c over time was not significantly different from zero for the sample as a whole , there was significant within - person variation in a1c change over time : slopes for 95% of the sample ranged from 0.122 to 0.128 , with some decreasing and some increasing systematically over time . table 2 shows that three t1 variables independently predicted change in a1c over time : older patients and those having diabetes longer displayed significantly greater decreases in a1c over time than younger patients and those with a more recent diagnosis of diabetes . also , those with more comorbidities at t1 displayed greater increases in a1c over time than those with fewer comorbidities . also , none of the three reached significance when each was entered individually into separate regression models . these analyses added sets of time - varying covariates to the previous model to examine whether change in each characteristic was independently related to change in a1c over time . we included patient sex , ethnicity , age , education , and time with diabetes as covariates in these analyses but did not explore their time - varying relationships with a1c because these variables were viewed as being relatively fixed over time . these analyses provided statistical information only about the degree of time - concordant association between changes in a characteristic and changes in a1c over time ; they did not provide information about the causal linkages between the two . the first columns of table 3 show the coefficients for the time - varying associations among the three affective variables and a1c , with all other variables entered as controls . only dds , but not mdd or ces - d , displayed a significant time concordant association with a1c ( b = 0.024 , p = 0.001 ) . the right - hand columns of table 3 show the independent time - varying relationships for all variables in the model . again , only dds displayed a significant time concordant relationship with a1c ( b = 0.023 , p = 0.001 ) . when each of the affective variables was entered individually into separate models , only dds reached significance ( p = 0.001 ) . time - covarying models predicting change in glycemic control over time ( a1c ) data are unstandardized regression coefficients . patient sex , race , age , education , and time since diagnosis also were included in the model . those with mdd , high depressive affect , or diabetes distress did not miss a wave or drop out more often than those without these conditions . all three affective variables were significantly intercorrelated at t1 , although the relationship between ces - d and dds ( r = 0.48 , p < 0.001 ) was notably higher than the relationship of these two variables with mdd ( mdd with ces - d , r = 0.29 , p < 0.001 ; mdd with dds , r = 0.15 , p < 0.001 ) . both ces - d ( r = 0.14 ; p = 0.002 ) and dds ( r = 0.17 ; p = 0.001 ) were significantly correlated with a1c , whereas mdd was not ( r = 0.05 ) . of the 12 other variables in the multivariate model ( table 2 ) , 8 displayed significant zero - order correlations with a1c : race / ethnicity ( r = 0.19 , p < 0.001 ) , age ( r = 0.08 , p < 0.05 ) , education ( r = 0.16 , p < 0.001 ) , time with diabetes ( r = 0.27 , p < 0.001 ) , insulin use ( r = 0.29 , p < 0.001 ) , complications ( r = 0.17 , p < 0.001 ) , life events ( r = 0.13 , p < 0.01 ) , and diet ( r = 0.09 , p < 0.05 ) . cross - sectional and prospective models predicting glycemic control ( a1c ) data are unstandardized regression coefficients . the cross - sectional model uses data from t1 ; the prospective model uses t1 predictors of change in a1c over time . table 2 shows the cross - sectional relationships between each variable in the model and a1c at t1 , with controls for all other variables . patients who were nonwhite , had more comorbidities , had diabetes longer , and were receiving insulin had higher a1c at t1 than those who were white , had few comorbidities , had diabetes a shorter time , and were not receiving insulin . of the three affective variables , however , a significant positive relationship with a1c was found only for dds but not for mdd or ces - d . not shown are models in which each of the three affective variables was entered into separate equations individually . although the average change in a1c over time was not significantly different from zero for the sample as a whole , there was significant within - person variation in a1c change over time : slopes for 95% of the sample ranged from 0.122 to 0.128 , with some decreasing and some increasing systematically over time . table 2 shows that three t1 variables independently predicted change in a1c over time : older patients and those having diabetes longer displayed significantly greater decreases in a1c over time than younger patients and those with a more recent diagnosis of diabetes . also , those with more comorbidities at t1 displayed greater increases in a1c over time than those with fewer comorbidities . also , none of the three reached significance when each was entered individually into separate regression models . these analyses added sets of time - varying covariates to the previous model to examine whether change in each characteristic was independently related to change in a1c over time . we included patient sex , ethnicity , age , education , and time with diabetes as covariates in these analyses but did not explore their time - varying relationships with a1c because these variables were viewed as being relatively fixed over time . these analyses provided statistical information only about the degree of time - concordant association between changes in a characteristic and changes in a1c over time ; they did not provide information about the causal linkages between the two . the first columns of table 3 show the coefficients for the time - varying associations among the three affective variables and a1c , with all other variables entered as controls . only dds , but not mdd or ces - d , displayed a significant time concordant association with a1c ( b = 0.024 , p = 0.001 ) . the right - hand columns of table 3 show the independent time - varying relationships for all variables in the model . again , only dds displayed a significant time concordant relationship with a1c ( b = 0.023 , p = 0.001 ) . when each of the affective variables was entered individually into separate models , only dds reached significance ( p = 0.001 ) . time - covarying models predicting change in glycemic control over time ( a1c ) data are unstandardized regression coefficients . patient sex , race , age , education , and time since diagnosis also were included in the model . ours is one of the few observational , noninterventional studies that explored both the cross - sectional and longitudinal relationships of mdd , depressive symptoms , and diabetes distress with glycemic control using well - established scales that specifically addressed each of the three affective constructs . with a comprehensive battery of controls in the models , we found no statistically significant cross - sectional , prospective , or time - concordant relationship between mdd and a1c or between depressive symptoms and a1c . only distress specifically linked to diabetes displays both cross - sectional and time - varying longitudinal relationships with a1c . congruent with prior research , we find no evidence of a statistically significant relationship between mdd and glycemic control or between depressive symptoms and glycemic control ( 8,21 ) . a similar finding is provided by a recent study with both type 1 and type 2 diabetic patients that showed that improvements in depressive symptoms after cognitive behavior therapy were not associated with changes in a1c ( 7 ) . thus , in both prospective studies , in which changes in mdd or in depressive symptoms occur after behavioral or pharmacological intervention , and in noninterventional studies , in which changes in symptoms or mdd are recorded over time , we see little or no concomitant changes in glycemic control . we conclude from these studies that the association between mdd and depressive symptoms with glycemic control is most likely modest at best and may be an artifact of the complex pattern of frequently uncontrolled interrelationships often found among a host of mood , diabetes status , treatment , behavioral , and life context variables ( 22 ) . if mdd and glycemic control are linked , it may be that depressive states have to be of sufficient intensity and duration to demonstrate the effect , or it may be that there are multiple pathways between mdd and depressive symptoms with glycemic control and that they operate differently for different patients under different life contexts . in contrast , we find that emotional distress specifically tied to diabetes and its management displays both cross - sectional and time - concordant relationships with a1c . these results do not necessarily imply a causative relationship between the two , especially because no significant prospective linkages between dds and a1c were found . we suspect that each most likely influences the other over time , suggesting a bidirectional relationship ( 5 ) within the context of other co - occurring diabetes and life context variables ( 22 ) . for example , for some patients , high disease distress can influence self - management and medication adherence with subsequent effects on glycemic control , and for other patients , poor control can lead to distress , which can influence disease management ( 23 ) . this formulation of the relationship between diabetes distress and glycemic control does not assume the direct involvement of any physiological process but instead emphasizes the ongoing negative subjective experience of emotional distress around the management of a significant chronic condition that has implications for ongoing disease - related behavior , motivation , self - efficacy , and problem solving . it is also likely that some depressive symptoms partly reflect the negative emotional experience that surrounds disease - specific distress ( 26 ) . this factor may explain the significant association between ces - d and dds ( r = 0.48 ) , coupled with the finding that only dds , but not ces - d , displays both independent cross - sectional and time concordant relationships with a1c . in an effort to clarify and be more precise about what has been called depression in diabetes , it may be helpful clinically to consider and assess two relatively common conditions : mdd and diabetes - specific distress . for example , it has been shown that most individuals who are distressed about their chronic disease are not clinically depressed ( 27 ) , that distress can be conceptually and empirically differentiated from depression and depressive symptoms , and that distress has stronger linkages with common psychological , behavioral , and social factors than clinical depression or depressive symptoms ( 26,28 ) . diabetes distress is about twice as prevalent as mdd in this population , is more persistent over time than mdd and high depressive symptoms , and is significantly and independently associated with a host of diabetes - related variables , e.g. , bmi , complications , comorbidities , and self - management behaviors ( 26 ) . both mdd and diabetes distress are serious , treatable , and worthy of clinical concern . third , the failure to observe a relationship between mdd and glycemic control may be partially due to a statistical issue : mdd is a dichotomous variable , whereas dds , ces - d , and a1c are continuous variables , and correlations between continuous variables generally will be higher than correlations between a continuous variable and a binary variable . fourth , some of the findings from the time - covarying analyses may have been influenced by patient knowledge of their a1c level . in contrast , the strengths of the study include a diverse community - based sample with high rates of participation and retention , and the use of sophisticated data analytic procedures that permit maximum flexibility and power in analyzing both cross - sectional and longitudinal data . in summary , we found no cross - sectional , prospective , or time - concordant associations between mdd and depressive symptoms with glycemic control , whereas significant cross - sectional and time - concordant relationships were found between diabetes distress and glycemic control . given the linkages between diabetes distress and a host of diabetes management variables , we emphasize the importance of exploring further with empirical studies the interactive relationship between diabetes distress and glycemic control , screening for both mdd and disease - related distress in the clinical setting , and development of interventions for nondepressed but distressed patients with diabetes .

uterine transplantation ( utn ) has been proposed as a possible solution to absolute uterine factor infertility ( aufi ) untreatable by any other option . the inability to experience a pregnancy , give birth , and bring up a child because of infertility issues may be one of the most traumatic and devastating situations to affect a woman or couple , with the capacity to have a severely detrimental effect on the quality of life of both parties [ 24 ] . the term infertile is an all - encompassing term and includes women with aufi . causes which render women unconditionally infertile are either congenital , namely , mllerian duct anomalies , or acquired causes ( such as leiomyomata , radiation damage , intrauterine adhesions , or premenopausal hysterectomy for obstetric bleeding or cervical / endometrial cancer ) . tremendous advances have been made during the last decades in the fields of transplantation and reproductive medicine , in particular , the first reports of successful transplantation of a solid organ , the kidney [ 5 , 6 ] , and live birth after ivf . transplantation surgery today includes types of organ / tissue transplantation that will enhance the quality - of - life as exemplified by transplantation of the hand , the abdominal wall , the larynx , and the face , with the aim to add utn to the list . utn was first performed in a human in 2000 on a 26-year - old female who had previously lost her uterus as a result of postpartum haemorrhage . the event granted much needed impetus for research into utn , which since then has been a slow , methodical process within an animal setting involving multiple institutions and disciplines over several continents . research has focused on several important areas , mainly surgical , immunological , and reproductive aspects . the second utn attempt in a human model is anticipated in the not - too - distant future . a major obstacle the process of rejection in allogeneic utn was first described by two studies in 1969 [ 12 , 13 ] , the decade in which utn research was first published . at that time , utn commonly involved en bloc autotransplantation of a combination of the different reproductive organs : uterus , fallopian tubes , and ovaries . since then , assisted reproductive technologies such as in vitro fertilization ( ivf ) have addressed many of the causes of infertility . however , for women who suffer from aufi , surrogacy , adoption , or lifetime infertility remains the only option . the hope is that , with continued advances in transplantation surgery and control of tissue rejection , utn can bring an end to aufi by allowing a select group of women to become mothers . the treatment of choice for certain end - stage organ diseases , such as kidney and liver failure , is organ transplantation . since 1956 and joseph murray 's first successful solid - organ transplant , involving the transfer of a kidney between genetically identical twins , this field has progressed rapidly because of considerable improvements in two vital areas : surgical methodology and immunology . with respect to the latter , it is important to recognise that without full understanding of the risks posed by the rejection process and subsequent development of immunosuppressants , transplantation as we understand it today would not be possible . it would certainly involve a smaller group of possible donors ( only syngeneic ) , resulting in a greatly reduced number of potential beneficiaries . research of graft rejection between two allogeneically different individuals resulted in the development of immunosuppressive agents in the 1960s . 5-mercaptopurine / azathioprine , in combination with corticosteroids , was the first to be marketed for use . next came the discovery of the calcineurin inhibitor cyclosporine a ( csa ) in the late 1970s . these drugs , especially csa , represented at the time a widely sought panacea and caused a rapid advancement in clinical organ transplantation . the risks posed by immunosuppressants are well known , and the potential teratogenicity of certain agents still poses a considerable threat . at the time of the first healthy birth to a transplant recipient in 1958 , however , approximately 14,000 births ( more since this result was first published in 2002 ) among transplant women have been reported worldwide , resulting in a change of opinion , whereby restored fertility is now a recognized desirable outcome following transplantation [ 19 , 20 ] . sir peter brian medawar first introduced the idea of immunosuppressants as possible agents that could be used to prevent and treat allograft rejection . currently short - term effects have been very positive but chronic rejection is still a major issue and long - term use has its drawbacks . these are mainly related to nonspecific suppression of the host immune system , which may cause any of the following : bacterial and viral infections , nephrotoxicity and neurotoxicity with chronic renal failure , newly diagnosed diabetes mellitus , hyperlipidaemia , leucopoenia , and even cancer [ 15 , 21 ] . deciding on the type , dose , and monitoring of immunosuppressants prescribed is best made within a multidisciplinary team setting including an obstetrician , so as to maximize the efficacy and minimize potential toxicity . as immunosuppressants can cross the human placental barrier and enter the fetal circulation , possibly affecting the immune system of the fetus , obstetric input is paramount . which immunosuppressant is administered depends very much on the age and comorbidities of the patient , type of transplanted organ , length of time that the patient is expected to live postoperatively , and the transplant centre . medications used to suppress immunologic activity are chosen based on the stage of the transplant process . in the induction phase , intravenous immunoglobulin is usually combined with a cornerstone immunosuppressant such as a cytokine modulator ( csa ) , purine - related analogue ( azathioprine ) , and a purine synthesis inhibitor ( mycophenolate mofetil ) . csa or tacrolimus are then usually continued as maintenance therapy , in combination with glucocorticoids , mycophenolate mofetil / sodium , or azathioprine . in addition , alkylating agents ( cyclophosphamide and chlorambucil ) and biologic response modifiers ( tnf antagonists ) can be used [ 15 , 24 ] . the first record of the use of immunosuppressants in pregnancy was in 1967 by board et al . the risk that immunosuppressants pose to both the mother and fetus , with respect to potential birth defects , needs to be fully investigated and understood . this is especially important as the number of female transplant recipients of child - bearing age is increasing . furthermore , problems related to thalidomide exposure in the 1960s clearly portray the devastating consequences of the release of unknown drug into the general population . the level of risk is dependent on whether an animal or human is receiving the immunosuppressant as well as the dose and route of administration of the immunosuppressant . fetal risk ranges from severe abnormalities related to structural development that are diagnosable either at any stage of pregnancy or only in the third trimester , to those that are only subtle and apparent after delivery . csa is a cyclic nonribosomal decapeptide derived from the fungus , tolypocladium inflatum , first isolated in norway in 1969 . it was first used to successfully prevent kidney and liver transplant rejection , with official approval for csa use arriving in 1983 . apart from transplant medicine , csa is also used in a number of autoimmune diseases : dermatological - psoriasis ; severe atopic dermatitis ; pyoderma gangrenosum ; rheumatologic - rheumatoid arthritis and gastrointestinal such as ulcerative colitis and crohn 's disease . the main role of csa is to interfere with signaling pathways important for the clonal expansion of immunocompetent lymphocytes . it does this by forming complexes through binding to an intracellular cytosolic protein called cyclophilin . the csa - cyclophilin complex inhibits the phosphatase activity of a calcium - activated enzyme called calcineurin . calcineurin is responsible for transmitting signals from the t - cell receptor to the nucleus . it is activated following the rise of intracellular ca levels in response to t - cell receptor binding . calcineurin then dephosphorylates the transcription factor nf - at ( nuclear factor of activated t cells ) in the cytoplasm , allowing for its migration to the nucleus which leads to the induction of transcription of genes coding for il-2 , cd40 ligand , and fas ligand . therefore , by inhibiting the function of calcineurin , the csa - cyclophilin complex manages to block clonal t - cell proliferation in response to the host immune system recognizing a specific antigen as being foreign . this results in reduced function of effector t cells . csa also acts on other immunopotent cells and has a large variety of other immunological effects ( table 2 ) . research into utn restarted before its afore - mentioned milestone in 2000 , that is , the first human attempt . this attempt may have been premature , with little characterization or understanding of the rejection response prior to the operation . csa was administered 6 hours prior to surgery and postoperatively ( 4 mg / kg / body weight divided into two doses to assure a serum trough level of 200 ng ) along with azathioprine , prednisolone and a boost by antithymocyte globulin . cd4/cd8 ratio in blood and doppler of uterine blood flow were the only techniques employed to monitor possible rejection patterns . the first rejection episode occurred on the ninth postoperative day and the patient was treated by increasing the oral doses of csa and azathioprine and administering an intravenous dose of prednisolone . the rejection resolved after 2 days but only after antithymocytic globulin was given . on the 99th day , histopathologic examination confirmed thrombosis of uterine vessels , and , interestingly , there was no signs of rejection with apparent viability of both tubes . to date , csa has not been used as an immunosuppressant in the few cases where the primary objective was to bring about pregnancy following utn . 4 cases involved a syngeneic model which removed the need for an immunosuppressant [ 3235 ] . in october of the last year , an allogeneic uterine transplant reported resorbed pregnancies in 4 out of 5 pregnant rats , with only tacrolimus used as an immunosuppressant . however , csa has been tried as an immunosuppressant in utn studies where achieving pregnancy was not the primary objective . described below are all studies where csa was administered as an immunosuppressant in utn . its first recorded use was in 1986 when confino et al . described unilateral nonvascular utn in 18 rabbit models . out of those , three rabbits developed pelvic abscesses while in three other rabbits both the endometrial and myometrial layers survived for four weeks . brnnstrm group went on to describe the acute rejection response in a mouse model , with signs of rejection from day 2 to day 5 , represented by increased density of cd3 + t cells in the myometrium and endometrium , and full rejection with massive necrosis and fibrosis by day 28 . having demonstrated that predominately neutrophils , macrophages , and cd4/cd8 t cells were responsible for acute rejection , csa was first tried as a potential immunosuppressant in a mouse utn model . 5 mice acted as control ( no csa given ) and 5 received either 10 or 20 mg / kg / day of csa . as expected apoptosis and inflammation were less prominent in grafts taken from the recipient mice that had received a higher csa dose . therefore , the authors concluded that administration of csa can evidently delay the progress of rejection of grafted uteri . however rather surprisingly , t - cell infiltration was not suppressed with the cd8 + count significantly higher in the two allogeneic groups receiving csa compared with the control group . this outcome may be explained by csa inhibition of activation - induced cell death ( aicd ) of cytotoxic t cells by major histocompatibility complex - peptide complexes [ 40 , 41 ] . a single - agent csa was effective for maintaining a uterus transplant in several nonhuman primate models . clinical signs , serum analysis , and ultrasound examination of the uterus and vascular flow through the anastomosis were all used to monitor rejection and graft viability . in the first , the target csa trough was 150 ng / ml , with a similar dose response to other solid organ transplants ( 5 mg / kg / day ; dose mean : 15.5 ng / ml ; 8 mg / kg / day mean : 191.8 59.3 ng / ml ) . however , during the second postoperative month , the following symptoms were reported : recipient weight loss and pelvic oedema . an apparent rejection episode was managed with additional immunosuppressants , high - dose steroids , and tacrolimus . on examination of the uterus a year after utn , minimal signs of rejection were revealed with intact anastomosis [ 1 , 43 ] . csa and tacrolimus were administered to the same sheep model with successful results . initially , tacrolimus was given for 12 days intravenously , followed by csa only orally . the uterus and ovary were harvested en bloc , flushed ex vivo , and transplanted in an orthotopic position by vascular anastomosis , either between individuals ( allotransplantation , n = 9 ) or back to the same animal ( autotransplantation , n = 7 ) . immunosuppression after allotransplantation consisted of administration of intravenous csa ( 25 mg / kg ) or oral tacrolimus ( 0.30.5 mg / kg ) for 3 weeks , and the animals were then examined and euthanized . autotransplanted ewes were evaluated at 7 weeks and then introduced to a ram ( n = 5 ) for assessment of transplant function . in allotransplanted animals the ovary and uterus showed normal appearance in 33% ( 2 of 6 ) of csa - treated and 66% ( 2 of 3 ) of tacrolimus - treated ewes at 3 weeks after transfusion . analysis of uteri that diverged from normal gross appearance showed different degrees of inflammation , infiltration of leukocytes and necrosis . in the autotransplanted group , two animals were sacrificed 7 weeks after transplantation showing normal uteri and ovaries . vaginal examination and analysis of serum progesterone confirmed satisfactory healing in the remaining 5 animals before a ram was introduced . four ewes mated and three conceived , bearing one twin and two singleton pregnancies with normal foeti at termination on day 90 ( n = 1 ) and day 140 ( n = 2 ) of pregnancy . in conclusion , a model for utn has been developed in the ewe with results indicating that immunosuppression by tacrolimus , rather than cyclosporine , is to be preferred in this model . furthermore , avison et al . ten transplants were performed , with 5 animals alive and healthy at the end of followup ( 0.512 months ) . immunosuppression given was as follows : 1020 mg / day of methylprednisolone for a month and oral csa ( 10 mg / kg / day ) as maintenance . rejection episodes during the 2nd and 3rd months after transplantation were treated successfully with increased doses of oral csa . the above findings suggest that csa administered at higher doses or used in combination with another type of immunosuppressant , as is the case in other organ transplants ( kidney , liver , and heart ) , would be an ideal way of preventing utn rejection . the effects of immunosuppressants on the maternal immune system have been well documented , and , therefore , viral serology for cytomegalovirus as well as microbiological cultures of vaginal smears repeated monthly may be helpful . levels of csa and other immunosuppressive drugs in the blood should be monitored , thus permitting adjustments of drug dose relative to graft function and physiological changes of pregnancy . importantly , the risks to mother and fetus will be not different from those faced by renal , hepatic , or cardiac transplant patients undergoing pregnancy . all utn patients should be managed within a multidisciplinary team setting , including a transplant immunologist . the care pathway would be very similar to any pregnant patient with a transplanted organ . visual inspection of the transplanted cervix would of course likely provide clinical clues of the graft 's condition . in order to create a controlled environment during the labour process , a caesarean section will be advisable . for csa to become the immunosuppressant of choice in utn , its role in pregnancy and , therefore , its effect on both mother and fetus would have to be better understood . we describe below the different ways in which csa can impact during the course of a pregnancy . the aim of organ transplantation is to cure or at least reverse end - stage organ failure . severe forms of both kidney and liver disease are associated with infertility . a significant proportion of all kidney and liver end - organ ( thought to be higher than 50% ) damage sufferers experience menstrual cycle - associated pathology , ranging from amenorrhoea to dysmenorrhoea and irregular cycles . it has been demonstrated that following successful solid organ transplantation , as the graft begins to slowly function , normal endocrine and fertility function returns to the patient , allowing her to attempt pregnancy [ 47 , 48 ] . both renal and liver transplanted women report a restoration of their ovarian and menstrual function within an average of six to ten months following transplantation [ 4951 ] . the hypothalamo - pituitary - ovarian axis in a transplant patient is thought to be functioning as normally as it would in a nontransplant female , with serum gonadotrophin and prolactin levels very similar in the two groups . higher levels of oestradiol are reported in renal graft patients in comparison to nontransplant women . therefore , restoration of fertility is often used as a variable to assess the function of the transplanted graft , with the two outcomes tending to mirror each other [ 47 , 53 ] . data on pregnancy outcomes of transplant patients is derived from voluntary registries , case reports , and retrospective centre studies . davison and baylis have summarised the outcomes of 2040 live births among female solid organ transplant recipients recorded in three major registries ( the european dialysis and transplantation association registry , the uk transplant pregnancy registry , and the national transplantation pregnancy registry ( ntpr ) in the usa ) . overall conclusions , particularly when analysing the ntpr data , indicate a higher incidence of maternal hypertension and preeclampsia in transplanted patients , with the number varying with the organ transplanted . ntpr has reported hypertension rates of 4773% in pregnant kidney - transplant recipients , a much higher rate than those demonstrated in pregnant women who have received liver , heart , or lung transplants . csa is known to induce hypertension , and its more frequent use in kidney transplantation may explain such a high percentage . armenti et al . [ 5658 ] revealed a hypertension incidence increase from 52% to 5663% when nonemulsified csa was administered and 6873% when emulsified csa ( neoral ) was used . a similar picture is found with preeclampsia whose incidence varies from 25 to 45% with csa use [ 5558 ] , with a third of all pregnant women receiving kidney or pancreas - kidney transplants reporting this diagnosis and only 25% of liver , heart , or lung recipients [ 18 , 46 , 52 , 59 ] . the two main markers of preeclampsia , hypertension and proteinuria , are found in many transplant patients ( especially kidney ) prior to pregnancy . furthermore , csa increases uric acid levels , a blood marker for diagnosing preeclampsia , and therefore causes it to be less reliable [ 18 , 60 ] . indirectly , they lead to premature membrane rupture and subsequent preterm delivery and low birth weight [ 56 , 57 , 60 ] . this may explain why up to half of all pregnancies in transplant recipients , especially with renal grafts , end in preterm delivery [ 18 , 46 , 61 , 62 ] . a dose must be low enough not to bring about any toxicity within the mother or the neonate but at the same time with respect to csa , there may be no perfect dose but a trial and error approach which accepts the considerable variation in circulating drug levels associated with the increase in a pregnant woman 's extracellular volume and altered pharmacokinetics . thomas et al . assessed the effects of pregnancy on csa levels in six renal allograft patients and found that , after adjusting for dose , five of the six patients had declines in csa level during pregnancy . frustratingly , there is very limited evidence , especially when searching for case - control studies of immunosuppressive levels in pregnant transplant women , to determine whether a drop in the blood concentration of an immunosuppressant below the prepregnancy level invariably leads to rejection . csa levels dropped during the pregnancy which was complicated by acute and chronic allograft rejection , resulting in irreversible loss of lung function . furthermore , according to ntpr outcomes , pregnant kidney - transplant recipients who took higher doses of csa before and during pregnancy maintained normal graft function in comparison to those patients who had smaller doses [ 18 , 65 ] . long - term data following long - term csa use with regards to allograft loss , maternal survival after pregnancy ( due to an increased risk of viral diseases and neoplasm secondary to immunosuppressants ) , and offspring outcomes of transplant recipients is rather limited . long - term outcomes focusing on physical and mental development of offspring and immunological and oncological pathologies later in life are necessary . the comparison of pregnancy outcomes in a single group of women before and after transplantation can be of use currently , only one study by klln et al . has carried out such work . it concluded that despite an initial increased risk for preeclampsia , growth restriction , preterm birth , and the risk of miscarriage , the odds of these 4 outcomes were all statistically similar before and after transplantation . in fact , smoking and mother 's comorbidity were the single most important determinants of fetal and neonatal well - being . the use of csa throughout pregnancy exposes the fetus to potential fetotoxic and teratogenic agents . csa , like other immunosuppressants , is required during the entire gestational period , with maintenance of appropriate csa dose . it must not be reduced without reason or even worse , believing that natural nonspecific maternal immunosuppression can prevent allogeneic graft rejection . any random reduction in csa dosing may lead to rejection of transplanted organs , with two maternal deaths reported as due to discontinuation of immunosuppressive medications during pregnancy . what makes this issue particularly challenging is the fact that such effects , if any , of csa on the neonate with regards to phenotype and growth may be difficult to determine and may not be obvious at birth . in addition , underling maternal comorbidity , together with coexisting use of other types of immunosuppression and/or general medication , may act as a confounding factor and can therefore confuse the issue of whether it is the actual immunosuppressant responsible for the maternal or fetal effects . first , the molecular targets of calcineurin inhibitors such as csa are found in other cells types , which means that csa may act on other tissues . . intrauterine csa exposure at each stage of development can induce permanent nephron and hepatic damage in the offspring . injected 30 mg / kg body weight of csa into pregnant mice on days 68 or 1012 of gestation . this dose did not raise the maternal mortality rate but histological examination revealed pathological alterations in the maternal thymus , liver , kidney , and spleen . interestingly , most changes had disappeared by 1 week following the last injection . with respect to pregnancy , csa reduced the number of viable embryos and increased the number of embryos resorbed . organogenesis was not affected by the drug but csa had an apparent embryotoxic effect . two studies by tendron - franzin et al . evaluated the impact of csa on embryonic renal development in a rabbit model [ 71 , 72 ] . initially , twenty - one pregnant rabbits were injected with 10 mg / kg body weight of csa for 5 days , either from days 1418 or days 2024 of gestation . the latter group demonstrated a reduced number of living pups , which were also growth - retarded . the former group exhibited normal fetal growth , and blood concentrations of csa matched human data . examinations of kidneys at birth suggested nephron mass and number reduction by 25 and 33% in both groups . despite compensatory adaptation of the existing nephrons , the second study was also conducted in a rabbit model to assess the long - term systemic and renal effects of a csa - induced ( 10 mg / kg body weight injected ) nephron reduction . csa intrauterine exposure led to ( a ) permanent nephron deficit , ( b ) glomerular , tubular and intrarenal haemodynamic dysfunction , ( c ) enlarged kidneys with numerous tubular and glomerular lesions , and ( d ) an endothelin - dependent systemic hypertension that worsened with age . this resulted in systemic hypertension and progressive chronic renal insufficiency in adulthood , suggesting that infants who are born to mothers treated with csa during pregnancy must have long - term surveillance of kidney function in the form of blood tests and imaging . for comparative purposes , we should emphasise that the dosages of csa given in the animal models are applicable to those used in humans . csa dosage depends on whether the drug is used at the induction stage or maintenance stage as well as on the institution and country . in the uk , adult dosage for induction therapy varies from 10 to 20 mg / kg . in humans , the story is rather different with no similar effects identified [ 73 , 74 ] . investigated renal function with the latter using inulin clearance , para - aminohippuric acid clearance , microalbuminuria , and electrolyte reabsorption rate in the offspring of transplant patients from birth to the age of seven . they concluded that , in children born to transplanted women taking csa , renal function develops normally despite prolonged exposure in utero . csa can cross the placenta and thus enter fetal circulation , easily detected in the placenta , amniotic fluid as well as fetal tissues . the level of csa in fetal blood tends to be half the csa level in the mother [ 75 , 79 ] but even this level is enough to pose significant immunosuppression to the fetus . higher levels in the placenta and umbilical cord than in maternal blood have been recorded [ 18 , 77 , 79 ] . according to the food and drug administration ( fda ) , csa belongs to the category c designation which indicates that human risk can not be ruled out because studies in humans are lacking and studies in animals are either positive for risk or lacking all together . until now , no consistent congenital malformations in the offspring of rodents exposed to csa during pregnancy have been noted ; however , a small number of reports have recorded : cataracts , growth delay , and fetotoxic effects at high doses [ 69 , 80 , 81 ] . there have been isolated reports of birth defects in humans but because of such small numbers and inconsistent patterns of defects it is difficult to conclude whether the incidence is secondary to csa exposure or another factor . the prevalence of major structural malformations in pregnant women without genetic history or disease is around 3 percent which was approximately the same as the figure reported by ntpr ( 4 - 5 percent ) . a meta - analysis of 15 studies by bar et al . concluded that csa does not appear to be a major human teratogen but may be associated with increased rates of prematurity . as discussed above , apart from downgrading the maternal immune system , csa manages to also enter fetal circulation during pregnancy . one would assume that this would result in csa interrupting normal fetal t - cell development . however , only a limited number of studies have included the fetal and neonatal immune system as an end - point . they suggest that immunosuppressants can have a profound effect on the development of an immune system . exposed pregnant mice to a calcineurin inhibitor ( csa and tacrolimus ) and , by doing so , brought about dysfunctional t - cell reactivity and prevented the generation of single positive mature t cells in newborn mice . drugs were administered during the final third of the gestational period which is an important time for immune development . also noticeable was the formation of hypoplastic peripheral lymphatic organs [ 84 , 85 ] . another similar study involving a rat model showed a series of immune perturbations including a decreased delayed type hypersensitivity response , splenic b cell number , and functional impairment during postnatal maturation . some have reported normal immunologic function in infants exposed to immunosuppressants in utero [ 87 , 88 ] . another study of continuous exposure of csa in utero in six infants born to female kidney transplant recipients demonstrated a seemingly impaired t- , b- , and nk - cell development and/or maturation ( reduction in numbers and expression of cd25 and hla - dr on t cells and cd5 on b cells ) , with most effects still apparent at one year . takhaashi et al . investigated the lymphocyte subpopulations in cord blood of six newborn infants born to mothers following renal transplantation . the number of b cells and the percentage of b cell in total mononuclear cells were significantly lower in these infants at one and three months of age , with no significant decrease between numbers of cd2 + , cd4 + , or cd8 + cells . this may suggest that the b - cell line is more sensitive to immunosuppressants in utero than the t - cell line . another threat posed by csa and immunosuppression of the fetal immune system in general is a risk of autoimmune disease in the offspring of transplant recipients [ 88 , 91 ] . in a particular study , csa was administered to pregnant mice to study the effects of passively transferred csa on the developing immune system which clearly altered the developing immune system . embryos were partially depleted of cd4 + cd8 cells and 11 of 50 offspring born to csa - treated mothers developed significant levels of igg autoantibodies to gastric antigens . two animals developed an extensive mononuclear cell infiltrate in the gastric mucosa resembling autoimmune gastritis . however , in the motherisk program in canada , the incidence of autoimmune diseases in the offspring of mothers with kidney transplants included one child with insulin - dependent diabetes mellitus and two children with asthma . part of the decision making process as to which immunosuppressant should be used depends on the effect of that immunosuppressant on pregnancy specific hormones . published the first study assessing reproductive health in animals exposed to immunosuppressive drugs ( csa ) in utero . their results demonstrated that direct maternal and in utero exposure to high doses of csa reduced implantation rates , fetal survival , and adolescent growth but did not affect offspring fertility . this correlation was dose dependent , with higher doses exhibiting a more pronounced negative effect . the reduced implantation rates have been reported in previous similar experiments involving murine models [ 69 , 70 ] . in a particular study by mason et al . , female lewis rats were given 1025 mg / kg / day of csa from the time of mating to 20 days after coitus . higher csa doses caused a powerful fetotoxic effect , resulting in a high incidence of fetal mortality or miscarriage , suggesting that csa can affect the endometrium or decidua in a negative way . in fact there is evidence which reveals a possible link between csa and suboptimal ovarian function , as a result of hindrance to sex steroid secretion at the time of ovulation [ 95 , 96 ] . this in turn leads to issues with conception which tends to occur at the time of ovulation . the endometrial layer depends on the sex steroids for its maintenance , and , if their secretion is impaired , the lining is also dysfunctional . as a result conception is impaired , an effect even more pronounced as a result of the role csa plays in both t - cell and nk - cell activation . finally , it is worth noting that the effect of csa upon the fetus continues after delivery if the mother breastfeeds . data regarding breast feeding by mothers taking immunosuppressive medication is still relatively sparse . according to the american academy of paediatrics , steroids such as prednisolone no recommendations regarding azathioprine or tacrolimus exist in the literature . measuring csa levels in breast milk is currently not a recognised tool as levels vary greatly between different transplant recipients , from undetectable to equal to those in the maternal serum . flechner et al . reported a term pregnancy in a csa and prednisone - treated female cadaveric renal allograft recipient . a male child , small for gestational age at 2370 g , was born at 38 weeks of gestation with neither congenital anomalies nor nephrotoxicity or hepatotoxicity . csa determined by a radioimmunoassay was present in the fetal circulation during gestation and maternal breast milk at similar concentrations to those in the mother . interestingly , fetal serum at birth displayed 25% suppression of a third - party mixed lymphocyte culture compared with control incubations , adding to the body of evidence which advises against the breastfeeding of children by csa - treated mothers . whether exposure to csa through breast milk poses a big enough risk to outweigh the benefits of breast feeding remains an unknown topic . in utn , maintaining a healthy pregnancy is the most important marker of graft function . monitoring of the patient and the fetus should be managed by a multidisciplinary team , including a transplant surgeon , high - risk obstetrician , and neonatologist . the pregnant transplant recipient must consult frequently and well in advance of the labour process , with the neonatologist and the paediatrician so that both the patient and team are prepared for any untoward outcomes . csa serum levels should be monitored closely with attention paid to graft function including visual inspection of the cervix . csa is a widely used immunosuppressant by transplant recipients , required to maintain adequate graft and maternal survival . its effect on the fetus and the variation of its influence upon the graft during pregnancy has been of major concern . continuing advances and modifications in csa therapy , coupled with an increasing number of successful pregnancy outcomes after all types of solid organ transplantation , have resulted in a greater confidence in csa use during pregnancy . when used in combination with other immunosuppressants , csa has demonstrated a more potent protective influence on the transplanted graft , with minimal side effects and a negligible teratogenic effect . finally , it is worth highlighting that despite the theoretical risks of csa and other types of immunosuppressants to mother and fetus , successful pregnancies are now considered the norm in transplant recipients . importantly , successful pregnancy is defined as one without evidence of malformations in the newborn , as well as worsening of graft function ( because of gestational factors or prepregnancy morbidity ) in comparison to the prepregnancy graft state .

uterine transplantation has been proposed as a possible solution to absolute uterine factor infertility untreatable by any other option . since the first human attempt in 2000 , various teams have tried to clarify which immunosuppressant would be most suitable for protecting the allogeneic uterine graft while posing a minimal risk to the fetus . cyclosporine a ( csa ) is an immunosuppressant widely used by transplant recipients . it is currently being tested as a potential immunosuppressant to be used during utn . its effect on the mother and fetus and its influence upon the graft during pregnancy have been of major concern . we review the role of csa in utn and its effect on pregnant transplant recipients and their offspring .

1. Background 2. Transplant Surgery 3. Immunosuppression in General 4. Cyclosporine 5. Use of Cyclosporine in Uterine Transplantation 6. Cyclosporine and Pregnancy 7. Conclusion

uterine transplantation ( utn ) has been proposed as a possible solution to absolute uterine factor infertility ( aufi ) untreatable by any other option . the inability to experience a pregnancy , give birth , and bring up a child because of infertility issues may be one of the most traumatic and devastating situations to affect a woman or couple , with the capacity to have a severely detrimental effect on the quality of life of both parties [ 24 ] . the term infertile is an all - encompassing term and includes women with aufi . tremendous advances have been made during the last decades in the fields of transplantation and reproductive medicine , in particular , the first reports of successful transplantation of a solid organ , the kidney [ 5 , 6 ] , and live birth after ivf . transplantation surgery today includes types of organ / tissue transplantation that will enhance the quality - of - life as exemplified by transplantation of the hand , the abdominal wall , the larynx , and the face , with the aim to add utn to the list . utn was first performed in a human in 2000 on a 26-year - old female who had previously lost her uterus as a result of postpartum haemorrhage . the event granted much needed impetus for research into utn , which since then has been a slow , methodical process within an animal setting involving multiple institutions and disciplines over several continents . the second utn attempt in a human model is anticipated in the not - too - distant future . with respect to the latter , it is important to recognise that without full understanding of the risks posed by the rejection process and subsequent development of immunosuppressants , transplantation as we understand it today would not be possible . 5-mercaptopurine / azathioprine , in combination with corticosteroids , was the first to be marketed for use . next came the discovery of the calcineurin inhibitor cyclosporine a ( csa ) in the late 1970s . at the time of the first healthy birth to a transplant recipient in 1958 , however , approximately 14,000 births ( more since this result was first published in 2002 ) among transplant women have been reported worldwide , resulting in a change of opinion , whereby restored fertility is now a recognized desirable outcome following transplantation [ 19 , 20 ] . currently short - term effects have been very positive but chronic rejection is still a major issue and long - term use has its drawbacks . deciding on the type , dose , and monitoring of immunosuppressants prescribed is best made within a multidisciplinary team setting including an obstetrician , so as to maximize the efficacy and minimize potential toxicity . as immunosuppressants can cross the human placental barrier and enter the fetal circulation , possibly affecting the immune system of the fetus , obstetric input is paramount . which immunosuppressant is administered depends very much on the age and comorbidities of the patient , type of transplanted organ , length of time that the patient is expected to live postoperatively , and the transplant centre . medications used to suppress immunologic activity are chosen based on the stage of the transplant process . in the induction phase , intravenous immunoglobulin is usually combined with a cornerstone immunosuppressant such as a cytokine modulator ( csa ) , purine - related analogue ( azathioprine ) , and a purine synthesis inhibitor ( mycophenolate mofetil ) . in addition , alkylating agents ( cyclophosphamide and chlorambucil ) and biologic response modifiers ( tnf antagonists ) can be used [ 15 , 24 ] . the first record of the use of immunosuppressants in pregnancy was in 1967 by board et al . the risk that immunosuppressants pose to both the mother and fetus , with respect to potential birth defects , needs to be fully investigated and understood . this is especially important as the number of female transplant recipients of child - bearing age is increasing . the main role of csa is to interfere with signaling pathways important for the clonal expansion of immunocompetent lymphocytes . it is activated following the rise of intracellular ca levels in response to t - cell receptor binding . calcineurin then dephosphorylates the transcription factor nf - at ( nuclear factor of activated t cells ) in the cytoplasm , allowing for its migration to the nucleus which leads to the induction of transcription of genes coding for il-2 , cd40 ligand , and fas ligand . therefore , by inhibiting the function of calcineurin , the csa - cyclophilin complex manages to block clonal t - cell proliferation in response to the host immune system recognizing a specific antigen as being foreign . research into utn restarted before its afore - mentioned milestone in 2000 , that is , the first human attempt . this attempt may have been premature , with little characterization or understanding of the rejection response prior to the operation . the first rejection episode occurred on the ninth postoperative day and the patient was treated by increasing the oral doses of csa and azathioprine and administering an intravenous dose of prednisolone . on the 99th day , histopathologic examination confirmed thrombosis of uterine vessels , and , interestingly , there was no signs of rejection with apparent viability of both tubes . to date , csa has not been used as an immunosuppressant in the few cases where the primary objective was to bring about pregnancy following utn . in october of the last year , an allogeneic uterine transplant reported resorbed pregnancies in 4 out of 5 pregnant rats , with only tacrolimus used as an immunosuppressant . however , csa has been tried as an immunosuppressant in utn studies where achieving pregnancy was not the primary objective . described below are all studies where csa was administered as an immunosuppressant in utn . having demonstrated that predominately neutrophils , macrophages , and cd4/cd8 t cells were responsible for acute rejection , csa was first tried as a potential immunosuppressant in a mouse utn model . 5 mice acted as control ( no csa given ) and 5 received either 10 or 20 mg / kg / day of csa . therefore , the authors concluded that administration of csa can evidently delay the progress of rejection of grafted uteri . in the first , the target csa trough was 150 ng / ml , with a similar dose response to other solid organ transplants ( 5 mg / kg / day ; dose mean : 15.5 ng / ml ; 8 mg / kg / day mean : 191.8 59.3 ng / ml ) . the uterus and ovary were harvested en bloc , flushed ex vivo , and transplanted in an orthotopic position by vascular anastomosis , either between individuals ( allotransplantation , n = 9 ) or back to the same animal ( autotransplantation , n = 7 ) . in allotransplanted animals the ovary and uterus showed normal appearance in 33% ( 2 of 6 ) of csa - treated and 66% ( 2 of 3 ) of tacrolimus - treated ewes at 3 weeks after transfusion . in conclusion , a model for utn has been developed in the ewe with results indicating that immunosuppression by tacrolimus , rather than cyclosporine , is to be preferred in this model . the above findings suggest that csa administered at higher doses or used in combination with another type of immunosuppressant , as is the case in other organ transplants ( kidney , liver , and heart ) , would be an ideal way of preventing utn rejection . the effects of immunosuppressants on the maternal immune system have been well documented , and , therefore , viral serology for cytomegalovirus as well as microbiological cultures of vaginal smears repeated monthly may be helpful . levels of csa and other immunosuppressive drugs in the blood should be monitored , thus permitting adjustments of drug dose relative to graft function and physiological changes of pregnancy . importantly , the risks to mother and fetus will be not different from those faced by renal , hepatic , or cardiac transplant patients undergoing pregnancy . the care pathway would be very similar to any pregnant patient with a transplanted organ . visual inspection of the transplanted cervix would of course likely provide clinical clues of the graft 's condition . for csa to become the immunosuppressant of choice in utn , its role in pregnancy and , therefore , its effect on both mother and fetus would have to be better understood . a significant proportion of all kidney and liver end - organ ( thought to be higher than 50% ) damage sufferers experience menstrual cycle - associated pathology , ranging from amenorrhoea to dysmenorrhoea and irregular cycles . it has been demonstrated that following successful solid organ transplantation , as the graft begins to slowly function , normal endocrine and fertility function returns to the patient , allowing her to attempt pregnancy [ 47 , 48 ] . the hypothalamo - pituitary - ovarian axis in a transplant patient is thought to be functioning as normally as it would in a nontransplant female , with serum gonadotrophin and prolactin levels very similar in the two groups . therefore , restoration of fertility is often used as a variable to assess the function of the transplanted graft , with the two outcomes tending to mirror each other [ 47 , 53 ] . davison and baylis have summarised the outcomes of 2040 live births among female solid organ transplant recipients recorded in three major registries ( the european dialysis and transplantation association registry , the uk transplant pregnancy registry , and the national transplantation pregnancy registry ( ntpr ) in the usa ) . csa is known to induce hypertension , and its more frequent use in kidney transplantation may explain such a high percentage . furthermore , csa increases uric acid levels , a blood marker for diagnosing preeclampsia , and therefore causes it to be less reliable [ 18 , 60 ] . a dose must be low enough not to bring about any toxicity within the mother or the neonate but at the same time with respect to csa , there may be no perfect dose but a trial and error approach which accepts the considerable variation in circulating drug levels associated with the increase in a pregnant woman 's extracellular volume and altered pharmacokinetics . assessed the effects of pregnancy on csa levels in six renal allograft patients and found that , after adjusting for dose , five of the six patients had declines in csa level during pregnancy . frustratingly , there is very limited evidence , especially when searching for case - control studies of immunosuppressive levels in pregnant transplant women , to determine whether a drop in the blood concentration of an immunosuppressant below the prepregnancy level invariably leads to rejection . furthermore , according to ntpr outcomes , pregnant kidney - transplant recipients who took higher doses of csa before and during pregnancy maintained normal graft function in comparison to those patients who had smaller doses [ 18 , 65 ] . long - term data following long - term csa use with regards to allograft loss , maternal survival after pregnancy ( due to an increased risk of viral diseases and neoplasm secondary to immunosuppressants ) , and offspring outcomes of transplant recipients is rather limited . the use of csa throughout pregnancy exposes the fetus to potential fetotoxic and teratogenic agents . any random reduction in csa dosing may lead to rejection of transplanted organs , with two maternal deaths reported as due to discontinuation of immunosuppressive medications during pregnancy . what makes this issue particularly challenging is the fact that such effects , if any , of csa on the neonate with regards to phenotype and growth may be difficult to determine and may not be obvious at birth . in addition , underling maternal comorbidity , together with coexisting use of other types of immunosuppression and/or general medication , may act as a confounding factor and can therefore confuse the issue of whether it is the actual immunosuppressant responsible for the maternal or fetal effects . injected 30 mg / kg body weight of csa into pregnant mice on days 68 or 1012 of gestation . evaluated the impact of csa on embryonic renal development in a rabbit model [ 71 , 72 ] . initially , twenty - one pregnant rabbits were injected with 10 mg / kg body weight of csa for 5 days , either from days 1418 or days 2024 of gestation . the former group exhibited normal fetal growth , and blood concentrations of csa matched human data . this resulted in systemic hypertension and progressive chronic renal insufficiency in adulthood , suggesting that infants who are born to mothers treated with csa during pregnancy must have long - term surveillance of kidney function in the form of blood tests and imaging . for comparative purposes , we should emphasise that the dosages of csa given in the animal models are applicable to those used in humans . csa dosage depends on whether the drug is used at the induction stage or maintenance stage as well as on the institution and country . investigated renal function with the latter using inulin clearance , para - aminohippuric acid clearance , microalbuminuria , and electrolyte reabsorption rate in the offspring of transplant patients from birth to the age of seven . the level of csa in fetal blood tends to be half the csa level in the mother [ 75 , 79 ] but even this level is enough to pose significant immunosuppression to the fetus . higher levels in the placenta and umbilical cord than in maternal blood have been recorded [ 18 , 77 , 79 ] . according to the food and drug administration ( fda ) , csa belongs to the category c designation which indicates that human risk can not be ruled out because studies in humans are lacking and studies in animals are either positive for risk or lacking all together . until now , no consistent congenital malformations in the offspring of rodents exposed to csa during pregnancy have been noted ; however , a small number of reports have recorded : cataracts , growth delay , and fetotoxic effects at high doses [ 69 , 80 , 81 ] . there have been isolated reports of birth defects in humans but because of such small numbers and inconsistent patterns of defects it is difficult to conclude whether the incidence is secondary to csa exposure or another factor . the prevalence of major structural malformations in pregnant women without genetic history or disease is around 3 percent which was approximately the same as the figure reported by ntpr ( 4 - 5 percent ) . concluded that csa does not appear to be a major human teratogen but may be associated with increased rates of prematurity . as discussed above , apart from downgrading the maternal immune system , csa manages to also enter fetal circulation during pregnancy . they suggest that immunosuppressants can have a profound effect on the development of an immune system . exposed pregnant mice to a calcineurin inhibitor ( csa and tacrolimus ) and , by doing so , brought about dysfunctional t - cell reactivity and prevented the generation of single positive mature t cells in newborn mice . drugs were administered during the final third of the gestational period which is an important time for immune development . another study of continuous exposure of csa in utero in six infants born to female kidney transplant recipients demonstrated a seemingly impaired t- , b- , and nk - cell development and/or maturation ( reduction in numbers and expression of cd25 and hla - dr on t cells and cd5 on b cells ) , with most effects still apparent at one year . another threat posed by csa and immunosuppression of the fetal immune system in general is a risk of autoimmune disease in the offspring of transplant recipients [ 88 , 91 ] . in a particular study , csa was administered to pregnant mice to study the effects of passively transferred csa on the developing immune system which clearly altered the developing immune system . part of the decision making process as to which immunosuppressant should be used depends on the effect of that immunosuppressant on pregnancy specific hormones . published the first study assessing reproductive health in animals exposed to immunosuppressive drugs ( csa ) in utero . their results demonstrated that direct maternal and in utero exposure to high doses of csa reduced implantation rates , fetal survival , and adolescent growth but did not affect offspring fertility . the reduced implantation rates have been reported in previous similar experiments involving murine models [ 69 , 70 ] . , female lewis rats were given 1025 mg / kg / day of csa from the time of mating to 20 days after coitus . in fact there is evidence which reveals a possible link between csa and suboptimal ovarian function , as a result of hindrance to sex steroid secretion at the time of ovulation [ 95 , 96 ] . the endometrial layer depends on the sex steroids for its maintenance , and , if their secretion is impaired , the lining is also dysfunctional . as a result conception is impaired , an effect even more pronounced as a result of the role csa plays in both t - cell and nk - cell activation . finally , it is worth noting that the effect of csa upon the fetus continues after delivery if the mother breastfeeds . according to the american academy of paediatrics , steroids such as prednisolone no recommendations regarding azathioprine or tacrolimus exist in the literature . measuring csa levels in breast milk is currently not a recognised tool as levels vary greatly between different transplant recipients , from undetectable to equal to those in the maternal serum . csa determined by a radioimmunoassay was present in the fetal circulation during gestation and maternal breast milk at similar concentrations to those in the mother . interestingly , fetal serum at birth displayed 25% suppression of a third - party mixed lymphocyte culture compared with control incubations , adding to the body of evidence which advises against the breastfeeding of children by csa - treated mothers . whether exposure to csa through breast milk poses a big enough risk to outweigh the benefits of breast feeding remains an unknown topic . in utn , maintaining a healthy pregnancy is the most important marker of graft function . monitoring of the patient and the fetus should be managed by a multidisciplinary team , including a transplant surgeon , high - risk obstetrician , and neonatologist . the pregnant transplant recipient must consult frequently and well in advance of the labour process , with the neonatologist and the paediatrician so that both the patient and team are prepared for any untoward outcomes . csa is a widely used immunosuppressant by transplant recipients , required to maintain adequate graft and maternal survival . its effect on the fetus and the variation of its influence upon the graft during pregnancy has been of major concern . continuing advances and modifications in csa therapy , coupled with an increasing number of successful pregnancy outcomes after all types of solid organ transplantation , have resulted in a greater confidence in csa use during pregnancy . when used in combination with other immunosuppressants , csa has demonstrated a more potent protective influence on the transplanted graft , with minimal side effects and a negligible teratogenic effect . finally , it is worth highlighting that despite the theoretical risks of csa and other types of immunosuppressants to mother and fetus , successful pregnancies are now considered the norm in transplant recipients . importantly , successful pregnancy is defined as one without evidence of malformations in the newborn , as well as worsening of graft function ( because of gestational factors or prepregnancy morbidity ) in comparison to the prepregnancy graft state .

one ambition of synthetic biology is the creation of man - made enzymes with prescribed physical characteristics and performance specifications engineered for work in vitro and eventually in living cells . as with any practical machine , a natural enzyme molecule comprises a consortium of component parts reflecting production , assembly , and service engineering that supports the functionally active parts . the sharing of residues , structural motifs , and domains by these parts contributes to the complexity of natural proteins . this complexity is amplified by the unplanned modifications from repeated blind natural selection over evolutionary time , which confounds reliable extraction of essential elements of natural enzyme activity for translation into man - made reproductions . our approach to the creation of man - made oxidoreductases and related proteins thus seeks to minimize complexity by combining first principles established for protein folding with those for engineering electron - transfer and oxidation reduction systems . we avoid the common practice of designing man - made proteins by mimicking natural proteins . our method to functionalize oxidoreductase maquettes endeavors to keep the cofactor assembly process much simpler than is evident in natural systems . in the cell , the import and ligation of the large , complex mg / zn and fe tetrapyrrole structures of chlorins and hemes , respectively , of photosynthesis , respiration , and oxidative and reductive metabolism employ elaborate engineering to regulate tetrapyrrole production and delivery into the apoprotein . in the laboratory , the analogous in vitro equipping of man - made apoprotein designs with tetrapyrroles has to date been a matter of empirically finding satisfactory conditions of mixing tetrapyrroles in organic solution with the apoprotein in aqueous media with minor attention to the essential details of the assembly process . as maquettes , man - made 4--helical proteins designed with simple oxidoreductase functions that match key characteristics of their natural counterparts , continue to advance to include multi - cofactor combinations and more sophisticated catalysis , it becomes important to learn how the assembly of tetrapyrrole and protein proceeds and to develop strategies to ensure competent integration of all cofactors into a designed host protein . the staged , step - by - step developmental approach toward construction of heme maquettes displaying oxidoreductive activities has generated an extensive family of related 4--helical proteins . five of these are selected for their markedly different oligomeric and topological forms and the degree of structuring of their apo and holo heme - ligated states ( table 1 ) . together with the bacterial 4--helix hemoprotein cytochrome b562 , they offer a useful palette with which to determine the time scales and barriers governing tetrapyrrole partition from aqueous medium into the hydrophobic interior of the protein followed by positioning at a preselected location by bis - his ligation . in the present work , we begin comparing tetrapyrrole binding using the common natural cofactor fe - protoporphyrin ix ( heme b ) , which binds to all the selected proteins with similar nanomolar affinities ( dissociation constants , kd ) . the study is then extended beyond heme b to examine time scales of binding of a series of related heme analogues differing in peripheral substitutions and hydrophobicity to clarify the influence of cofactor physical chemistry on the assembly process . absence of structure refers to nmr characterization of a molten globular state for the five maquettes and a random coil for the natural cytochrome b562 . iron protoporphyrin ix ( hemin ) and the other porphyrins were purchased from frontier scientific . all porphyrins were dissolved in dimethyl sulfoxide ( dmso ) with the concentration determined via weight , except for hemin whose concentration was determined via the hemochrome assay utilizing the extinction coefficient at 556 nm . proteins e and f were prepared at 0.1 mmol scale on a cem liberty microwave peptide synthesizer using standard fluorenylmethyloxycarbonyl ( fmoc)/tbu protection protocols . the side chain protecting groups were as follows : cys ( trt ) , lys ( boc ) , his ( boc ) , and asp / glu ( otbu ) . after synthesis , the protein was cleaved from the resin by incubating it with a mixture of trifluoroacetic acid ( tfa ) , ethanedithiol , anisole , and thioanisole in a 9:0.2:0.5:0.3 ratio for 2.5 h protected from light . after excess reagent removal via rotovap , protein was precipitated with methyl t - butyl ether and purified using a reverse - phase waters hplc equipped with a c18 column running a linear gradient of acetonitrile with 0.1% ( v / v ) tfa and water with 0.1% ( v / v ) tfa . the genes for the protein were ordered from dna2.0 containing a his - tag , a linker with a tev site , and the protein in pj414 vector . dna was transformed into bl-21de3 strain escherichia coli cells ( new england biolabs ) . these bl-21de3 cells were grown in 4 l of media ( 12 g / l bacto - peptone , 24 g / l bacto - yeast extract , 4 ml / l glycerol , and 14 g / l kh2po4 , ph 7.5 ) until the od600 was at 0.6 au . overexpression was induced by addition of iptg to a final concentration of 1 mm . the cells overexpressed the protein for 4.5 h and were spun down to a pellet . the pellet was then resuspended in lysis buffer ( 300 mm nacl , 50 mm nah2po4 , 10 mm imidazole , ph 7 ) and sonicated at an amplitude of 90 ( 5 times , 20 s each time ) in a misonix sonicator ( s-4000 with microtip attachment ) . the histidine tag was removed by incubating the eluate with tev protease ( 1 mm dithiothreitol , 1 unit tev enzyme purchased from invitrogen per 100 l volume , overnight ) and rerun on the histrap column to separate cleaved from uncleaved . the flowthrough of this second column was dialyzed into n - cyclohexyl-2-aminoethanesulfonic acid ( ches ) buffer ( 20 mm ches , 150 mm kcl , ph 9 ) . the concentration for all protein variants was determined by the monitoring absorbance at 280 nm using an extinction coefficient of 22 500 m cm . all proteins in this paper were purified by hplc , and the masses were verified by maldi as described above ( supplemental figures 112 ) . millisecond time scale measurements were made on an olis rsm 1000 stopped flow spectrophotometer , which takes a full visible spectrum every millisecond . the tetrapyrrole and protein were added to separate syringes and shot together , and the absorbance was monitored in a 2 cm flow cell . temperature was controlled with a fischer - scientific isotemp 3031 water bath attached . all experiments were performed in triplicate , and the data were averaged together for further analysis . initial rates were determined by fitting the first 20 ms to a linear regression and dividing this slope by the extinction coefficient . pseudothermodynamic parameters of the transition state were derived using eq 1 for the temperature - dependent rates.1where k is the rate of the reaction at a certain temperature , kb is boltzmann s constant , t is the temperature in kelvin , and h is planck s constant ; is the transmission coefficient , which is set to 1 for all reactions in this paper , indicating all reactions that reach the transition state proceed to completion . s is the entropy change between the transition state and the reactants , h is the enthalpy change between the transition state and the reactants , and r is the gas constant in units of cal k mol . the linearized form ( eq 2 ) is used in the eyring plot of figure 2:2 a varian cary-500 spectrophotometer measured slower than millisecond kinetics . partition coefficients were determined by first dissolving the chromophore in 1-octanol and taking a spectrum . this solution was then mixed with an equal volume 50 mm ches , 150 mm kcl buffer , ph 9 , vortexed , and allowed to sit for 1 h at room temperature . the concentration of porphyrin in the aqueous layer was calculated as the concentration in octanol before incubation minus the concentration in octanol after incubation . the partition coefficient ( log p ) was then determined as the log of the ratio of the concentration of porphyrin in the octanol layer to the aqueous layer . there are many structural factors that can govern speed of cofactor assembly in proteins . in this work , we focus on the effect of structural elements at the higher tertiary and quaternary levels without changing the lower primary and secondary structural elements . specifically , our maquette selections maintain the same positioning of bis - his heme ligation sites and the similarity of surrounding amino acids ( see supporting information table 1 ) . the -helical secondary structure content of maquettes remains similar and comparable to b562 ( see table 1 ) . differences at the tertiary and quaternary levels for our set of six selected comparison sequences are illustrated in table 1 . the tertiary structure is altered by ( a ) helical constraint via connecting loops , ( b ) the ability or inability of helices to orient parallel versus antiparallel ( syn vs anti conformations ) , and ( c ) the relative mobility or rigidity of interior amino acids packing between helices in the apo state , as assayed by nmr and x - ray crystallography . to test for the effects of differences in quaternary structure , we vary the nature of the connection of the helical pair units , that is , whether or not they are linked to other helical pair units through covalent loops and/or disulfide bonds and whether helical pair units associate tightly or relatively loosely in the apo state . specifically , maquettes b , c , and d have identical helical sequences but differ in the number and position of interhelix loops and the presence or absence of a disulfide link between loops . maquettes d , e , and f are unlinked and capable of dissociating into separate helical pair subunits and associating in two distinct syn or anti forms based on the relative positions of the connecting loops to one another ( table 1 ) though remain as 4-helix units when in the apo state . in this regard , maquette d has the greatest tendency to dissociate under the conditions used here . table 1 shows that , despite the differences in tertiary and quaternary structure evident in the set of proteins , all sequences display kd values for heme binding in the tight nanomolar range , which makes the binding effectively irreversible . the heme binding reaction is monitored by a characteristic spectral shift of the ferric heme soret absorption from 395 nm in the aqueous phase to 412 nm on bis - his ligation . the heme binding process is straightforward and the heme - bound state stable beyond 20 h. under all experimental conditions used , the assembly process can be regarded as a movement of the heme b from the aqueous phase into the apoprotein and ending after the heme has found and bis - his - ligated at the site . figure 1a presents time courses of heme b ligation to cytochrome b562 ( a ) and maquettes b f under comparable conditions of one heme b per ligation site . rate constants at 30 c ( figure 1 ) are summarized in table 2 . heme b ligation to the six proteins covers a broad range of time , proceeding to completion following time courses characteristic of a second - order ( figure 1b ) , but not a first - order , reaction with equimolar reactants ( supplementary figures 13 and 14 ) . the linear dependence of the initial rate of binding for 20 m excess heme combined with variable protein concentrations of 1 , 2 , 3 , and 4 m confirmed a second - order process in all cases except for the untethered d , which was ambiguous ( supplementary figure 15 ) . similarly , the linear dependence of the initial rate for 30 m excess maquette b protein with variable heme b concentrations of 6 , 9 , 12 , and 18 m confirmed a second - order process ( supplementary figure 16 ) . the single - chain monomer maquette b , unstructured in the apo state and structured in the holo state , displays the most rapid time course of heme ligation , proceeding to completion on a subsecond time scale with a second - order rate constant of 3.04 10 m s ( see table 2 and figure 1 ) , the same rate as natural protein a. rates for binding heme at different sites are equivalent for this maquette , as well ( supplemental figure 17 ) . all data were collected at 30 c in 20 mm ches , 150 mm buffer , ph 9 . ( b ) heme binding of maquette a at various temperatures fit to both a first - order ( dashed lines ) and second - order ( solid lines ) kinetic time course . the bold / italic numbers denote the predominant barrier for each protein . compared to natural protein a , the temperature dependence for heme binding to single - chain maquette b ( presented in eyring plots of figure 2 ) balances a slightly smaller enthalpic ( 10.6 vs 11.5 kcal / mol ) with a slightly larger entropic ( 14.0 vs 13.0 kcal / mol at 30 c ) component . despite markedly different primary sequences , natural sequence a and maquette b appear to present a common barrier height to heme entry , ligation , and establishment of a singular structure . eyring plot of the temperature dependence of the initial rates of the various proteins studied . a ( gray ) , b ( green ) , c ( red ) , and e ( pink ) share similar slopes and intercepts of their temperature dependence . f ( black ) has a higher transition state enthalpy of formation and d ( blue ) a higher transition state entropy of formation : 20 mm ches , 150 mm kcl buffer , ph 9 ; protein at 5 m , heme at 10 m . dimeric maquette c shares the same sequence as monomeric maquette b and also assumes a singular structure in the holo state but has a different tertiary topology of helical tethering that involves removal of the loop connecting helices 2 and with the addition of a disulfide bond connecting the loops at the opposite end of the bundle ( table 1 ) . the maquette c has a more exaggerated enthalpic and entropic counterbalancing ( 8.7 and 16.2 kcal / mol ) with a net effect of only a 2-fold slowing of the second - order rate constant . the enhanced mobility at one end of the helical bundle in c appears to increase the entropic term while lowering the activation enthalpy . the site farthest away from the tether ( h42f ) assembles at a rate 3 times faster than the site nearest the tether ( h7f ) . this could also be due to a combination of cofactor access to the indivdual sites and differences in the level of apo structure of maquette c. this also manifests in the affinity , showng two separate kd values . the simple removal of the disulfide tether in c to create the untethered helix loop helix homodimer d substantially lowers the second - order rate constant by 50-fold . the eyring plot reveals a dramatically different rate - limiting barrier for the dominant slow phase . figure 2 and table 2 show that the heme b ligation process for d slows slightly with increasing temperature , yielding a modest enthalpic term of 1.54 kcal / mol , with a dominating entropic term ( 28.8 kcal / mol ) . this suggests a distinctly different rate - limiting process that may be responding to the freedom of the apo - maquette helical pairs to pack parallel or antiparallel in a syn or anti conformation and/or the repelling effect of the high net charge on each of the homodimers ( 8 charge per one helix loop the untethered homodimer e with neutral net charge has a similar enthalpic but larger entropic contribution than natural sequence a. this slows the second - order rate constant 10-fold . although also net - neutral charge , the untethered homodimer f , the only sequence that is structured ( x - ray and nmr ) in the apo form , reverses the thermodynamic trend , raising the enthalpic term ( 17.9 kcal / mol ) and lowering the entropic term ( 8.7 kcal / mol ) , for a net slowing of the binding rate by 40-fold . these results separate d and f from the others as clearly rate - limited , respectively , by diffusion between heme b and the weakly coupled homodimers or a higher thermal activation barrier to access the heme into a singular structured apoprotein . in complementary trials , we kept the maquette sequence constant ( b ) but varied the fe - porphyrin to test whether the transfer of cofactor from the aqueous phase into the maquette interior limits the rapid time course of ligation and to assess the effect of porphyrin aggregation in aqueous solution . figure 3a presents a family of synthetic fe - porphyrins with marked variance in their overall degree of hydrophobicity or hydrophilicity and in the pattern of polar and nonpolar groups around the porphyrin macrocycle ( affinity data in supplemental table 2 ) . we used [ n - octanol]/[water ] partition coefficients p(24,34 ) as a guide to the effect of the porphyrin macrocycle substitution on the relationship between the aqueous phase and the hydrophobic interior of the maquette b ( figure 3a ) . figure 3b displays typical spectra of three heme variants covering the experimental range of log p values at the concentrations of aqueous solutions used for the binding kinetic measurement . we assayed the solubility of the hemes and their tendency to form multimers in aqueous solution by uv visible spectroscopy . in the left panel of figure 3b , the visible spectrum of heme in ph 9 aqueous buffer ( green ) is dominated by the broad 350400 nm soret peak and the 605 nm peak characteristic of the dimer described by asher et al . the approximately 0.5 m aqueous dimer to monomer dissociation constant reported by this group indicates that a substantial fraction of heme monomer is also present . there is no indication of the -oxo heme dimer , reported decades ago by brown et al . the left panel also shows that heme dissolved in dmso ( black ) has the narrow soret absorption band at 405 nm characteristic of heme in monomeric form . the soret absorption remains narrow and red shifts to 412 nm on bis - his ligation in the maquette ( blue ) . heme variants with higher log p , such as protoporphyrin ix dimethyl ester , show the broad spectra of multimer formation in aqueous solution ( figure 3b , middle ) , while hemes equipped with highly polar substituents and low log p values ( diacetyl deuteroporphyrin ix ) exhibit simple monomeric solutions in water ( figure 3b , right ) . ( a ) various protoporphyrin ix analogues used to depict the effect of changing the ring substituents on the assembly rate . partition coefficient ( log p ) ( b ) spectra of three selected porphyrin in dmso ( black ) , 20 mm ches , 150 mm kcl , ph 9 , buffer ( green ) , and bound to protein b ( blue ) . heme b ( left ) , protoporphyrin ix dimethyl ester ( middle ) , and diacetyl deuteroporphyrin ix ( right ) . figure 4 shows time courses of binding of members of the family of fe - porphyrins to maquette b , while figure 5 summarizes the effect of log p on the binding rate ( linear time scale in supplemental figure 18 ) . compared to heme b , the slightly less polar deuteroporphyrin and mesoporphyrin bind to maquette b several - fold faster . on the other hand , making the fe - porphyrin more polar by substituting the heme b vinyls with structurally comparable nitriles ( 2,6-dinitrileporphyrin ) or adding polar hydroxyls ( isohematoporphyrin ) or carbonyls ( diacetyl deuteroprotoporphyrin ) slows the rate of binding . this suggests that the rate - limiting step of heme ligation to maquette b lies in the initial steps of partitioning into the hydrophobic interior of the maquette . assembly kinetics for the above series of fe - porphyrins with the monomer protein fit to a second - order reaction ( orange line ) . rates were collected upon rapid mixing of protein b ( 24 m ) with porphyrin ( 5 m ) at 25 c in 20 mm ches , 150 mm kcl buffer , ph 9 . comparison of protein b assembly rates with n - octanol / water log p values at ph 9 for the above series of fe - porphyrins . on the other hand , increasing the log p above a threshold value of about 0.75 by either removing the heme b propionates ( etioporphyrin ) or esterifying them ( protoporphyrin ix dimethyl ester ) leads to greater spectral evidence of porphyrin aggregation and significantly lowers the binding rates . this could reflect a lowering of the heme monomer concentration free in solution and/or a limiting rate of porphyrin disaggregation . scheme 1 summarizes the hurdles that can stand in the way of fe - porphyrin cofactor self - assembly into protein , starting in an aqueous medium and ending in a binding site in the interior of a protein that provides strong , effectively irreversible bishistidine ligation for the heme iron . the first barrier is associated not with the protein but with the polarity balance and aggregation state of the fe - porphyrin cofactor . increased log p , indicating higher hydrophobic character , shifts the equilibrium toward the aggregated fe - porphyrin in solution , a form unable to ligate to the proteins . decreased log p , indicating an increase in hydrophilic character , slows the rate by making partition of the unligated fe - porphyrin in the hydrophobic core less favorable . heme association across our range of maquettes shows assembly on the 100 ms to tens of seconds time scale , much faster than the tens of minutes time scale reported by kuzelova et al . kuzelova interpreted the observed rates around 0.001 s to reflect rate - limiting heme dimer dissociation . our distinctly faster assembly rates with all maquettes suggest that heme dimer dissociation is in fact much faster . while the assembly of most heme variants is well fit by a second - order time course , fe - tetracarboxyphenyl porphyrin ( tcp ) displays unusual biphasic kinetics , with a burst phase extent of 20% of unknown origin ( supplemental figure 19 ) . this may reflect some heterogeneity in the tcp dimer charge resulting from the pk values of 9.6 reported by stong et al . the various proteins are limited in their assembly by either entropic or enthalpic barriers related to protein mobility , while the porphyrin is limited by amphiphilicity . the entropic barrier of d is shown as flipping in this scheme , while f is shown as a tightly packed cartoon that opens up for assembly . splaying open ( e and c ) is used as a pictorial representation of an open state capable of heme binding . a and b are not thought to be limited substantially by the protein structure . the first way is to impose a large entropic reorganization barrier , best demonstrated by protein d. entropic barriers associated with minimal temperature dependence of the rate is a characteristic of structural rearrangements . helix halves associating from solution to form the four - helix dimer that favorably buries the hydrophobic core of each monomer and enables heme binding . at extremely low nanomolar concentrations of protein however , at micromolar concentrations , there is a conspicuous rapid burst phase of heme binding that indicates preformed dimers are available . on the other hand , the untethered monomers are free to associate in a helical bundle geometry ( syn ) that does not bring the histidines close enough together to form a bis - his heme ligation site . similar geometry changes are seen in the h10s24 protein maquette of grosset et al . her work clearly shows a protein capable of reorganizing between syn and anti conformations on seconds time scale initiated by a change in heme redox states . in the case of maquette d , conformational change would impose a barrier wherein the maquette must search structural space in order to find one state suitable for cofactor assembly . the addition of a simple tether , the disulfide bond linking loops in c , for example , can restrict the conformational freedom of the heme binding segments such that the two monomers can not rearrange or diffuse away from one another , lowering the entropic barrier to moderate levels and speeding the rate of binding ( table 2 ) . protein c still retains residual conformational freedom associated with the smaller entropic barrier at the unlinked end of the bundle , while the increased structuring imposed by the tether adds to an enthalpic barrier . the second way protein structure can slow cofactor self - assembly is to impose a large enthalpic barrier associated with apoprotein structuring . protein f is highly structured in the apo state as seen in the crystal structure . this structuring interferes with heme entry to the core as various packing interactions and internal hydrogen bonds must be broken . such structural destabilization requires substantial amounts of thermal energy , thus giving protein f its strong temperature dependence . these foregoing conclusions clearly demonstrate the power of maquettes to uncover the relevant physical chemical principles of protein design without complexity brought on by evolution . the stepwise redesign of maquettes from early multichain forms to a final single chain form that are sufficiently malleable in the apo state has increased the rate of cofactor self - assembly to a point comparable to natural proteins and limited by the physical chemical properties of the porphyrin itself . like the maquette b , natural cytochromes b562 and b5 and oxygen transport proteins such as myoglobin are predominantly unstructured in the apo state , becoming substantially more structured when heme is bound . in all cases , there is a small amount of structure in the apo state that restricts the conformational freedom of the unstructured regions . apocytochrome b562 retains two intact helices , while the helices that bind heme are random coil . moreover , in apocytochrome b5 , only a small region has any secondary structure , while the majority of the protein is random coil and unstructured . though myoglobin is expressed as a single - chain protein , there is no published apo structure to reveal if there are minor structural elements restricting the conformational freedom . despite this , nmr analysis of apomyoglobin has allowed eliezer and wright to construct a theoretical model of this protein without heme b showing , when combined with other literature describing its folding , that it too adheres to the principles outlined above : a conformationally restricted structure in which the binding site is malleable to allow access to heme b. the principles outlined in this article agree with the work of shoemaker and wolynes who describe the effect of molecular disorder on the binding of cofactors . the authors from this work predict that disordered apo states have greater rates of binding in part due to their capture radius , the distance at which the protein will come into contact with a binding partner . this concept , referred to as the fly casting mechanism , can be easily applied to the maquettes in this paper owing to helical fraying and the molten globular nature of the apo state . however , difficulty in measuring structure in any localized fashion , due to the repetition in maquette sequence and minimal change in cd signal between apo and holo states , makes proving this difficult . beyond insights into natural proteins , the principles here can be readily applied to the design of cofactor binding proteins . rate limitations can be uncovered through the use of temperature dependencies , and the structure can be addressed accordingly based on the information above . optimizing the ability of proteins to self - assemble with cofactors rapidly is as important to natural expression as increasing the affinity . rapid cofactor binding can be of critical importance in the cell for cofactor modification and customization . it also minimizes the cytotoxicity of free heme b , which generates reactive oxygen species when unbound in solution . future man - made protein designs that seek to integrate with and exploit natural biochemical pathways in vivo will benefit by using these principles to diminish assembly barriers and effectively compete with host natural proteins for available cofactors described in this paper . indeed , recent work utilizing a variant of maquette b has already been shown to co - opt the natural c - type machinery to covalently link heme b to an artificial protein .

timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes , including the oxidoreductases and allied transport and signaling proteins . likewise , ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man - made , first - principle protein constructs intended to operate in vitro or in vivo . as one of the most common catalytic cofactors in biology , we have chosen heme b , along with its chemical analogues , to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4--helix proteins . we compare five elementary synthetic designs ( maquettes ) and the natural cytochrome b562 that differ in oligomeric forms , apo- and holo - tertiary structural stability ; qualities that we show can either assist or hinder assembly . the cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic . with progressive removal of identified barriers , we achieve maquette assembly rates as fast as native cytochrome b562 , paving the way to in vivo assembly of man - made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways .

Introduction Materials and Methods Results Discussion

one ambition of synthetic biology is the creation of man - made enzymes with prescribed physical characteristics and performance specifications engineered for work in vitro and eventually in living cells . this complexity is amplified by the unplanned modifications from repeated blind natural selection over evolutionary time , which confounds reliable extraction of essential elements of natural enzyme activity for translation into man - made reproductions . our approach to the creation of man - made oxidoreductases and related proteins thus seeks to minimize complexity by combining first principles established for protein folding with those for engineering electron - transfer and oxidation reduction systems . we avoid the common practice of designing man - made proteins by mimicking natural proteins . our method to functionalize oxidoreductase maquettes endeavors to keep the cofactor assembly process much simpler than is evident in natural systems . in the cell , the import and ligation of the large , complex mg / zn and fe tetrapyrrole structures of chlorins and hemes , respectively , of photosynthesis , respiration , and oxidative and reductive metabolism employ elaborate engineering to regulate tetrapyrrole production and delivery into the apoprotein . in the laboratory , the analogous in vitro equipping of man - made apoprotein designs with tetrapyrroles has to date been a matter of empirically finding satisfactory conditions of mixing tetrapyrroles in organic solution with the apoprotein in aqueous media with minor attention to the essential details of the assembly process . as maquettes , man - made 4--helical proteins designed with simple oxidoreductase functions that match key characteristics of their natural counterparts , continue to advance to include multi - cofactor combinations and more sophisticated catalysis , it becomes important to learn how the assembly of tetrapyrrole and protein proceeds and to develop strategies to ensure competent integration of all cofactors into a designed host protein . five of these are selected for their markedly different oligomeric and topological forms and the degree of structuring of their apo and holo heme - ligated states ( table 1 ) . together with the bacterial 4--helix hemoprotein cytochrome b562 , they offer a useful palette with which to determine the time scales and barriers governing tetrapyrrole partition from aqueous medium into the hydrophobic interior of the protein followed by positioning at a preselected location by bis - his ligation . in the present work , we begin comparing tetrapyrrole binding using the common natural cofactor fe - protoporphyrin ix ( heme b ) , which binds to all the selected proteins with similar nanomolar affinities ( dissociation constants , kd ) . absence of structure refers to nmr characterization of a molten globular state for the five maquettes and a random coil for the natural cytochrome b562 . iron protoporphyrin ix ( hemin ) and the other porphyrins were purchased from frontier scientific . after synthesis , the protein was cleaved from the resin by incubating it with a mixture of trifluoroacetic acid ( tfa ) , ethanedithiol , anisole , and thioanisole in a 9:0.2:0.5:0.3 ratio for 2.5 h protected from light . the genes for the protein were ordered from dna2.0 containing a his - tag , a linker with a tev site , and the protein in pj414 vector . the pellet was then resuspended in lysis buffer ( 300 mm nacl , 50 mm nah2po4 , 10 mm imidazole , ph 7 ) and sonicated at an amplitude of 90 ( 5 times , 20 s each time ) in a misonix sonicator ( s-4000 with microtip attachment ) . all proteins in this paper were purified by hplc , and the masses were verified by maldi as described above ( supplemental figures 112 ) . the tetrapyrrole and protein were added to separate syringes and shot together , and the absorbance was monitored in a 2 cm flow cell . all experiments were performed in triplicate , and the data were averaged together for further analysis . pseudothermodynamic parameters of the transition state were derived using eq 1 for the temperature - dependent rates.1where k is the rate of the reaction at a certain temperature , kb is boltzmann s constant , t is the temperature in kelvin , and h is planck s constant ; is the transmission coefficient , which is set to 1 for all reactions in this paper , indicating all reactions that reach the transition state proceed to completion . the partition coefficient ( log p ) was then determined as the log of the ratio of the concentration of porphyrin in the octanol layer to the aqueous layer . there are many structural factors that can govern speed of cofactor assembly in proteins . in this work , we focus on the effect of structural elements at the higher tertiary and quaternary levels without changing the lower primary and secondary structural elements . specifically , our maquette selections maintain the same positioning of bis - his heme ligation sites and the similarity of surrounding amino acids ( see supporting information table 1 ) . to test for the effects of differences in quaternary structure , we vary the nature of the connection of the helical pair units , that is , whether or not they are linked to other helical pair units through covalent loops and/or disulfide bonds and whether helical pair units associate tightly or relatively loosely in the apo state . specifically , maquettes b , c , and d have identical helical sequences but differ in the number and position of interhelix loops and the presence or absence of a disulfide link between loops . maquettes d , e , and f are unlinked and capable of dissociating into separate helical pair subunits and associating in two distinct syn or anti forms based on the relative positions of the connecting loops to one another ( table 1 ) though remain as 4-helix units when in the apo state . the heme binding reaction is monitored by a characteristic spectral shift of the ferric heme soret absorption from 395 nm in the aqueous phase to 412 nm on bis - his ligation . the heme binding process is straightforward and the heme - bound state stable beyond 20 h. under all experimental conditions used , the assembly process can be regarded as a movement of the heme b from the aqueous phase into the apoprotein and ending after the heme has found and bis - his - ligated at the site . figure 1a presents time courses of heme b ligation to cytochrome b562 ( a ) and maquettes b f under comparable conditions of one heme b per ligation site . heme b ligation to the six proteins covers a broad range of time , proceeding to completion following time courses characteristic of a second - order ( figure 1b ) , but not a first - order , reaction with equimolar reactants ( supplementary figures 13 and 14 ) . the linear dependence of the initial rate of binding for 20 m excess heme combined with variable protein concentrations of 1 , 2 , 3 , and 4 m confirmed a second - order process in all cases except for the untethered d , which was ambiguous ( supplementary figure 15 ) . similarly , the linear dependence of the initial rate for 30 m excess maquette b protein with variable heme b concentrations of 6 , 9 , 12 , and 18 m confirmed a second - order process ( supplementary figure 16 ) . the single - chain monomer maquette b , unstructured in the apo state and structured in the holo state , displays the most rapid time course of heme ligation , proceeding to completion on a subsecond time scale with a second - order rate constant of 3.04 10 m s ( see table 2 and figure 1 ) , the same rate as natural protein a. rates for binding heme at different sites are equivalent for this maquette , as well ( supplemental figure 17 ) . ( b ) heme binding of maquette a at various temperatures fit to both a first - order ( dashed lines ) and second - order ( solid lines ) kinetic time course . despite markedly different primary sequences , natural sequence a and maquette b appear to present a common barrier height to heme entry , ligation , and establishment of a singular structure . eyring plot of the temperature dependence of the initial rates of the various proteins studied . dimeric maquette c shares the same sequence as monomeric maquette b and also assumes a singular structure in the holo state but has a different tertiary topology of helical tethering that involves removal of the loop connecting helices 2 and with the addition of a disulfide bond connecting the loops at the opposite end of the bundle ( table 1 ) . the maquette c has a more exaggerated enthalpic and entropic counterbalancing ( 8.7 and 16.2 kcal / mol ) with a net effect of only a 2-fold slowing of the second - order rate constant . the enhanced mobility at one end of the helical bundle in c appears to increase the entropic term while lowering the activation enthalpy . the simple removal of the disulfide tether in c to create the untethered helix loop helix homodimer d substantially lowers the second - order rate constant by 50-fold . figure 2 and table 2 show that the heme b ligation process for d slows slightly with increasing temperature , yielding a modest enthalpic term of 1.54 kcal / mol , with a dominating entropic term ( 28.8 kcal / mol ) . this suggests a distinctly different rate - limiting process that may be responding to the freedom of the apo - maquette helical pairs to pack parallel or antiparallel in a syn or anti conformation and/or the repelling effect of the high net charge on each of the homodimers ( 8 charge per one helix loop the untethered homodimer e with neutral net charge has a similar enthalpic but larger entropic contribution than natural sequence a. this slows the second - order rate constant 10-fold . although also net - neutral charge , the untethered homodimer f , the only sequence that is structured ( x - ray and nmr ) in the apo form , reverses the thermodynamic trend , raising the enthalpic term ( 17.9 kcal / mol ) and lowering the entropic term ( 8.7 kcal / mol ) , for a net slowing of the binding rate by 40-fold . these results separate d and f from the others as clearly rate - limited , respectively , by diffusion between heme b and the weakly coupled homodimers or a higher thermal activation barrier to access the heme into a singular structured apoprotein . we used [ n - octanol]/[water ] partition coefficients p(24,34 ) as a guide to the effect of the porphyrin macrocycle substitution on the relationship between the aqueous phase and the hydrophobic interior of the maquette b ( figure 3a ) . we assayed the solubility of the hemes and their tendency to form multimers in aqueous solution by uv visible spectroscopy . in the left panel of figure 3b , the visible spectrum of heme in ph 9 aqueous buffer ( green ) is dominated by the broad 350400 nm soret peak and the 605 nm peak characteristic of the dimer described by asher et al . there is no indication of the -oxo heme dimer , reported decades ago by brown et al . the soret absorption remains narrow and red shifts to 412 nm on bis - his ligation in the maquette ( blue ) . heme b ( left ) , protoporphyrin ix dimethyl ester ( middle ) , and diacetyl deuteroporphyrin ix ( right ) . figure 4 shows time courses of binding of members of the family of fe - porphyrins to maquette b , while figure 5 summarizes the effect of log p on the binding rate ( linear time scale in supplemental figure 18 ) . compared to heme b , the slightly less polar deuteroporphyrin and mesoporphyrin bind to maquette b several - fold faster . on the other hand , making the fe - porphyrin more polar by substituting the heme b vinyls with structurally comparable nitriles ( 2,6-dinitrileporphyrin ) or adding polar hydroxyls ( isohematoporphyrin ) or carbonyls ( diacetyl deuteroprotoporphyrin ) slows the rate of binding . this suggests that the rate - limiting step of heme ligation to maquette b lies in the initial steps of partitioning into the hydrophobic interior of the maquette . on the other hand , increasing the log p above a threshold value of about 0.75 by either removing the heme b propionates ( etioporphyrin ) or esterifying them ( protoporphyrin ix dimethyl ester ) leads to greater spectral evidence of porphyrin aggregation and significantly lowers the binding rates . this could reflect a lowering of the heme monomer concentration free in solution and/or a limiting rate of porphyrin disaggregation . scheme 1 summarizes the hurdles that can stand in the way of fe - porphyrin cofactor self - assembly into protein , starting in an aqueous medium and ending in a binding site in the interior of a protein that provides strong , effectively irreversible bishistidine ligation for the heme iron . decreased log p , indicating an increase in hydrophilic character , slows the rate by making partition of the unligated fe - porphyrin in the hydrophobic core less favorable . while the assembly of most heme variants is well fit by a second - order time course , fe - tetracarboxyphenyl porphyrin ( tcp ) displays unusual biphasic kinetics , with a burst phase extent of 20% of unknown origin ( supplemental figure 19 ) . the first way is to impose a large entropic reorganization barrier , best demonstrated by protein d. entropic barriers associated with minimal temperature dependence of the rate is a characteristic of structural rearrangements . at extremely low nanomolar concentrations of protein however , at micromolar concentrations , there is a conspicuous rapid burst phase of heme binding that indicates preformed dimers are available . the addition of a simple tether , the disulfide bond linking loops in c , for example , can restrict the conformational freedom of the heme binding segments such that the two monomers can not rearrange or diffuse away from one another , lowering the entropic barrier to moderate levels and speeding the rate of binding ( table 2 ) . protein c still retains residual conformational freedom associated with the smaller entropic barrier at the unlinked end of the bundle , while the increased structuring imposed by the tether adds to an enthalpic barrier . this structuring interferes with heme entry to the core as various packing interactions and internal hydrogen bonds must be broken . the stepwise redesign of maquettes from early multichain forms to a final single chain form that are sufficiently malleable in the apo state has increased the rate of cofactor self - assembly to a point comparable to natural proteins and limited by the physical chemical properties of the porphyrin itself . like the maquette b , natural cytochromes b562 and b5 and oxygen transport proteins such as myoglobin are predominantly unstructured in the apo state , becoming substantially more structured when heme is bound . in all cases , there is a small amount of structure in the apo state that restricts the conformational freedom of the unstructured regions . moreover , in apocytochrome b5 , only a small region has any secondary structure , while the majority of the protein is random coil and unstructured . despite this , nmr analysis of apomyoglobin has allowed eliezer and wright to construct a theoretical model of this protein without heme b showing , when combined with other literature describing its folding , that it too adheres to the principles outlined above : a conformationally restricted structure in which the binding site is malleable to allow access to heme b. the principles outlined in this article agree with the work of shoemaker and wolynes who describe the effect of molecular disorder on the binding of cofactors . this concept , referred to as the fly casting mechanism , can be easily applied to the maquettes in this paper owing to helical fraying and the molten globular nature of the apo state . however , difficulty in measuring structure in any localized fashion , due to the repetition in maquette sequence and minimal change in cd signal between apo and holo states , makes proving this difficult . rate limitations can be uncovered through the use of temperature dependencies , and the structure can be addressed accordingly based on the information above . it also minimizes the cytotoxicity of free heme b , which generates reactive oxygen species when unbound in solution . future man - made protein designs that seek to integrate with and exploit natural biochemical pathways in vivo will benefit by using these principles to diminish assembly barriers and effectively compete with host natural proteins for available cofactors described in this paper . indeed , recent work utilizing a variant of maquette b has already been shown to co - opt the natural c - type machinery to covalently link heme b to an artificial protein .

since the original observation of motionally restricted boundary lipid in progressively delipidated preparations of cytochrome oxidase by griffith and colleagues ( jost et al . 1973 ) , electron paramagnetic resonance ( epr ) of spin - labelled lipids has developed into an extremely versatile method for studying lipid protein interactions in biological membranes ( marsh et al . 1982 , 2002a ; marsh 1983 , 1987 , 1990 , 1995 , 1996 , 2008b ; marsh and powell 1988 ; knowles and marsh 1991 ; marsh and horvth 1998 ; marsh and pli 2004 ; esmann and marsh 2006 ) . the spin - label epr method is based primarily on the spectral resolution of lipids directly in contact with the intramembranous sector of the protein from those exhibiting the characteristic chain fluidity gradient in the bulk membrane ( cf . the fraction , fb , of spin - labelled lipids contacting the protein is related directly to the stoichiometry and selectivity of interaction with the protein ( marsh 1985 ; brotherus et al . 1981):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { { \frac{{1 - f_{\text{b } } } } { { f_{\text{b } } } } } } \right ) = { \frac{1}{{k_{\text{r } } } } } \left ( { { \frac{{n_{\text{t } } } } { { n_{\text{b } } } } } - 1 } \right ) $ $ \end{document}where nt is the total lipid / protein mole ratio . in eq . 1 , nb is the number of lipid association sites on the protein and kr is the association constant of spin - labelled lipid with the protein , relative to that of the unlabelled background host lipid , according to the lipid exchange reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{p}}\cdot{\text{l}}_{{n_{\text{b } } } } + { \text{l}}^ { * } \overset { k_{\text{r } } } \longleftrightarrow { \text{p}}\cdot{\text{l}}_{{n_{\text{b } } -1 } } { \text{l}}^ { * } + { \text{l } } $ $ \end{document}where p is protein , l is lipid and the asterisk ( * ) indicates spin label . material balance relates the fraction of protein - associated spin - labelled lipids to the kinetics of lipid exchange in the above reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{b } } \tau_{\text{b}}^ { - 1 } = ( 1 - f_{\text{b } } ) \tau_{\text{f}}^ { - 1 } $ $ \end{document}where f1 is the on - rate and b1 is the off - rate for spin - labelled lipids l * interacting with the protein . these dynamic parameters governing lipid exchange also can be determined by spin - label epr . in addition to the systematic dependences on lipid protein ratio and lipid selectivity that are observed , and invariance with respect to different spin - label configurations ( esmann et al . the latter techniques demonstrate unambiguously that the two - component nature of the spin - label spectra is preserved under quite different conditions of g - shift anisotropy or dynamic window of observation ( see also watts et al . 1981 ) . amongst the various available techniques , spin - label epr remains unique in yielding directly the stoichiometry of the protein - associated lipid , a quantity that is related to the intramembranous structure and assembly of integral proteins ( see fig . 1 ; marsh 1997 ; pli et al . representative data on the numbers of lipids , nb , that contact the intramembranous perimeter of different membrane proteins are presented in table 1.fig . 1first shell of lipids surrounding the crystal structure of escherichiacoli outer membrane protein fhua ( pdb code 2fcp ) ( ferguson et al . part of the shell of energy - minimised dic14:0ptdcho lipids ( space - filling representation ) is cut away to show the protein in ribbon and wire - frame representation . in total , 34 lipids contact the intramembrane perimeter of the protein ( pli et al . the stoichiometry of motionally restricted lipids observed by epr is nb = 32 ( ramakrishnan et al . 2004)table 1stoichiometries , nb of first - shell lipids surrounding integral proteins and peptides from spin - label epr , model building with x - ray protein structures ( pli et al . 2006 ) , and geometrical predictions for -helical bundles and -sheets ( marsh 1997)proteinnb ( mol / mol)referenceseprx - raygeometry-helical : rhodopsin222527 224.515adp - atp carrier2528 222.56ca - atpase222428 4317 , 8na , k - atpase323333911cytochrome reductase38 346 43712nicotinic acetylcholine receptor41 752 45213 , 14cytochrome oxidase56 557 468.51543 46316phospholamban a11.310174.0 0.1417m13 coat protein45418myelin proteolipid10 2101916-kda proteolipid561020-barrel / sheet : ompa111121ompg20 11922foma2323fhua323021m13 phage coat protein418k27 , k - channel peptide2.224k27-2.525-helix : gramicidin a3.6 0.3261 ( 2006)value per monomer deduced from structure of dimer ( 27)in dic14:0ptdgroin c16:0c18:1cptdchopredictions for pentamer : nagg = 5predictions for hexamer : nagg = 6-sheet form of m13 coat proteinsequence : klealyilmvlgffgfftlgimlsyir . both k27 and k27- are in the -sheet form first shell of lipids surrounding the crystal structure of escherichiacoli outer membrane protein fhua ( pdb code 2fcp ) ( ferguson et al . part of the shell of energy - minimised dic14:0ptdcho lipids ( space - filling representation ) is cut away to show the protein in ribbon and wire - frame representation . in total , the stoichiometry of motionally restricted lipids observed by epr is nb = 32 ( ramakrishnan et al . 2004 ) stoichiometries , nb of first - shell lipids surrounding integral proteins and peptides from spin - label epr , model building with x - ray protein structures ( pli et al . 2006 ) , and geometrical predictions for -helical bundles and -sheets ( marsh 1997 ) 1 . ( 2006 ) value per monomer deduced from structure of dimer ( 27 ) predictions for pentamer : nagg = 5 predictions for hexamer : nagg = 6 -sheet form of m13 coat protein sequence : klealyilmvlgffgfftlgimlsyir . both k27 and k27- are in the -sheet form an extension of this method is to investigate the partial penetration of surface - associated proteins and peptides into the membrane ( grrissen et al . 1986 ; rietveld et al . 1985 , 1986 ; jordi et al . 1989 ; snel and marsh 1994 ; sankaram et al . 1995 , 1996 ; snel et al . 1995 ; montich and marsh 1995 ; montich et al . 1995 ; ramakrishnan et al . 2001 ; swamy et al . the lipid stoichiometries are relatively low and the degree of motional restriction of the lipid chains is less than that for truly transmembrane proteins . a wide variety of spin - labelled species is available ( see marsh 2008a ) that can be used together with the epr method to determine the selectivity patterns of interaction with different transmembrane proteins ( see fig . 2 ) . representative values of the mean relative association constants for different lipid species are listed for a variety of transmembrane proteins in table 2 . the free energy of association , gass(l * ) , that characterises the lipid selectivity is related to the relative association constant by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \updelta \updelta g_{\text{ass } } = \updelta g_{\text{ass } } \left ( { { \text{l}}^ { * } } \right ) - \updelta g_{\text{ass } } \left ( { \text{l } } \right ) = - rt \ln \left ( { k_{\text{r } } } \right ) $ $ \end{document}where t is the absolute temperature and r is the ideal gas constant . selectivities are observed for certain negatively charged lipids that are partly , but not exclusively , of electrostatic origin ( see , e.g. , fig . the spin - label nitroxyl ring is rigidly attached to the c - n atom of the lipid hydrocarbon chain ( n = 14 in the figure ) , or to the steroid nucleus . for transmembrane proteins , two - component spectra are detected with lipids spin - labelled at the c14-position of the hydrocarbon chain ( 14-pxsl and 14-sasl ) . two - component spectra are also resolved with the spin - labelled steroids , cholestane and androstanol ( csl and asl , respectively ) . n - sasl , stearic acid ; n - pcsl , -pesl , -pgsl , -pssl , -pisl , and -pasl : phosphatidylcholine , phosphatidylethanolamine , phosphatidylglycerol , phosphatidylserine , phosphatidylinositol and phosphatidic acid , respectively . n - smsl , sphingomyelin ; n - gm1sl , monosialoganglioside gm1 ; n - gm2sl , monosialoganglioside gm2 ; n - gm3sl , monosialoganglioside gm3 ; n - gd1bsl , disialoganglioside gd1b ; n - mgdgsl , monogalactosyl diglyceride ; csl , cholestane ; and asl , androstanoltable 2relative association constants kr , referred to phosphatidylcholine or phosphatidylglycerol ( kr , o 1 ) , for various spin - labelled phospholipid species interacting with different transmembrane proteins or peptidesproteinkr / kr , oreferencesclptdstptdserptdgroptdetnptdcho-helix : plp10.46.52.21.81.01 dm-202.32.01.11.01.01 myelin proteolipid1.52.97.01.41.10.51.023.02.42.91.42.01.71.03 na , k - atpase3.81.51.71.70.90.91.04 , 5 na , k - atpase trypsinised1.5 , 2.81.91.06 cytochrome c oxidase5.41.91.01.01.01.07 adp - atp - carrier3.84.34.12.40.81.08 nicotinic acetylcholine receptor5.14.92.71.70.51.09 m13 phage coat protein1.61.21.21.11.01.010 16-kda proteolipid2.41.51.41.011 phospholamban phospholamban-a1.11.81.01.00.81.012 galp1.0 , 2.91.9 , 4.31.00.91.01.013 rhodopsin1.01.01.01.01.01.01.014 , 15-barrel / sheet : ompa2.5~0.20.61.00.50.616 ompg1.20.71.10.91.017 foma1.24.11.61.11.11.018 fhua1.53.01.41.00.61.119 m13 phage coat protein4.2 4.22.32.11.60.91.020 k27 , k - channel peptide3.32.02.01.1 - 1.121 k27-4.34.32.51.0 - 1.022-helix : gramicidin a1.20.7 , 1.41.10.80.81.0231 . ( 2004)cl , cardiolipin ( ptd2gro ) ; ptd , phosphatidic acid ; st , stearic acid ; ptdser , phosphatidylserine ; ptdgro , phosphatidylglycerol ; ptdetn , phosphatidylethanolamine ; ptdcho , phosphatidylcholine ; plp , myelin proteolipid proteinnatural mixture of the proteolipid protein ( plp ) and the dm-20 isoform ( plp-)in dic14:0ptdchoin dic14:0ptdgrovalues for protonated and charged forms , respectively-helical ( partly ) form of the protein in dic14:0ptdchorelative to ptdgro-sheet form of the protein in dic14:0ptdcho / dic14:0ptdgro ( 80:20 mol / mol)sequence : klealyilmvlgffgfftlgimlsyir . 3clusters of basic amino - acids that give rise to the pronounced selectivity of fhua for anionic lipids ( phosphatidic acid , phosphatidylserine and stearic acid ) . electrostatic surfaces are coloured with blue positive , red negative and white neutral ( ramakrishnan et al . 2004 ) spin - labelled lipids used for investigating lipid protein interactions . the spin - label nitroxyl ring is rigidly attached to the c - n atom of the lipid hydrocarbon chain ( n = 14 in the figure ) , or to the steroid nucleus . for transmembrane proteins , two - component spectra are detected with lipids spin - labelled at the c14-position of the hydrocarbon chain ( 14-pxsl and 14-sasl ) . two - component spectra are also resolved with the spin - labelled steroids , cholestane and androstanol ( csl and asl , respectively ) . n - sasl , stearic acid ; n - pcsl , -pesl , -pgsl , -pssl , -pisl , and -pasl : phosphatidylcholine , phosphatidylethanolamine , phosphatidylglycerol , phosphatidylserine , phosphatidylinositol and phosphatidic acid , respectively . n - smsl , sphingomyelin ; n - gm1sl , monosialoganglioside gm1 ; n - gm2sl , monosialoganglioside gm2 ; n - gm3sl , monosialoganglioside gm3 ; n - gd1bsl , disialoganglioside gd1b ; n - mgdgsl , monogalactosyl diglyceride ; csl , cholestane ; and asl , androstanol relative association constants kr , referred to phosphatidylcholine or phosphatidylglycerol ( kr , o 1 ) , for various spin - labelled phospholipid species interacting with different transmembrane proteins or peptides 1 . ( 2004 ) cl , cardiolipin ( ptd2gro ) ; ptd , phosphatidic acid ; st , stearic acid ; ptdser , phosphatidylserine ; ptdgro , phosphatidylglycerol ; ptdetn , phosphatidylethanolamine ; ptdcho , phosphatidylcholine ; plp , myelin proteolipid protein natural mixture of the proteolipid protein ( plp ) and the dm-20 isoform ( plp- ) values for protonated and charged forms , respectively -helical ( partly ) form of the protein in dic14:0ptdcho -sheet form of the protein in dic14:0ptdcho / dic14:0ptdgro ( 80:20 mol / mol ) sequence : klealyilmvlgffgfftlgimlsyir . -sheet forms of k27 and k27- clusters of basic amino - acids that give rise to the pronounced selectivity of fhua for anionic lipids ( phosphatidic acid , phosphatidylserine and stearic acid ) . electrostatic surfaces are coloured with blue positive , red negative and white neutral ( ramakrishnan et al . 2004 ) selectivity studies have also been performed with spin - labelled glycolipids ( li et al . 2003 ) , and a variety of lyso and acyl derivatives of cl ( powell et al . 1987 ; abramovitch et al . 1990 ; esmann et al . in addition to the selective interaction with lipids , the method may also be extended to spin - labelled drugs : for example , local anaesthetics ( miyazaki et al . 1990a ) , and inhibitors of the osteoclast v - atpase ( dixon et al . 2004 , 2008 ) whereas the great power of the spin - label method lies in the determination of structural parameters ( i.e. , stoichiometries ) and thermodynamic parameters ( i.e. , selectivities ) , the dynamic parameters that can be determined by epr spectroscopy are equally of interest . of especial relevance is the rate at which lipids in direct contact with the protein exchange with those in the bulk fluid bilayer regions of the membrane . resolution of two distinct components in the conventional epr spectra of spin - labelled lipids in lipid protein systems implies that exchange between the two lipid populations is slower than the difference in their spectral frequencies ( horvth et al . the critical rate is ~5 10 s ( see , e.g. , marsh et al . 2002b ) , which exceeds that for translational diffusion of free lipids in fluid bilayer membranes ( sachse et al . thus , even if spin - labelled lipids at the protein interface exchange at rates comparable to those of translation in the bulk , they are resolved as a separate component of slower rotational mobility . the exchange is slow on the conventional spin - label epr timescale , but is still sufficiently rapid to affect transverse relaxation , i.e. , linewidths . the rate of exchange of spin - labelled lipids at the intramembranous surface of the protein can be estimated by simulating the two - component epr lineshapes with the bloch equations that incorporate two - site exchange:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text { l}}_{\text{f}}^ { * } \begin{array}{*{20}c } { \mathop\rightleftarrows\limits^{{{{\uptau}}_{\text{f}}^ { - 1 } } } _ { { { { \uptau}}_{\text{b}}^ { - 1 } } } } \\ f refer to protein - associated and bilayer - associated environments , respectively , of the lipid spin label . the steady - state rate equations for the complex transverse magnetisations : mb = ub + ivb and mf = uf + ivf in the rotating frame , can be written as ( horvth et al . 1988a , 1994):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ [ ( \omega - \omega_{\text{b } } ) + i\tau_{\text{b}}^ { - 1 } ] m_{\text{b } } - i\tau_{\text{f}}^ { - 1 } m_{\text{f } } = - \gamma_{\text{e } } b_{1 } m_{\text{o } } f_{\text{b } } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ [ ( \omega - \omega_{\text{f } } ) + i\tau_{\text{f}}^ { - 1 } ] m_{\text{f } } - i\tau_{\text{b}}^ { - 1 } m_{\text{b } } = - \gamma_{\text{e } } b_{1 } m_{\text{o } } ( 1 - f_{\text{b } } ) $ $ \end{document}where b = o , b it2,b1 and f = o , f it2,f1 are the complex angular resonance frequencies of the protein - associated and bilayer - associated spin - label components , respectively , and t2,b and t2,f are the corresponding transverse relaxation times . in eqs . 4 and 5 , e is the electron gyromagnetic ratio , b1 is the microwave magnetic field intensity and mo is the equilibrium z - magnetisation . the slow - exchange solution for the epr absorption lineshape is then given by:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v(\omega ) \propto { \frac{{f_{\text{b } } ( t_{{2,\,{\text{b}}}}^ { - 1 } + \tau_{\text{b}}^ { - 1 } ) } } { { ( \omega_{{{\text{o,}}\,{\text{b } } } } - \omega ) ^{2 } + ( t _ { { 2 , \,{\text{b}}}}^ { - 1 } + \tau_{\text{b}}^ { - 1 } ) ^{2 } } } } + { \frac{{(1 - f_{\text{b } } ) \,(t_{{2,\,{\text{f}}}}^ { - 1 } + \tau_{\text{f}}^ { - 1 } ) } } { { ( \omega_{{{\text{o,}}\,{\text{f } } } } - \omega ) ^{2 } + ( t_{{2,\,{\text{f}}}}^ { - 1 } + \tau_{\text{f}}^ { - 1 } ) ^{2 } } } } $ $ \end{document}when b1 , f1 < < ( o , b o , f ) . the two rate constants are related by eq . 2 , where b1 is the intrinsic exchange rate that depends on the affinity of l * for the protein ( i.e. , on kr ) , and is independent of the lipid protein ratio . the on - rate , on the other hand , is diffusion - controlled and depends on the size of the free pool of lipid according to eq . 2 . data on the intrinsic off - rate constants ( b,01 ) for exchange of phosphatidylcholine ( a lipid that does not express selectivity for the protein ) at the interface with different transmembrane proteins are listed in table 3 . these are obtained from simulation of the spectral lineshapes by using the exchange - coupled bloch equations . for comparison , the intrinsic diffusional lipid lipid exchange rates in fluid phosphatidylcholine bilayers are diff1 8 10 s ( sachse et al . the rates of exchange at the protein interface are such that h - labelled lipids would be in fast exchange on the quadrupolar nmr timescale , explaining why only single - component lipid spectra are observed in lipid 1987).table 3off - rate constants ( b , o1 ) and activation energies ( ea , o ) for exchange of spin - labelled phosphatidylcholine ( 14-pcsl ) at the intramembranous surface of different transmembrane proteinsprotein / lipidt ( c)b , o1 ( s)ea , o ( kj / mol)referencesmyelin proteolipid protein / dic14:0ptdcho301.6 10201 , 2myelin dm-20 protein / dic14:0ptdcho301.5 103adp - atp carrier / egg ptdcho101.4 104m13 coat protein(-helix)/dic18:1cptdcho243.0 105m13 coat protein(-helix)/dic14:0ptdcho302.3 105m13 coat protein(-sheet)/dic14:0ptdcho305.3 106rhodopsin / dic14:0ptdcho301.6 102071 . ( 1987 ) off - rate constants ( b , o1 ) and activation energies ( ea , o ) for exchange of spin - labelled phosphatidylcholine ( 14-pcsl ) at the intramembranous surface of different transmembrane proteins 1 . ( 1987 ) because the on - rate is diffusion - controlled for all lipids , the off - rates must reflect the relative affinities of the different lipids for the protein . combining eqs . 1 and 2 for any fixed total lipid / protein ratio , one sees that the intrinsic off - rates of lipids a and b depend inversely on their relative association constants ( horvth et al . 1988a):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{\tau_{\text{b}}^ { - 1 } ( a)}}{{\tau_{\text{b}}^ { - 1 } ( b ) } } } = { \frac{{k_{\text{r } } ( b)}}{{k_{\text{r } } ( a ) } } } $ $ \end{document}this reciprocal relation is found to hold for most systems investigated ( horvth et al . 1988b , 1990b , c ; wolfs et al . , 1989 ; peelen et al . 1992 ) , with few exceptions that must reflect the existence of highly specific sites ( horvth et al . slow - exchange simulations , which depend on transverse relaxation rates , lie at the limits of dynamic sensitivity for conventional spin - label epr spectroscopy . more sensitive determination of the exchange rate comes from epr saturation experiments because these depend on longitudinal ( i.e. , spin - lattice ) relaxation rates , which are optimally matched in timescale to lipid exchange processes ( pli et al . the effect of two - site exchange on spin - lattice relaxation can be determined from the rate equations for the population differences ( n ) between the up and down spin orientations ( see fig . the steady - state rate equations when spin transitions are induced at rate 2w by b1-irradiation of the protein - associated component b are ( horvth et al . 1993a):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - 2wn_{\text{b } } + { \frac{{n_{\text{b}}^{\text{o } } - n_{\text{b } } } } { { t_{{1,{\text{b}}}}^{\text{o } } } } } - n_{\text{b } } \tau_{\text{b}}^ { - 1 } + n_{\text{f } } \tau_{\text{f}}^ { - 1 } = 0 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{n_{\text{f}}^{\text{o } } - n_{\text{f } } } } { { t_{{1,{\text{f}}}}^{\text{o } } } } } - n_{\text{f } } \tau_{\text{f}}^ { - 1 } + n_{\text{b } } \tau_{\text{b}}^ { - 1 } = 0 $ $ \end{document}where nbo and nfo are the spin population differences at boltzmann equilibrium , and t1,bo and t1,fo are the spin - lattice relaxation times in the absence of exchange , for protein - associated and bilayer - associated spin - labelled lipids , respectively , in both cases . solution of these rate equations , together with eq . 2 ( which also holds for population differences ) , yields the standard expression for saturation of the spin system at the protein - associated site b ( see , e.g. , slichter 1978):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{b } } = { \frac{{n_{\text{b}}^{\text{o } } } } { { 1 + 2wt_{{1,\,{\text{b}}}}^{\text{eff } } } } } $ $ \end{document}where t1,beff is the effective spin - lattice relaxation time in the presence of exchange . 1993a):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{t_{{1,{\text{b}}}}^{\text{o } } } } { { t_{{1,{\text{b}}}}^{\text{eff } } } } } = 1 + { \frac{{\left ( { 1 - f_{\text{b } } } \right)t_{{1,\,{\text{b}}}}^{\text{o } } \tau_{\text{b}}^ { - 1 } } } { { 1 - f_{\text{b } } + f_{\text{b } } t_{{1,\,{\text{f}}}}^{\text{o } } \tau_{\text{b}}^ { - 1 } } } } $ $ \end{document}from which the intrinsic off - rate , b1 , for exchange is obtained.fig . 4energy levels , spin populations ( nb , nf ) , and transitions for two spin - label sites , b and f. the spin population difference is given by : nb = nb nb+ . the transition rate for spin - lattice relaxation is : 2we = 1/t1o . the rate of exchange between the two sites is : nbb1 = nff1 , for both spin populations and population differences . the rate of heisenberg exchange between spins b and f is : 2kxnbnf ( marsh 1993 ) energy levels , spin populations ( nb , nf ) , and transitions for two spin - label sites , b and f. the spin population difference is given by : nb = nb nb+ . the transition rate for spin - lattice relaxation is : 2we = 1/t1o . the rate of exchange between the two sites is : nbb1 = nff1 , for both spin populations and population differences . the rate of heisenberg exchange between spins b and f is : 2kxnbnf ( marsh 1993 ) figure 5 shows progressive saturation curves for a spin - labelled lipid in lipid bilayers alone , associated with a delipidated protein ( plp ) , and in lipid membranes containing plp . data are given for temperatures both below and above the chain - melting transition of the lipid . in both cases , spin - labelled lipids associated with the delipidated protein saturate more readily than those in membranes of the lipid alone , indicating a pronounced difference in the lipid chain dynamics at the protein interface ( livshits et al . 1998 , 2003 ) . in the fluid phase , this mobility difference is clear from the two - component lineshapes , i.e. , occurs on the timescale of t2-relaxation . the saturation properties show that this distinction extends also to the gel phase , where differences in mobility are found on the longer timescale of t1-relaxation.fig . 5progressive saturation curves for the integrated intensity of the conventional epr spectra of spin - labelled phosphatidylcholine 14-pcsl in : dic14:0ptdcho membranes ( solid circles ) , delipidated myelin proteolipid protein plp ( open circles ) , and plp / dic14:0ptdcho membranes of lipid / protein ratio 24:1 mol / mol ( solid squares ) . left in the lipid gel phase ( 4c ) , solid lines are fits of eq . 12 for saturation of the single components ( fb = 0 or 1 ) , and dotted lines are predictions for saturation of the lipid protein membranes assuming no exchange between the two components ( fb = 0.40 ) ( horvth et al . 1993a ) progressive saturation curves for the integrated intensity of the conventional epr spectra of spin - labelled phosphatidylcholine 14-pcsl in : dic14:0ptdcho membranes ( solid circles ) , delipidated myelin proteolipid protein plp ( open circles ) , and plp / dic14:0ptdcho membranes of lipid / protein ratio 24:1 mol / mol ( solid squares ) . left in the lipid gel phase ( 4c ) , solid lines are fits of eq . 12 for saturation of the single components ( fb = 0 or 1 ) , and dotted lines are predictions for saturation of the lipid protein membranes assuming no exchange between the two components ( fb = 0.40 ) ( horvth et al . 1993a ) the saturation behaviour in the protein - containing membranes is intermediate between that in the lipid - alone and protein - alone environments . however , in the fluid phase , the saturation curve is strongly biassed towards that from the lipid alone . the dependence of the double - integrated intensity of a two - component spectrum on the microwave field strength , b1 , is given by ( snel and marsh 1994 ; pli et al . 1993):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s(b_{1 } ) = s_{\text{o } } b_{1 } \left ( { { \frac{{f_{\text{b } } } } { { \sqrt { 1 + \sigma_{\text{b } } } } } } + { \frac{{1 - f_{\text{b } } } } { { \sqrt { 1 + \sigma_{\text{f } } } } } } } \right ) $ $ \end{document}where the saturation parameter for the protein - associated lipids is b = e2b12t1,bt2,b and similarly for f ( e = 1.76086 10 rad s 12 illustrates the double advantage of using the integrated epr intensity , instead of spectral lineheights , for constructing saturation curves : contributions from different components in multi - component systems are strictly additive , and the saturation curves do not depend on the degree of inhomogeneous broadening of any of the components . ) the dotted saturation curves in fig . 12 by using the saturation parameters determined from the corresponding single - component systems . in the gel phase , where the lipid mobility is low , the spectrum from the lipid protein membrane saturates exactly as predicted for a linear combination of the two isolated environments . in the fluid phase , on the other hand , the spectrum saturates less readily than predicted from the isolated components . alleviation of saturation arises from lipid exchange between the two environments that must take place on the timescale of spin - lattice relaxation ( i.e. , in the microsecond regime ) , in fluid membranes . table 3 ) , although still relatively rapid , confirms that the two - component lineshapes observed by conventional epr correspond to slow exchange . of the various non - linear epr methods ( marsh 1993 ) , out - of - phase detection ( as in saturation transfer epr ) has proved to be an especially powerful tool for probing lipid exchange in protein - containing membranes . in the absence of exchange between components on the t1-timescale , the first integral ist of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v^{\prime}_{2 } $ $ \end{document}-stepr spectrum ( second harmonic detected in phase quadrature ) is additive between the different components ( horvth and marsh 1983):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ i_{\text{st } } = \left ( { 1 - f_{\text{b } } } \right)i_{{{\text{st}},\,{\text{f}}}}^{\text{o } } + f_{\text{b } } i_{{{\text{st}},\,{\text{b}}}}^{\text{o } } $ $ \end{document}where ist , bo and ist , fo are the values of ist for components b and f , respectively , and fb is the fraction of component figure 6 gives the integrated \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v^{\prime}_{2 } $ $ \end{document}-stepr intensity as a function of the fraction of spin - labelled lipid that is associated with the plp protein in reconstituted membranes . samples all have the same total lipid / protein ratio ; fb is varied by using spin - labelled lipid species with differing affinities for plp . the value of fb is determined by spectral subtraction with the two - component conventional v1-epr spectra , as for the data in tables 1 and 2 . below the lipid chain - melting temperature ( tt ) , ist depends linearly on fb , as predicted by eq . 13 , showing that in the gel phase any lipid exchange is extremely slow , relative to the microsecond timescale . in the fluid lipid phase , above tt , however , the dependence of ist on fb lies below the straight ( dashed ) line expected for no exchange . saturation is partially alleviated by exchange between sites on and off the protein at rates comparable to the spin - lattice relaxation time . this result , which is based on differential selectivities of various lipid species for plp ( cf . table 2 ) , complements that from the progressive saturation experiments given in the right panel of fig . 6dependence of the integrated v2 saturation transfer epr intensity , ist , from different spin - labelled lipids on the fraction , fb , of each lipid species associated with the myelin proteolipid protein in dic14:0ptdcho membranes at fixed lipid / protein ratio ( 37:1 mol / mol ) . measurements correspond to the gel - phase ( t < tt ) and the fluid phase ( t > tt ) at 4 and 30c , respectively . straight lines are predictions for zero exchange rate ( b1 = 0 ) at the two temperatures , and the curved line is a non - linear least - squares fit of eq . 14 with eq . 11 ( and equivalent ) giving a constant lipid on - rate of t1,bof1 = 2.9 in the fluid phase at 30c ( horvth et al . 1993a ) dependence of the integrated v2 saturation transfer epr intensity , ist , from different spin - labelled lipids on the fraction , fb , of each lipid species associated with the myelin proteolipid protein in dic14:0ptdcho membranes at fixed lipid / protein ratio ( 37:1 mol / mol ) . measurements correspond to the gel - phase ( t < tt ) and the fluid phase ( t > tt ) at 4 and 30c , respectively . straight lines are predictions for zero exchange rate ( b1 = 0 ) at the two temperatures , and the curved line is a non - linear least - squares fit of eq . 14 with eq . 11 ( and equivalent ) giving a constant lipid on - rate of t1,bof1 = 2.9 in the fluid phase at 30c ( horvth et al . 1993a ) both theoretical simulations and experiment indicate that the normalised saturation transfer intensity , ist , is approximately proportional to t1 , over a considerable range of spin - lattice relaxation times ( pli et al . the net st - epr intensity in the presence of exchange between components b and f is thus given by ( cf . 1992):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ i_{\text{st } } = \left ( { 1 - f_{\text{b } } } \right)i_{{{\text{st}},\,{\text{f}}}}^{\text{o } } .{\frac{{t_{{1,\,{\text{f } } } } } } { { t_{{1,\,{\text{f}}}}^{\text{o } } } } } + f_{\text{b } } i_{{{\text{st}},\,{\text{b}}}}^{\text{o } } { \frac{{t_{{1,\,{\text{b } } } } } } { { t_{{1,\,{\text{b}}}}^{\text{o } } } } } $ $ \end{document}where t1,b and t1,f are the spin - lattice relaxation times at lipid locations , respectively , on and off the protein , and t1,bo , t1,fo are the corresponding values in the absence of exchange . combining eq . 14 with eq . 11 and its equivalent for t1,f / t1,fo gives the predicted dependence of ist on fb in the presence of exchange . the non - linear least - squares fit to the data shown for the fluid phase in fig . 6 yields a normalised on - rate constant for lipid exchange of t1,bof1 = 2.9 ( at fixed lipid / protein ratio of 37:1 mol / mol and t = 30c ) . with this on - rate , the off - rates b1 for the lipids with different affinities for plp are determined from the values of fb by material balance , i.e. , eq . 2 ( see table 4).table 4normalised off - rates for exchange of different spin - labelled lipid species at the protein interface in myelin proteolipid ( plp)/dic14:0ptdcho membranes at 30c ( horvth et al . 1993a)lipidist 10t1,bob1stearic acid0.190.67phosphatidic acid0.270.86phosphatidylserine0.202.45phosphatidylglycerol0.134.9phosphatidylcholine0.144.9intrinsic off - rates , b1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the protein - associated lipids , t1,bo ~ 1 s . ist is the difference in normalised st - epr intensity from that predicted by linear additivity normalised off - rates for exchange of different spin - labelled lipid species at the protein interface in myelin proteolipid ( plp)/dic14:0ptdcho membranes at 30c ( horvth et al . 1993a ) intrinsic off - rates , b1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the protein - associated lipids , t1,bo ~ 1 s . ist is the difference in normalised st - epr intensity from that predicted by linear additivity the extent of membrane penetration by a spin - labelled segment of a surface - associated or transmembrane protein can be assessed from spin spin interactions with spin labels located at different positions in a lipid molecule ( snel and marsh 1994 ; pli et al . spin exchange in the double - labelled system is analogous to molecular exchange at the lipid protein interface ( marsh 1992a , b ; and see fig . 4 ) . in the presence of heisenberg spin - exchange between the two distinguishable spin - labelled species , the spin - lattice relaxation time of the spin - labelled protein is given by ( snel and marsh 1994):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{t_{{1,\,{\text{p}}}}^{\text{o } } } } { { t_{{1,\,{\text{p}}}}^{\text{eff } } } } } = 1 + { \frac{{\left ( { 1 - f_{\text{p } } } \right)t_{{1,\,{\text{p}}}}^{\text{o } } \tau_{\text{ex}}^ { - 1 } } } { { 1 + f_{\text{p } } t_{{1,\,{\text{l}}}}^{o } \tau_{\text{ex}}^ { - 1 } } } } $ $ \end{document}where subscripts p and l refer to spin - labelled protein and lipid , respectively , and fp is the fraction of the total spin - label intensity that is associated with the protein . the mutual spin exchange frequency ex1 is proportional to the total spin label concentration and is a measure of the collision frequency between the two spin - labelled species . table 5 gives the spin exchange frequencies between a spin label on cys or cys in the n - terminal region of apocytochrome c bound to phosphatidylglycerol membranes and spin labels in the polar group ( t - pasl ) or at different positions in the acyl chains ( 5-pgsl and 14-pgsl ) of the membrane lipids . ( note that cys and cys are the sites of covalent attachment of the haem group in the holoprotein and , at labelling levels of 1:1 mol / mol , either one or other of these residues in the apoprotein bears the spin label . ) the maximum exchange frequency is found with a spin label on the c-14 atom of the lipid chain . this indicates that the n - terminal section of apocytochrome c penetrates deeply into the membrane , consistent with lipid protein interactions facilitating the translocation of this haem - less precursor into the mitochondrion ( grrissen et al . in contrast , for the mature holocytochrome c protein spin labelled on cys close to the c - terminal , the largest exchange frequency is found with a lipid spin labelled in the polar headgroup , as expected for a native peripheral protein bound at the outer surface of the inner mitochondrial membrane ( kostrzewa et al . spin - labelled at different positions and spin - labelled proteins ( cytochrome c and apocytochrome c ) bound to phosphatidylglycerol bilayer membranes ( snel and marsh 1994)spin - labelled lipid(1/t1t2)10 ( s)t1,poex1apocytochrome c / dic14:0ptdgro : t - pasl00 5-pgsl0.30.2 14-pgsl8.55apocytochrome c / dic18:1cptdgro : t - pasl5.65 5-pgsl00 14-pgsl5.423cytochrome c / dic18:1cptdgro : t - pasl1.60.8 5-pgsl0.20.1 14-pgsl00spin exchange frequencies , ex1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the spin - labelled protein , t1,po ~ 1 s . (1/t1t2 ) is the difference in 1/t1t2 of the double - labelled system from that predicted by eq . 12 with saturation parameters from the single spin - labelled systems . t - pasl is phosphatidic acid spin - labelled on the phosphate headgroup normalised spin exchange frequencies between lipids spin - labelled at different positions and spin - labelled proteins ( cytochrome c and apocytochrome c ) bound to phosphatidylglycerol bilayer membranes ( snel and marsh 1994 ) spin exchange frequencies , ex1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the spin - labelled protein , t1,po ~ 1 s . (1/t1t2 ) is the difference in 1/t1t2 of the double - labelled system from that predicted by eq . 12 with saturation parameters from the single spin - labelled systems . t - pasl is phosphatidic acid spin - labelled on the phosphate headgroup the vertical depth of the dccd - reactive glutamate that is essential for proton translocation by the 16-kda proteolipid subunit of the vacuolar atpase ( vma3p in yeast ) was mapped in a similar way by using different spin - labelled lipids and a nitroxyl analogue of dccd ( pli et al . most importantly , the stepr intensities of the double - labelled system demonstrate that the essential glutamate residue is exposed to lipid , as is required by the rotary mechanism for proton transport by stepwise interaction with the vph1p subunit of the yeast v - atpase . spin - label epr has provided a wealth of data on both stoichiometry and selectivity of lipid protein interactions in biological membranes . the experiments are , however , mostly restricted to spin - labelled lipids at probe concentrations , which means that a small number of high - affinity sites are not distinguished from a smaller generalised selectivity for all lipid sites at the intramembranous perimeter of the protein . in one instance , competition with exogenous cardiolipin failed to yield evidence for a very high - affinity site on cytochrome oxidase ( powell et al . 1985 ) , where endogenous cardiolipin had been replaced by phosphatidylcholine ( watts et al . both x - ray crystallography and fluorescence experiments have located non - annular lipid at intersubunit sites . more data are also needed on the dynamics of lipid exchange : the methods are in place but the current database is of relatively modest size . nevertheless , a specific site for cardiolipin on the adp - atp translocator is already indicated by comparison of exchange rates and relative association constants for this lipid ( horvth et al .

conventional electron paramagnetic resonance ( epr ) spectra of lipids that are spin - labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane . this allows determination of both the stoichiometry of lipid protein interaction ( i.e. , number of lipid sites at the protein perimeter ) and the selectivity of the protein for different lipid species ( i.e. , association constants relative to the background lipid ) . spin - label epr data are summarised for 20 or more different transmembrane peptides and proteins , and 7 distinct species of lipids . lineshape simulations of the two - component conventional spin - label epr spectra allow estimation of the rate at which protein - associated lipids exchange with those in the bulk fluid regions of the membrane . for lipids that do not display a selectivity for the protein , the intrinsic off - rates for exchange are in the region of 10 mhz : less than 10 slower than the rates of diffusive exchange in fluid lipid membranes . lipids with an affinity for the protein , relative to the background lipid , have off - rates for leaving the protein that are correspondingly slower . non - linear epr , which depends on saturation of the spectrum at high radiation intensities , is optimally sensitive to dynamics on the timescale of spin - lattice relaxation , i.e. , the microsecond regime . both progressive saturation and saturation transfer epr experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale . the sensitivity of non - linear epr to low frequencies of spin exchange also allows the location of spin - labelled membrane protein residues relative to those of spin - labelled lipids , in double - labelling experiments .

Introduction Stoichiometry and selectivity of lipidprotein interaction Lipid exchange dynamics at the protein interface Sensitivity of spin-lattice relaxation to exchange of spin-labelled lipids Accessibility of penetrant proteins to spin exchange with labelled lipids Conclusion

1973 ) , electron paramagnetic resonance ( epr ) of spin - labelled lipids has developed into an extremely versatile method for studying lipid protein interactions in biological membranes ( marsh et al . the spin - label epr method is based primarily on the spectral resolution of lipids directly in contact with the intramembranous sector of the protein from those exhibiting the characteristic chain fluidity gradient in the bulk membrane ( cf . the fraction , fb , of spin - labelled lipids contacting the protein is related directly to the stoichiometry and selectivity of interaction with the protein ( marsh 1985 ; brotherus et al . 1 , nb is the number of lipid association sites on the protein and kr is the association constant of spin - labelled lipid with the protein , relative to that of the unlabelled background host lipid , according to the lipid exchange reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{p}}\cdot{\text{l}}_{{n_{\text{b } } } } + { \text{l}}^ { * } \overset { k_{\text{r } } } \longleftrightarrow { \text{p}}\cdot{\text{l}}_{{n_{\text{b } } -1 } } { \text{l}}^ { * } + { \text{l } } $ $ \end{document}where p is protein , l is lipid and the asterisk ( * ) indicates spin label . material balance relates the fraction of protein - associated spin - labelled lipids to the kinetics of lipid exchange in the above reaction:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{\text{b } } \tau_{\text{b}}^ { - 1 } = ( 1 - f_{\text{b } } ) \tau_{\text{f}}^ { - 1 } $ $ \end{document}where f1 is the on - rate and b1 is the off - rate for spin - labelled lipids l * interacting with the protein . amongst the various available techniques , spin - label epr remains unique in yielding directly the stoichiometry of the protein - associated lipid , a quantity that is related to the intramembranous structure and assembly of integral proteins ( see fig . of especial relevance is the rate at which lipids in direct contact with the protein exchange with those in the bulk fluid bilayer regions of the membrane . resolution of two distinct components in the conventional epr spectra of spin - labelled lipids in lipid protein systems implies that exchange between the two lipid populations is slower than the difference in their spectral frequencies ( horvth et al . thus , even if spin - labelled lipids at the protein interface exchange at rates comparable to those of translation in the bulk , they are resolved as a separate component of slower rotational mobility . the rate of exchange of spin - labelled lipids at the intramembranous surface of the protein can be estimated by simulating the two - component epr lineshapes with the bloch equations that incorporate two - site exchange:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text { l}}_{\text{f}}^ { * } \begin{array}{*{20}c } { \mathop\rightleftarrows\limits^{{{{\uptau}}_{\text{f}}^ { - 1 } } } _ { { { { \uptau}}_{\text{b}}^ { - 1 } } } } \\ f refer to protein - associated and bilayer - associated environments , respectively , of the lipid spin label . 1988a , 1994):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ [ ( \omega - \omega_{\text{b } } ) + i\tau_{\text{b}}^ { - 1 } ] m_{\text{b } } - i\tau_{\text{f}}^ { - 1 } m_{\text{f } } = - \gamma_{\text{e } } b_{1 } m_{\text{o } } f_{\text{b } } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ [ ( \omega - \omega_{\text{f } } ) + i\tau_{\text{f}}^ { - 1 } ] m_{\text{f } } - i\tau_{\text{b}}^ { - 1 } m_{\text{b } } = - \gamma_{\text{e } } b_{1 } m_{\text{o } } ( 1 - f_{\text{b } } ) $ $ \end{document}where b = o , b it2,b1 and f = o , f it2,f1 are the complex angular resonance frequencies of the protein - associated and bilayer - associated spin - label components , respectively , and t2,b and t2,f are the corresponding transverse relaxation times . data on the intrinsic off - rate constants ( b,01 ) for exchange of phosphatidylcholine ( a lipid that does not express selectivity for the protein ) at the interface with different transmembrane proteins are listed in table 3 . the rates of exchange at the protein interface are such that h - labelled lipids would be in fast exchange on the quadrupolar nmr timescale , explaining why only single - component lipid spectra are observed in lipid 1987).table 3off - rate constants ( b , o1 ) and activation energies ( ea , o ) for exchange of spin - labelled phosphatidylcholine ( 14-pcsl ) at the intramembranous surface of different transmembrane proteinsprotein / lipidt ( c)b , o1 ( s)ea , o ( kj / mol)referencesmyelin proteolipid protein / dic14:0ptdcho301.6 10201 , 2myelin dm-20 protein / dic14:0ptdcho301.5 103adp - atp carrier / egg ptdcho101.4 104m13 coat protein(-helix)/dic18:1cptdcho243.0 105m13 coat protein(-helix)/dic14:0ptdcho302.3 105m13 coat protein(-sheet)/dic14:0ptdcho305.3 106rhodopsin / dic14:0ptdcho301.6 102071 . ( 1987 ) off - rate constants ( b , o1 ) and activation energies ( ea , o ) for exchange of spin - labelled phosphatidylcholine ( 14-pcsl ) at the intramembranous surface of different transmembrane proteins 1 . 1993a):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - 2wn_{\text{b } } + { \frac{{n_{\text{b}}^{\text{o } } - n_{\text{b } } } } { { t_{{1,{\text{b}}}}^{\text{o } } } } } - n_{\text{b } } \tau_{\text{b}}^ { - 1 } + n_{\text{f } } \tau_{\text{f}}^ { - 1 } = 0 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{n_{\text{f}}^{\text{o } } - n_{\text{f } } } } { { t_{{1,{\text{f}}}}^{\text{o } } } } } - n_{\text{f } } \tau_{\text{f}}^ { - 1 } + n_{\text{b } } \tau_{\text{b}}^ { - 1 } = 0 $ $ \end{document}where nbo and nfo are the spin population differences at boltzmann equilibrium , and t1,bo and t1,fo are the spin - lattice relaxation times in the absence of exchange , for protein - associated and bilayer - associated spin - labelled lipids , respectively , in both cases . in both cases , spin - labelled lipids associated with the delipidated protein saturate more readily than those in membranes of the lipid alone , indicating a pronounced difference in the lipid chain dynamics at the protein interface ( livshits et al . 5progressive saturation curves for the integrated intensity of the conventional epr spectra of spin - labelled phosphatidylcholine 14-pcsl in : dic14:0ptdcho membranes ( solid circles ) , delipidated myelin proteolipid protein plp ( open circles ) , and plp / dic14:0ptdcho membranes of lipid / protein ratio 24:1 mol / mol ( solid squares ) . 1993a ) progressive saturation curves for the integrated intensity of the conventional epr spectra of spin - labelled phosphatidylcholine 14-pcsl in : dic14:0ptdcho membranes ( solid circles ) , delipidated myelin proteolipid protein plp ( open circles ) , and plp / dic14:0ptdcho membranes of lipid / protein ratio 24:1 mol / mol ( solid squares ) . 1993):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s(b_{1 } ) = s_{\text{o } } b_{1 } \left ( { { \frac{{f_{\text{b } } } } { { \sqrt { 1 + \sigma_{\text{b } } } } } } + { \frac{{1 - f_{\text{b } } } } { { \sqrt { 1 + \sigma_{\text{f } } } } } } } \right ) $ $ \end{document}where the saturation parameter for the protein - associated lipids is b = e2b12t1,bt2,b and similarly for f ( e = 1.76086 10 rad s 12 illustrates the double advantage of using the integrated epr intensity , instead of spectral lineheights , for constructing saturation curves : contributions from different components in multi - component systems are strictly additive , and the saturation curves do not depend on the degree of inhomogeneous broadening of any of the components . ) alleviation of saturation arises from lipid exchange between the two environments that must take place on the timescale of spin - lattice relaxation ( i.e. of the various non - linear epr methods ( marsh 1993 ) , out - of - phase detection ( as in saturation transfer epr ) has proved to be an especially powerful tool for probing lipid exchange in protein - containing membranes . 2 ( see table 4).table 4normalised off - rates for exchange of different spin - labelled lipid species at the protein interface in myelin proteolipid ( plp)/dic14:0ptdcho membranes at 30c ( horvth et al . 1993a)lipidist 10t1,bob1stearic acid0.190.67phosphatidic acid0.270.86phosphatidylserine0.202.45phosphatidylglycerol0.134.9phosphatidylcholine0.144.9intrinsic off - rates , b1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the protein - associated lipids , t1,bo ~ 1 s . ist is the difference in normalised st - epr intensity from that predicted by linear additivity normalised off - rates for exchange of different spin - labelled lipid species at the protein interface in myelin proteolipid ( plp)/dic14:0ptdcho membranes at 30c ( horvth et al . 1993a ) intrinsic off - rates , b1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the protein - associated lipids , t1,bo ~ 1 s . in the presence of heisenberg spin - exchange between the two distinguishable spin - labelled species , the spin - lattice relaxation time of the spin - labelled protein is given by ( snel and marsh 1994):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{t_{{1,\,{\text{p}}}}^{\text{o } } } } { { t_{{1,\,{\text{p}}}}^{\text{eff } } } } } = 1 + { \frac{{\left ( { 1 - f_{\text{p } } } \right)t_{{1,\,{\text{p}}}}^{\text{o } } \tau_{\text{ex}}^ { - 1 } } } { { 1 + f_{\text{p } } t_{{1,\,{\text{l}}}}^{o } \tau_{\text{ex}}^ { - 1 } } } } $ $ \end{document}where subscripts p and l refer to spin - labelled protein and lipid , respectively , and fp is the fraction of the total spin - label intensity that is associated with the protein . t - pasl is phosphatidic acid spin - labelled on the phosphate headgroup normalised spin exchange frequencies between lipids spin - labelled at different positions and spin - labelled proteins ( cytochrome c and apocytochrome c ) bound to phosphatidylglycerol bilayer membranes ( snel and marsh 1994 ) spin exchange frequencies , ex1 , are normalised with respect to the intrinsic spin - lattice relaxation time of the spin - labelled protein , t1,po ~ 1 s . the experiments are , however , mostly restricted to spin - labelled lipids at probe concentrations , which means that a small number of high - affinity sites are not distinguished from a smaller generalised selectivity for all lipid sites at the intramembranous perimeter of the protein .

considering their poor prognosis and resistance to chemotherapy and radiotherapy , brain tumours in general , and high - grade astrogliomas in particular , are among the most devastating types of human cancer . at the cell population level , brain malignancies are typically heterogeneous lesions composed of cells with diverse morphologies that express a variety of neural lineage markers . prognosis and response to treatment differ significantly not only among histologically different brain tumours , but also among those that appear similar in terms of morphology and patterns of phenotypic markers . this notorious heterogeneity of brain tumours likely facilitates development of treatment - resistant clones , a process that is further fuelled by the ability of astroglioma cells to shift between quiescent and proliferating states . given such unusual plasticity and the frightening rate of treatment failure , it is now commonly believed that treatment outcome and hence patients ' survival can only improve if the biology of the brain cancer cell populations is better understood . classification of adult brain tumours is based on the world health organization ( who ) classification of nervous system tumours . the who grading of astrocytomas establishes a malignancy scale based on histologic features of the tumour . the histological grades are as follows : who grade i includes lesions with low proliferative potential , a frequently discrete nature , and the possibility of cure following surgical resection alone . who grade ii lesions are generally infiltrating and display low mitotic activity but recur . some lower - grade lesions tend to progress to higher grades of malignancy . who grade iii includes lesions with histological evidence of malignancy , generally in the form of mitotic activity , clearly expressed infiltrative capabilities , and anaplasia . who grade iv lesions are mitotically active , necrosis - prone and commonly associated with a rapid pre - operative and post - operative evolution of disease the most malignant types of astrogliomas are anaplastic astrocytoma ( grade iii astrocytoma ) and glioblastoma multiforme ( gbm , grade iv astrocytoma ) . in this article , we commonly use the general term astrogliomas for simplicity , yet our discussion mainly concerns the grade iii and particularly grade iv tumours . one of the key features of astrogliomas , affecting their biological and clinical behaviour , is dense vascularization . since the introduction of the concept of an angiogenic switch as a pivotal component of tumour growth and metastasis by folkman et al . , multiple therapies targeting the molecular regulators of this mechanism have been tested with variable clinical efficacy . despite such mixed initial results , anti - angiogenic therapy is regarded as a promising treatment strategy , and various pre - clinical experimental approaches targeting neovascularization turned out to be effective in vivo . for example , one study showed that systemic therapy with a mono- clonal antibody against vascular endothelial growth factor receptor 2 ( vegfr2 ) inhibited tumour growth in mice by some 80% . the ligand that operates through vegfrs to promote tumour angiogenesis is vegf , a versatile regulatory cytokine that is aberrantly up - regulated in a wide range of tumour types and acts as a potent modulator of tumour growth and metastasis in numerous pre - clinical tumour models . particularly relevant to this review , several pieces of evidence indicate prognostic significance of vegf in high - grade astrogliomas [ 68 ] . in this article , we highlight the emerging role(s ) of vegf signalling in astroglioma biology , including the possible links with dna damage checkpoints in response to radiation treatment . furthermore , we postulate and discuss a hypothetical function of autocrine vegf signalling in regulation of the so - called brain tumour - derived cancer stem - like cells ( btcsc ) and their intriguing resistance to therapy . unlike the early - stage embryonic brain harbouring large numbers of neural stem and progenitor cells , the adult brain is mainly composed of highly differentiated and specialized cell types , including neurons , glia ( oligodendrocytes , astrocytes , microglia , ependymal cells ) , vascular endothelium and meningeal cells . over the past century , the brain has traditionally been viewed as static with respect to its very limited turnover and regenerative capacity . however , it has recently become clear that neurogenesis continues into adult life in restricted germinal zones [ 1013 ] . a small percentage of quiescent cells present in the adult hippocampal dentate gyrus ( sub - granular zone , sgz ) , sub - ventricular zone of the lateral ventricles ( svz ) and olfactory bulbs are undifferentiated and multipotent neural stem cells ( nsc ) capable of self - renewal [ 1415 ] . these stem cells divide to generate rapidly cycling transitamplifying cells with a limited proliferation and differentiation potential . the transitamplifying cells give rise to restricted progenitor cells undergoing terminal differentiation . in the 1970s , an idea of specific anatomical locations termed niches. in the adult mammalian brain , we are just beginning to identify the cellular and molecular features that characterize the neurogenic niches in the svz and sgz , and the mechanisms by which the full range of adult nsc development is regulated . nscs are grown in vitro either as neurospheres in suspension or as an adherent monolayer . some of the cells within the neurospheres proliferate as multipotent , self - renewing nscs upon stimulation with either epidermal growth factor ( egf ) or basic fibroblast growth factor ( bfgf ) . the nscs residing in the adult human brain are attractive candidates for isolation , in vitro expansion and autologous transplantation to replace neurons lost to neurodegenerative diseases , stroke and traumatic brain injury . recently , a small population of cancer stem - like cells ( cscs ) has been identified in adult and paediatric brain tumours , as well as in established cell lines [ 2024 ] . these cells express a number of nsc markers including cd133 , a cell surface protein which is extensively exploited for enrichment of stem - like cells using fluorescence activated cell sorting ( facs ) and/or magnetic bead separation , thereby providing cells to be examined in diverse applications [ 2526 ] . the human cd133 antigen , also known as ac133 , was originally identified as a marker of haematopoietic stem cells , however its function has remained unclear . a characteristic feature of the cd133 protein is its rapid down - regulation during cell differentiation [ 2829 ] , which makes it a cell surface marker uniquely suited for the identification and isolation of stem - like and progenitor cells . besides expressing the cd133 antigen , cscs express mrnas for several additional recognized nsc markers , including bmi-1 , sox2 and musashi-1 . inspired by identification of cancer stem cells from leukaemia and breast cancer [ 2931 ] , btcscs were reportedly successfully cultured according to the following criteria : ( i ) expression of nsc markers cd133 and nestin ; ( ii ) generation of spheres morphologically indistinguishable from neurospheres ; ( iii ) self - renewal and proliferation and ( iv ) production of differentiated progeny in vitro or recapitulation of the parental tumour mass growth when implanted into immunodeficient animals [ 2022 , 24 , 3233 ] . as few as 100 cd133-positive human cells were sufficient to form brain tumours as xenografts in nod - scid mice . importantly , the cd133-positive btcscs also exhibit high resistance to current chemotherapy and radiotherapy , in contrast to their cd133-negative counterparts [ 3334 ] , an intriguing difference discussed in more detail later in this article . among the unresolved questions of btcscs ' biology is also whether , or to what extent , do the cd133-positive btcscs require interaction with the cd133-negative bulk sub - population of cells . another largely open issue concerns the emerging role of the local microenvironment in supporting the maintenance of these candidate cscs within tumour mass . as discussed below , identification of cd133-positive btcscs and attempts to understand their biology provide powerful new tools and approaches to better understand tumourigenesis in the cns , a research area likely to become crucial in developing novel therapies based on btcscs as a target . angiogenesis is a highly regulated process essential not only in early embryogenesis but also during tissue growth and repair , female reproductive cycle and diverse pathologies , such as inflammation or tumour development and progression [ 3536 ] . localized breakdown of extracellular matrix ( ecm ) precedes proliferation , migration and tissue infiltration of endothelial cells . in time these cells re - model back into capillary structures , and a new ecm is deposited . vegf expression strongly correlates with tumour aggressiveness , metastatic potential , a short time to relapse and , consequently , it commonly indicates poor prognosis in patients with cancer [ 6 , 3843 ] . recent work identified the btcscs to be a key source of angiogenic factors in brain cancer , suggesting that anti - angiogenic therapy targeting these stem - like tumour cell sub - populations might improve the therapy outcome . understanding angiogenesis and its relation to tumour growth and resistance to therapy is of considerable interest , particularly because diffusely infiltrating astrogliomas are mostly refractory to current surgical and adjuvant treatments . arrows indicate very week diffuse vegf staining in the cytoplasm of astroglial elements of a low grade astroglioma who grade ii ( a ) , in contrast to strong cytoplasmic positivity in astroglial elements in a high - grade astroglioma specimen who grade iv ( b ) . both tumour grades exhibit certain level of cytoplasmic positivity in endothelial elements ( arrowheads ) ( original magnification 200x ) . neovascularization in brain tumours correlates markedly with their enhanced aggressiveness , degree of malignancy and poor clinical prognosis and inversely with the post - operative survival time of patients [ 4951 ] . newly formed tumour blood vessels possess an ineffective blood brain barrier that contributes to the pathogenesis of tumour - associated oedema . the characteristic vascularity of astrogliomas has lead to a hypothesis that the formation of new blood vessels is crucial to tumour growth . astroglioma cells have angiogenic activities , in that they promote capillary morphogenesis and endothelial proliferation in vitro . as mentioned above , the pathologic features that distinguish gbm from lower grade astrogliomas are the presence of necrosis with pseudopalisades and a distinct form of angiogenesis , microvascular hyper - plasia [ 5456 ] . these palisades are in part caused by vessel regression and increased tumour cell proliferation [ 56 , 58 ] . analysis of the shapes and sizes of pseudopalisades suggests that these structures evolve and enlarge overtime , giving rise to a gradually expanding coagulative necrosis . such features inspired formulation of a vaso - occlusive and prothrombotic model of pseudopalisade formation in gbm . this emerging concept postulates hypoxic tumour cells migrating away from the central hypoxia that arises after a vascular insult . hypoxia resulting from such conditions is then thought to induce new blood vessels that supply the tumour with necessary metabolites . vegf is highly expressed in pseudopalisading tumour cells adjacent to necrotic zones and hyperplastic vessels in astroglioma . this phenomenon reflects an elevated transcriptional activity of hypoxia - inducible factors 1 and 2 ( hif-1 and -2 ) [ 6264 ] . secretion of vegf , in turn , causes endothelial cell proliferation and angiogenesis followed by microvascular hyperplasia and formation of glomeruloid bodies in gbm and other tumours [ 6566 ] . overexpression of vegf by tumour cells frequently occurs not only in response to hypoxia [ 62 , 67 ] , but also upon loss of function of certain tumour suppressor genes [ 6869 ] and oncogene activation . under hypoxic conditions , transcriptional regulation of vegf is dominated by hif-1 that , together with its target genes , plays a key role in astroglioma - induced angiogenesis [ 7172 ] . chronic oncogenic stimuli , such as activated ras and pi3k pathways appear to enhance hif-1 expression and likely contribute to gbm progression [ 60 , 73 ] . the protooncogene ets-1 and the transcription factor stat3 are also capable of vegf induction and/or activation . ets-1 activates genes for vegf receptors , matrix metalloprotease proteins ( mmps ) and urokinase plasminogen activator ( upa ) , all features that can promote angiogenesis and tumour progression . in addition , several cytokines and growth factors involved in astroglioma pathogenesis , including tgf- , egf , pdgf - b , bfgf , also up - regulate vegf . genetic alterations common in astrogliomas , such as mutational activation of the epidermal growth factor receptor ( egfr ) and loss - of - function mutations of the pten tumour suppressor , lead to enhanced vegf expression and increased angiogenic activity [ 7576 ] . astrogliomas frequently overexpress egfr , and its truncated mutant isoform egfrviii has been implicated in relapse and poor prognosis . enhanced egfr signalling can up - regulate vegf production in brain cancer , while blockage of egfr inhibits secretion of vegf and other angiogenic factors . overall , this accumulating evidence supports the notion that vegf plays a central role in the molecular pathogenesis of astrogliomas and strongly affects the biological behaviour of these tumours . the importance of microvasculature and proper local microenvironment ( niche ) including a plethora of regulatory cytokines and growth factors for the maintenance of stemness of the normal nscs is known . related critical issues for astrogliomas have been whether the btcscs also depend on their niche and , if yes , whether the mutual interdependencies of the niche microenvironment and the stem cells may differ between normal nscs and btcscs . if such differences exist , the specific features of the btcsc - niche interplay might offer potential targets for novel therapeutic interventions . there is little doubt that the nature of the niche microenvironment may represent an important factor in the behaviour of normal nscs and cscs in terms of their stem cell self - renewal and cell - fate decisions . the microenvironment composed of the ecm , stem and progenitor cells , as well as mature , differentiated cells secreting a range of growth factors seems to be important in such processes . balanced microenvironmental levels of various mitogens , including bfgf , egf and sonic hedgehog ( ssh ) , support the propagation of adult nsc in culture and appear to perform similar functions in vivo[8083 ] . histological and ex vivo cell culture studies of mouse tissues suggest that nsc lie within a vascular niche in which endothelial cells regulate stem cell self - renewal [ 8487 ] . growth factors , such as vegf , bfgf , pdgf and egf represent the mitogenic and trophic factors that regulate neurogenesis , while exerting direct neurotrophic and neuroprotective activities . bfgf together with egf are crucial growth factors necessary for nsc , as well as for csc proliferation and maintenance of their self - renewal properties in vitro[22 , 8990 ] . for example , vegf stimulates neurogenesis both in mouse brain cultures in vitro and in neuroproliferative regions ( sub - granular zone and sub - ventricular zone ) of the non - ischaemic mouse brain in vivo . naturally coupled to astrocytes through astrocytic end - feet , endothelial cells are also important components of the niche structure and closely co - operate with astrocytes to regulate adult neurogenesis [ 8485 ] . btcscs also interact with endothelial cells that secrete factors supporting the maintenance of these cells in stem - like cell state . while current evidence supports the idea of vegf as a regulator of neuronal cell fate and a key ingredient of such stem - cell niche in both , normal brain and astroglioma scenarios , one possibly important difference may reflect the plausible role of the vegf as an autocrine factor in astrogliomas , as opposed to only paracrine role under the normal niche conditions . tumours known to overexpress vegf have strong angiogenic potential which , in turn , could explain the documented importance of vegf in astroglioma progression . since vegf and both vegfrs are co - expressed in tumour cells in the majority of nascent primary gbm lesions ( fig . 2 ) , an autocrine role of the vegf in gbm might significantly contribute to tumour growth and invasivity [ 9395 ] . on the other hand , in contrast to vegfs well - established paracrine effects on endothelial cells , less is known about such potential autocrine role of vegf in glioblastoma . while some authors reported that exogenous vegf not only stimulates endothelial cells , but also directly enhances astroglioma cell motility , invasion and pro - liferation ( fig . known receptor types are indicated and identified ligands / ligand isoforms are listed directly in the figure above . cell membrane ( gc membrane ) receptors of the vegf tyrosine kinase receptor family consist of seven ig - like domains , transmembrane region and an intracellular tyrosine kinase binding domain interrupted by a kinase - insert sequence . the aberrant sflt-1 receptor form lacking one ig - like domain as a result of vegfr1 truncation is also shown . the neuropilin receptor acts as a co - receptor for vegfr2 , enhancing binding and biological activity of vegf165 . ligand binding through electrostatic interactions with specific sequences of sulphation within the hs chains leads to receptor dimerization and tyrosine domain phosphorylation , thereby activating the corresponding signalling pathway . ( a ) vegf secreted by tumour endothelial cells ( orange ) induces angiogenesis in an autocrine manner , while vegf secreted by astroglial tumour cells ( pink ) stimulates tumour angiogenesis in a paracrine manner . as reports on other biological effects of the autocrine vegf function in astroglial tumour cells ( b ) hypothetical distinct effects of vegf on the cd133-positive ( vegf secreting and radioresistant ) versus cd133-negative ( radiosensitive ) astroglial cell population . ir - induced vegf secretion by the cd133-positive astroglial cells ( cancer stem - like cells ) could enhance tumour angiogenesis and protection of endothelial cells against apoptosis . furthermore , ir - induced vegf could modulate proliferation of the cd133-positive cells , thereby regulating their sensitivity to ir . ir - induced vegf , massively secreted by the cd133-positive astroglial cell sub - population , might also help to protect these cells from apoptosis and increase their migration in an autocrine manner . despite our effort to understand the stem cell niche is in its infancy , particularly with regard to the btcsc niche and its potential aberrant features , recent studies in this area are encouraging and support a crucial role of a perivascular niche for brain tumour stem - like cells [ 92 , 98 ] . the emerging significance of this concept for development of new astroglioma treatment strategies is discussed in the ultimate section of this article . given the severity of the health problem posed by astrogliomas to the society , embarrassingly little is known about the biological basis and molecular mechanisms that allow brain tumours to survive treatment and recur . apart from surgery and some newly tested targeted therapies , the major modalities presently used in the clinic to treat astrogliomas are dna damaging treatments by ionizing radiation ( ir ) and chemotherapy . in this section , we briefly highlight how the cancer stem cell concept and recent advances in understanding the cellular machinery that responds to dna damage are beginning to converge to shed more light on the notoriously difficult issue of the treatment resistance . although chemotherapeutics , particularly dna alkylating drugs , such as temozolomide are currently widely used in combination with traditional radiotherapy to treat astrogliomas , the molecular basis of sensitivity versus resistance to such drugs is relatively well understood [ 99100 ] , and it will not be further discussed here . rather , we focus our discussion on ir and cellular responses to the most toxic ir - induced lesions , the dna double strand breaks ( dsbs ) . in response to dsb - causing insults such as ir , human cells activate their dna damage response machinery , a sophisticated network of signalling and effector pathways that co - ordinate cell cycle check - points with dna repair and cell death mechanisms [ 101102 ] . relevant for our discussion on cancer cells , dsbs can be generated not only through external genotoxic insults , but also from events within the cell itself , for example , due to metabolic reactive oxygen species , or errors during dna replication . the latter insult , often referred to as replication stress , is commonly caused by various activated oncogenes and loss of some tumour suppressors , leading to constitutively activated dna damage checkpoint signalling in tumours [ 102104 ] . such constitutive activation of the dna damage checkpoints is particularly apparent in borderline pre - malignant and early malignant lesions , resulting in enhanced apoptosis or induction of senescence as an inducible biological barrier against tumour progression [ 102107 ] . despite the existence of such physiological barrier response has been demonstrated for multiple types of human solid tumours , particularly carcinomas and melanomas , it is largely absent in testicular germ cell tumours , and its relevance for astrogliomas remains to be explored . what is important for our discussion about sensitivity versus resistance to dna - damaging therapies is the fact that during their progression , many malignant tumours , at least partially , disable the activated checkpoint barrier through mutations or epigenetic changes in relevant genes , such as p53 [ 102 , 109 ] . alternatively , malignant cells might progress in the face of constitutive dna damage by enhancing the efficiency of their dna repair pathways , in either case altering the overall sensitivity towards potential subsequent therapy by dna damaging modalities , such as ir . at the molecular level , the key element of the cellular dsb response is activation of the apical signalling kinase atm ( and also the atr and dna - pk kinases ) , which rapidly phosphorylate a wide range of substrates including the effector kinases chk2 and chk1 . chk2 and chk1 become activated upon their phosphorylation by atm and atr , respectively , and further propagate the dna damage alert signal to diverse effectors such as the tumour suppressor p53 , or other checkpoint and dna repair proteins . the severity of the dna damage , the effectiveness of the activated cell cycle checkpoint and dna repair mechanisms , as well as other parameters including the protective signals from the microenvironment , then jointly affect the final outcome and cell fate of the irradiated cell . in cancer cells , such a cell - fate decision may be widely variable due to the heterogeneity of tumour cell populations , and it may also reflect alterations in the dna damage machinery acquired during cancer progression ( see above ) . recent work on human glioblastomas now indicates that the cd133-positive stem - like cells may have an enhanced checkpoint response to radiation , and this may contribute to their selective survival and radioresistance . after irradiation , the cd133- positive fraction of human gbm cultures and xenografts was enriched up to fivefold compared with cd133-negative cells . this was not attributable to induction of cd133 expression in cd133-negative tumour cells , but to lower rates of apoptosis among the cd133-positive subset . analysis of the checkpoint machinery pointed to an augmented activation of the chk2 and chk1 kinases , faster repair of the ir - induced dsbs , and overall better survival and ability to recur among the cd133-enriched btcscs . furthermore , the authors used a chemical inhibitor to block the activities of the checkpoint kinases chk1 and chk2 after ir , and this treatment radiosensitized the cd133-positive subset more than the cd133- negative astroglioma cells from the same tumour . collectively , these intriguing results identify a possible mechanism that contributes to radioresistance of btcscs and thereby of the tumour , and suggest that targeting the dna damage checkpoints may be worth considering as an option in the gbm radiotherapy scenario . whether this mechanism may be in any way linked with the effects of vegf or other cytokines in the btcsc niche , and how to exploit these new insights into therapy resistance for in response to ir , vegf secretion by gbm cell lines was highly increased in a radiation dose - dependent manner [ 94 , 111 ] . among all the cell lines tested , the u87 mg is highly radioresistant and expresses the highest ir - induced vegf levels , a correlation that inspired a hypothesis about a potential involvement of ir - enhanced vegf secretion in radioresistance . based on observations of decreased gbm cell proliferation in response to exogenous vegf , it was proposed that expression of vegf would at the same time induce growth of new blood vessels ensuring a better supply of oxygen and nutrients and reduce gbm cell proliferation resulting in decreased sensitivity to ir . since proliferating cells are more sensitive to irradiation than quiescent cells and since vegf protects tumour blood vessels from irradiation- mediated toxicity , this suggests a possible mechanism through which gbm cells can escape the consequences of radiation treatment . after irradiation , the vegf gene promoter becomes stimulated via multiple mitogenactivated protein kinase ( mapk ) dependent pathways in both cultured normal human astrocytes and gbm cell lines . since hif-1 was not overexpressed under such conditions , hypoxia does n't seem to be involved in this mechanism . given the radioresistance of btcscs discussed in the previous section , could these data jointly indicate a connection between radiation - induced vegf secretion , increased angiogenesis and selective survival of the cd133- positive btcscs ? could ir - induced vegf secretion by btcscs selectively regulate their own migration and/or proliferation in an autocrine manner while protecting them from ir - induced apoptosis ( fig . 3b ) , possibly through some so - far unidentified link with dna damage checkpoint signalling or repair ? despite speculative at present , this idea seems indirectly supported by recent analysis of the so - called bystander effects of radiation . this study showed that conditioned media from irradiated human glioblastoma cells contained factors including cytokines , whose membrane - mediated signalling to non - irradiated cells resulted in activation of the checkpoint kinase atr and cellular dna damage response without direct exposure of such bystander cells to radiation . these results imply that secreted cytokines may be capable of inducing dna damage checkpoints , and that such bystander response differs in gbm cells compared with normal astrocytes , suggesting that these effects might be exploited through therapeutic targeting . the arguments discussed so far point to critical roles of vegf and btcsc niche , as well as the status of the dna damage response machinery , both of which show unique features in the treatment - resistant astroglioma stem - like cells . these accumulating results identify both vegf - mediated and dna damage signalling cascades as promising targets for treatment of astrogliomas , a notion that is supported also by recent successful attempts to experimentally target either vegf signalling alone [ 4 , 115 ] , or combine such vascular niche - targeting treatment with dna - damaging chemotherapy ( see below ) . formation of multiple vascular btcsc niches , each possibly capable of giving rise to a recurrent tumour , may strongly facilitate tumour growth and invasion [ 116118 ] . if btcsc are true tumour - initiating cells , then drugs selectively killing these cells could prove highly effective treatments for astrogliomas . encouraging are recent data providing proof of principle that selective targeting of cscs is possible , at least in some types of malignancies [ 34 , 44 , 119 ] . there is no doubt that additional factors of astroglioma biology may hinder attempts to successfully introduce combined vegf- and dna damage checkpoint - targeting treatment strategies . for example , infiltration of tumour cells into surrounding brain contributes to the treatment - refractory nature of malignant astrogliomas . moreover , the blood brain barrier represents a significant obstacle , preventing the delivery of large - molecular - weight polar compounds to brain tumour cells . in addition , brain tissue highly sensitive to cytotoxic treatments , and our own data show that inhibition of checkpoint kinases such as chk1 causes endogenous dna damage in proliferating human cells , thereby raising concerns as to the suitability of this approach for therapy . with the exception of the modest activity associated with temozolomid , there is no standard chemotherapy available for patients with high - grade astrogliomas , and resistance to chemotherapy is common . on the optimistic side bevacizumab ( avastin ) , a recombinant , humanized monoclonal antibody targeting vegf , has been recently approved for use in colorectal carcinoma - based on significant survival benefit observed following its addition to fluorouracil - based chemotherapy . preliminary results from single - arm phase ii study of bevacizumab with irinotecan ( cpt-11 ) , currently underway at the preston robert tisch brain tumor center at the duke university medical center for patients with recurrent malignant astroglioma , indicate that the most effective therapy of gbm identified to date could be a combined treatment by bevacizumab with a dna - damaging drug such as cpt-11 . thus , despite possible numerous obstacles that must not be underestimated , we believe that the available evidence justifies attempts to identify a clinically feasible , effective combination of therapeutic approaches that would allow complementary targeting of the vegf / niche , and the dna damage check - point aspects of malignant astrogliomas , with special emphasis on targeting the unique features of the candidate astroglioma stem cells ( fig . 4 ) . in general terms , such a combined strategy may include available or future drugs or antibodies to inhibit vegf and/or its receptor - mediated signalling , along with standard dna damaging treatment modalities , such as ir and alkylating drugs , complemented by selective inhibitors of checkpoint signalling or dna repair to counteract the mechanisms underlying astroglioma resistance to such treatments . in any case , given the close relationship between tumour microenvironment , vascular architecture and tumour response to therapy , it seems logical to investigate the potential role of vegf not only as an angiogenic factor stimulating formation of pathological vascular niche , but also as a potential regulator of btcsc cellfate . perhaps in concert with acquired defects within the apoptotic machinery and/or selective regulation of dna damage signalling / repair , vegf and vascular niche might help btcscs escape from the toxic effects of chemotherapy and radiotherapy by protecting them from dna damage - induced apoptosis [ 126129 ] . better mechanistic understanding of these biological processes appears to be more plausible based on the recent advances in the field , and such research will hopefully help to improve the presently dismal prognosis of astroglioma patients . potential therapeutic approaches targeting pathological vascular niche and dna damage checkpoint as unique features of btcscs . accumulating evidence suggests that the cd133-positive , candidate btcscs , harbour specific features that could be exploited in future combinatorial therapies of malignant astrogliomas . such potential treatment combination might include classical dna - damaging modalities : ionizing radiation ( ir ) , chemotherapy ( ct ) or both ( therapy 1 ) , a concomitant blockade of dna damage checkpoint signalling and/or dna repair ( therapy 2 ) , and inhibition of the cytokine - mediated paracrine / autocrine circuitry ( asterisk ) such as the vegf effects ( therapy 3 ) .

abstractmalignant astrogliomas are among the most aggressive , highly vascular and infiltrating tumours bearing a dismal prognosis , mainly due to their resistance to current radiation treatment and chemotherapy . efforts to identify and target the mechanisms that underlie astroglioma resistance have recently focused on candidate cancer stem cells , their biological properties , interplay with their local microenvironment or niche and their role in tumour progression and recurrence . both paracrine and autocrine regulation of astroglioma cell behaviour by locally produced cytokines such as the vascular endothelial growth factor ( vegf ) are emerging as key factors that determine astroglioma cell fate . here , we review these recent rapid advances in astroglioma research , with emphasis on the significance of vegf in astroglioma stem - like cell biology . furthermore , we highlight the unique dna damage checkpoint properties of the cd133-marker - positive astroglioma stem - like cells , discuss their potential involvement in astroglioma radioresistance and consider the implications of this new knowledge for designing combinatorial , more efficient therapeutic strategies .

Introduction Adult brain a dynamic structure with active stem cells? Cancer stem-like cells in brain tumours Angiogenesis in astrogliomas VEGF and the stem cell niche Resistance to DNA-damaging therapy in astrogliomas VEGF and DNA damage response: implications for astroglioma therapy

considering their poor prognosis and resistance to chemotherapy and radiotherapy , brain tumours in general , and high - grade astrogliomas in particular , are among the most devastating types of human cancer . this notorious heterogeneity of brain tumours likely facilitates development of treatment - resistant clones , a process that is further fuelled by the ability of astroglioma cells to shift between quiescent and proliferating states . classification of adult brain tumours is based on the world health organization ( who ) classification of nervous system tumours . who grade iv lesions are mitotically active , necrosis - prone and commonly associated with a rapid pre - operative and post - operative evolution of disease the most malignant types of astrogliomas are anaplastic astrocytoma ( grade iii astrocytoma ) and glioblastoma multiforme ( gbm , grade iv astrocytoma ) . one of the key features of astrogliomas , affecting their biological and clinical behaviour , is dense vascularization . , multiple therapies targeting the molecular regulators of this mechanism have been tested with variable clinical efficacy . for example , one study showed that systemic therapy with a mono- clonal antibody against vascular endothelial growth factor receptor 2 ( vegfr2 ) inhibited tumour growth in mice by some 80% . particularly relevant to this review , several pieces of evidence indicate prognostic significance of vegf in high - grade astrogliomas [ 68 ] . in this article , we highlight the emerging role(s ) of vegf signalling in astroglioma biology , including the possible links with dna damage checkpoints in response to radiation treatment . furthermore , we postulate and discuss a hypothetical function of autocrine vegf signalling in regulation of the so - called brain tumour - derived cancer stem - like cells ( btcsc ) and their intriguing resistance to therapy . a small percentage of quiescent cells present in the adult hippocampal dentate gyrus ( sub - granular zone , sgz ) , sub - ventricular zone of the lateral ventricles ( svz ) and olfactory bulbs are undifferentiated and multipotent neural stem cells ( nsc ) capable of self - renewal [ 1415 ] . in the adult mammalian brain , we are just beginning to identify the cellular and molecular features that characterize the neurogenic niches in the svz and sgz , and the mechanisms by which the full range of adult nsc development is regulated . some of the cells within the neurospheres proliferate as multipotent , self - renewing nscs upon stimulation with either epidermal growth factor ( egf ) or basic fibroblast growth factor ( bfgf ) . recently , a small population of cancer stem - like cells ( cscs ) has been identified in adult and paediatric brain tumours , as well as in established cell lines [ 2024 ] . these cells express a number of nsc markers including cd133 , a cell surface protein which is extensively exploited for enrichment of stem - like cells using fluorescence activated cell sorting ( facs ) and/or magnetic bead separation , thereby providing cells to be examined in diverse applications [ 2526 ] . the human cd133 antigen , also known as ac133 , was originally identified as a marker of haematopoietic stem cells , however its function has remained unclear . a characteristic feature of the cd133 protein is its rapid down - regulation during cell differentiation [ 2829 ] , which makes it a cell surface marker uniquely suited for the identification and isolation of stem - like and progenitor cells . inspired by identification of cancer stem cells from leukaemia and breast cancer [ 2931 ] , btcscs were reportedly successfully cultured according to the following criteria : ( i ) expression of nsc markers cd133 and nestin ; ( ii ) generation of spheres morphologically indistinguishable from neurospheres ; ( iii ) self - renewal and proliferation and ( iv ) production of differentiated progeny in vitro or recapitulation of the parental tumour mass growth when implanted into immunodeficient animals [ 2022 , 24 , 3233 ] . importantly , the cd133-positive btcscs also exhibit high resistance to current chemotherapy and radiotherapy , in contrast to their cd133-negative counterparts [ 3334 ] , an intriguing difference discussed in more detail later in this article . another largely open issue concerns the emerging role of the local microenvironment in supporting the maintenance of these candidate cscs within tumour mass . angiogenesis is a highly regulated process essential not only in early embryogenesis but also during tissue growth and repair , female reproductive cycle and diverse pathologies , such as inflammation or tumour development and progression [ 3536 ] . recent work identified the btcscs to be a key source of angiogenic factors in brain cancer , suggesting that anti - angiogenic therapy targeting these stem - like tumour cell sub - populations might improve the therapy outcome . understanding angiogenesis and its relation to tumour growth and resistance to therapy is of considerable interest , particularly because diffusely infiltrating astrogliomas are mostly refractory to current surgical and adjuvant treatments . neovascularization in brain tumours correlates markedly with their enhanced aggressiveness , degree of malignancy and poor clinical prognosis and inversely with the post - operative survival time of patients [ 4951 ] . overexpression of vegf by tumour cells frequently occurs not only in response to hypoxia [ 62 , 67 ] , but also upon loss of function of certain tumour suppressor genes [ 6869 ] and oncogene activation . under hypoxic conditions , transcriptional regulation of vegf is dominated by hif-1 that , together with its target genes , plays a key role in astroglioma - induced angiogenesis [ 7172 ] . genetic alterations common in astrogliomas , such as mutational activation of the epidermal growth factor receptor ( egfr ) and loss - of - function mutations of the pten tumour suppressor , lead to enhanced vegf expression and increased angiogenic activity [ 7576 ] . overall , this accumulating evidence supports the notion that vegf plays a central role in the molecular pathogenesis of astrogliomas and strongly affects the biological behaviour of these tumours . the importance of microvasculature and proper local microenvironment ( niche ) including a plethora of regulatory cytokines and growth factors for the maintenance of stemness of the normal nscs is known . related critical issues for astrogliomas have been whether the btcscs also depend on their niche and , if yes , whether the mutual interdependencies of the niche microenvironment and the stem cells may differ between normal nscs and btcscs . if such differences exist , the specific features of the btcsc - niche interplay might offer potential targets for novel therapeutic interventions . the microenvironment composed of the ecm , stem and progenitor cells , as well as mature , differentiated cells secreting a range of growth factors seems to be important in such processes . growth factors , such as vegf , bfgf , pdgf and egf represent the mitogenic and trophic factors that regulate neurogenesis , while exerting direct neurotrophic and neuroprotective activities . for example , vegf stimulates neurogenesis both in mouse brain cultures in vitro and in neuroproliferative regions ( sub - granular zone and sub - ventricular zone ) of the non - ischaemic mouse brain in vivo . btcscs also interact with endothelial cells that secrete factors supporting the maintenance of these cells in stem - like cell state . while current evidence supports the idea of vegf as a regulator of neuronal cell fate and a key ingredient of such stem - cell niche in both , normal brain and astroglioma scenarios , one possibly important difference may reflect the plausible role of the vegf as an autocrine factor in astrogliomas , as opposed to only paracrine role under the normal niche conditions . tumours known to overexpress vegf have strong angiogenic potential which , in turn , could explain the documented importance of vegf in astroglioma progression . 2 ) , an autocrine role of the vegf in gbm might significantly contribute to tumour growth and invasivity [ 9395 ] . on the other hand , in contrast to vegfs well - established paracrine effects on endothelial cells , less is known about such potential autocrine role of vegf in glioblastoma . while some authors reported that exogenous vegf not only stimulates endothelial cells , but also directly enhances astroglioma cell motility , invasion and pro - liferation ( fig . cell membrane ( gc membrane ) receptors of the vegf tyrosine kinase receptor family consist of seven ig - like domains , transmembrane region and an intracellular tyrosine kinase binding domain interrupted by a kinase - insert sequence . the aberrant sflt-1 receptor form lacking one ig - like domain as a result of vegfr1 truncation is also shown . as reports on other biological effects of the autocrine vegf function in astroglial tumour cells ( b ) hypothetical distinct effects of vegf on the cd133-positive ( vegf secreting and radioresistant ) versus cd133-negative ( radiosensitive ) astroglial cell population . ir - induced vegf secretion by the cd133-positive astroglial cells ( cancer stem - like cells ) could enhance tumour angiogenesis and protection of endothelial cells against apoptosis . furthermore , ir - induced vegf could modulate proliferation of the cd133-positive cells , thereby regulating their sensitivity to ir . despite our effort to understand the stem cell niche is in its infancy , particularly with regard to the btcsc niche and its potential aberrant features , recent studies in this area are encouraging and support a crucial role of a perivascular niche for brain tumour stem - like cells [ 92 , 98 ] . the emerging significance of this concept for development of new astroglioma treatment strategies is discussed in the ultimate section of this article . given the severity of the health problem posed by astrogliomas to the society , embarrassingly little is known about the biological basis and molecular mechanisms that allow brain tumours to survive treatment and recur . apart from surgery and some newly tested targeted therapies , the major modalities presently used in the clinic to treat astrogliomas are dna damaging treatments by ionizing radiation ( ir ) and chemotherapy . in this section , we briefly highlight how the cancer stem cell concept and recent advances in understanding the cellular machinery that responds to dna damage are beginning to converge to shed more light on the notoriously difficult issue of the treatment resistance . although chemotherapeutics , particularly dna alkylating drugs , such as temozolomide are currently widely used in combination with traditional radiotherapy to treat astrogliomas , the molecular basis of sensitivity versus resistance to such drugs is relatively well understood [ 99100 ] , and it will not be further discussed here . rather , we focus our discussion on ir and cellular responses to the most toxic ir - induced lesions , the dna double strand breaks ( dsbs ) . in response to dsb - causing insults such as ir , human cells activate their dna damage response machinery , a sophisticated network of signalling and effector pathways that co - ordinate cell cycle check - points with dna repair and cell death mechanisms [ 101102 ] . relevant for our discussion on cancer cells , dsbs can be generated not only through external genotoxic insults , but also from events within the cell itself , for example , due to metabolic reactive oxygen species , or errors during dna replication . the latter insult , often referred to as replication stress , is commonly caused by various activated oncogenes and loss of some tumour suppressors , leading to constitutively activated dna damage checkpoint signalling in tumours [ 102104 ] . such constitutive activation of the dna damage checkpoints is particularly apparent in borderline pre - malignant and early malignant lesions , resulting in enhanced apoptosis or induction of senescence as an inducible biological barrier against tumour progression [ 102107 ] . what is important for our discussion about sensitivity versus resistance to dna - damaging therapies is the fact that during their progression , many malignant tumours , at least partially , disable the activated checkpoint barrier through mutations or epigenetic changes in relevant genes , such as p53 [ 102 , 109 ] . alternatively , malignant cells might progress in the face of constitutive dna damage by enhancing the efficiency of their dna repair pathways , in either case altering the overall sensitivity towards potential subsequent therapy by dna damaging modalities , such as ir . at the molecular level , the key element of the cellular dsb response is activation of the apical signalling kinase atm ( and also the atr and dna - pk kinases ) , which rapidly phosphorylate a wide range of substrates including the effector kinases chk2 and chk1 . chk2 and chk1 become activated upon their phosphorylation by atm and atr , respectively , and further propagate the dna damage alert signal to diverse effectors such as the tumour suppressor p53 , or other checkpoint and dna repair proteins . the severity of the dna damage , the effectiveness of the activated cell cycle checkpoint and dna repair mechanisms , as well as other parameters including the protective signals from the microenvironment , then jointly affect the final outcome and cell fate of the irradiated cell . in cancer cells , such a cell - fate decision may be widely variable due to the heterogeneity of tumour cell populations , and it may also reflect alterations in the dna damage machinery acquired during cancer progression ( see above ) . recent work on human glioblastomas now indicates that the cd133-positive stem - like cells may have an enhanced checkpoint response to radiation , and this may contribute to their selective survival and radioresistance . this was not attributable to induction of cd133 expression in cd133-negative tumour cells , but to lower rates of apoptosis among the cd133-positive subset . analysis of the checkpoint machinery pointed to an augmented activation of the chk2 and chk1 kinases , faster repair of the ir - induced dsbs , and overall better survival and ability to recur among the cd133-enriched btcscs . furthermore , the authors used a chemical inhibitor to block the activities of the checkpoint kinases chk1 and chk2 after ir , and this treatment radiosensitized the cd133-positive subset more than the cd133- negative astroglioma cells from the same tumour . collectively , these intriguing results identify a possible mechanism that contributes to radioresistance of btcscs and thereby of the tumour , and suggest that targeting the dna damage checkpoints may be worth considering as an option in the gbm radiotherapy scenario . whether this mechanism may be in any way linked with the effects of vegf or other cytokines in the btcsc niche , and how to exploit these new insights into therapy resistance for in response to ir , vegf secretion by gbm cell lines was highly increased in a radiation dose - dependent manner [ 94 , 111 ] . among all the cell lines tested , the u87 mg is highly radioresistant and expresses the highest ir - induced vegf levels , a correlation that inspired a hypothesis about a potential involvement of ir - enhanced vegf secretion in radioresistance . based on observations of decreased gbm cell proliferation in response to exogenous vegf , it was proposed that expression of vegf would at the same time induce growth of new blood vessels ensuring a better supply of oxygen and nutrients and reduce gbm cell proliferation resulting in decreased sensitivity to ir . 3b ) , possibly through some so - far unidentified link with dna damage checkpoint signalling or repair ? this study showed that conditioned media from irradiated human glioblastoma cells contained factors including cytokines , whose membrane - mediated signalling to non - irradiated cells resulted in activation of the checkpoint kinase atr and cellular dna damage response without direct exposure of such bystander cells to radiation . the arguments discussed so far point to critical roles of vegf and btcsc niche , as well as the status of the dna damage response machinery , both of which show unique features in the treatment - resistant astroglioma stem - like cells . these accumulating results identify both vegf - mediated and dna damage signalling cascades as promising targets for treatment of astrogliomas , a notion that is supported also by recent successful attempts to experimentally target either vegf signalling alone [ 4 , 115 ] , or combine such vascular niche - targeting treatment with dna - damaging chemotherapy ( see below ) . if btcsc are true tumour - initiating cells , then drugs selectively killing these cells could prove highly effective treatments for astrogliomas . there is no doubt that additional factors of astroglioma biology may hinder attempts to successfully introduce combined vegf- and dna damage checkpoint - targeting treatment strategies . in addition , brain tissue highly sensitive to cytotoxic treatments , and our own data show that inhibition of checkpoint kinases such as chk1 causes endogenous dna damage in proliferating human cells , thereby raising concerns as to the suitability of this approach for therapy . with the exception of the modest activity associated with temozolomid , there is no standard chemotherapy available for patients with high - grade astrogliomas , and resistance to chemotherapy is common . preliminary results from single - arm phase ii study of bevacizumab with irinotecan ( cpt-11 ) , currently underway at the preston robert tisch brain tumor center at the duke university medical center for patients with recurrent malignant astroglioma , indicate that the most effective therapy of gbm identified to date could be a combined treatment by bevacizumab with a dna - damaging drug such as cpt-11 . thus , despite possible numerous obstacles that must not be underestimated , we believe that the available evidence justifies attempts to identify a clinically feasible , effective combination of therapeutic approaches that would allow complementary targeting of the vegf / niche , and the dna damage check - point aspects of malignant astrogliomas , with special emphasis on targeting the unique features of the candidate astroglioma stem cells ( fig . in general terms , such a combined strategy may include available or future drugs or antibodies to inhibit vegf and/or its receptor - mediated signalling , along with standard dna damaging treatment modalities , such as ir and alkylating drugs , complemented by selective inhibitors of checkpoint signalling or dna repair to counteract the mechanisms underlying astroglioma resistance to such treatments . perhaps in concert with acquired defects within the apoptotic machinery and/or selective regulation of dna damage signalling / repair , vegf and vascular niche might help btcscs escape from the toxic effects of chemotherapy and radiotherapy by protecting them from dna damage - induced apoptosis [ 126129 ] . better mechanistic understanding of these biological processes appears to be more plausible based on the recent advances in the field , and such research will hopefully help to improve the presently dismal prognosis of astroglioma patients . potential therapeutic approaches targeting pathological vascular niche and dna damage checkpoint as unique features of btcscs . such potential treatment combination might include classical dna - damaging modalities : ionizing radiation ( ir ) , chemotherapy ( ct ) or both ( therapy 1 ) , a concomitant blockade of dna damage checkpoint signalling and/or dna repair ( therapy 2 ) , and inhibition of the cytokine - mediated paracrine / autocrine circuitry ( asterisk ) such as the vegf effects ( therapy 3 ) .

heart performance and kidney function are closely interconnected , both in healthy and in disease conditions . it is also clear that there is a strong connection between renal and cardiovascular diseases . primary disorders of one of these two organs often result in secondary dysfunction or injury to the other . in this paper , we discuss about the adpkd and its relation with cardiorenal syndrome . ( crs ) was defined as the pathophysiological disorder of the heart and kidney in which acute or chronic dysfunction in one organ may induce acute or chronic dysfunction in the other organ . a large number of direct and indirect effects of each organ dysfunction can initiate and perpetuate the combined disorder of the two organs through a complex combination of neurohumoral feedback mechanisms . for this reason , it was necessary to classify and divide the cardiorenal syndrome into different subtypes to provide a more concise and logically correct approach to this condition ( see table 1 ) . patients with ckd are at higher risk for cardiovascular events , and they have a 10- to 20-fold increased risk of cardiac death compared with age - gender - matched control subjects without ckd . part of this problem may be related to the fact that such individuals are also less likely to receive risk - modifying interventions compared to their non - ckd counterparts . the association between reduced renal function and cardiovascular risk appears to consistently occur at estimated gfr levels below 60 ml / min/1.73 m . clinically , it is very difficult to distinguish between crs type 2 ( chronic cardiorenal syndrome ) and crs type 4 ( chronic renocardiac syndrome ) because often it is not clear whether the primary cause of the syndrome depends on the heart or the kidney . autosomal dominant polycystic kidney disease , a genetic disease that causes ckd , could be viewed as an ideal prototype of crs type 4 because it is certain that the kidney disease is the primary process . in this paper , we will briefly review the epidemiology of adpkd , conventional and novel biomarkers which may be useful in following the disease process , and prevention and treatment strategies . it accounts for ~10% of patients on renal replacement therapy representing an important cause of end - stage renal disease ( esrd ) worldwide . prevalence of the disease is higher than that of huntington disease , hemophilia , sickle cell disease , cystic fibrosis , myotonic dystrophy , and down syndrome combined , and it occurs in approximately 1 of every 400 to 1000 live births . epidemiological data on the prevalence of adpkd have been extensively reported , mainly in the united states and europe . adpkd is the fourth leading cause of ckd in the united states accounting for approximately 3% of cases . in europe , adpkd as etiology of ckd stage v has been reported as 7.8 and 6.0 per million for men and women , respectively . with the advent of renal replacement therapy , adpkd is a genetically heterogeneous disease identified by two phenotypically similar forms associated with several mutations in two genes : the pkd1 gene located on chromosome 16 ( 16p13.3 ) and the pkd2 gene mapped to chromosome 4 ( 4q13q23 ) . a variety of genetic defects have been described in adpkd patients , including deletions , frameshift , and missense mutations . mutations of pkd1 gene , encoding the polycystin-1 protein , result in adpkd type i ( adpkd1 ) which is responsible for approximately 85% of adpkd cases . gene pkd2 mutations , encoding the polycystin-2 protein , result in adpkd type ii ( adpkd2 ) , corresponding to 15% of adpkd cases . polycystin-1 is a large integral membrane protein with a domain architecture suggesting a function in cell - cell or cell - matrix interaction . polycystin-2 is a member of the calcium - permeable subfamily of transient receptor potential channels and forms a complex with polycystin-1 . polycystins are expressed in vascular smooth muscle and endothelia ; it suggests a direct role of these proteins in the vascular manifestations of adpkd [ 18 , 19 ] . mutations in these genes lead to abnormalities in cell proliferation , apoptosis , tubular basement membranes , and tubular fluid secretion , ultimately resulting in slowly expanding renal cysts . the precise processes leading to cyst formation and loss of renal function remain incompletely understood . several mechanisms contributing to the cyst formation have been identified , including a imbalance between epithelial cell proliferation and apoptosis , secretor defects , altered cell matrix interactions , cell polarity , ciliary dysfunction , and altered intracellular signaling . clinically , adpkd is an adult - onset disease characterized by progressive , bilateral renal cyst development and expansion of the kidneys . many patients with adpkd are completely asymptomatic and often are diagnosed because of their positive family history or the development of hypertension ( hp ) . whether adpkd2 patients are compared to adpkd1 cysts and kidney failure occur at an earlier age in adpkd1 ; the average age of ckd stage v is approximately 57 years in type i versus 69 years in type ii . adpkd is a disease with a variable clinical course not only among families with different mutations , but within families with a defined mutation as well ; it can be explained to the large extent by its genetic heterogeneity and modifier genes . liver cysts develop in more than 80% of patients , and the cysts are usually larger in women than in men . . other locations of cysts include the spleen , arachnoid membranes , and seminal vesicles in men . the new onset of hp in a patient at risk for polycystic kidney disease should prompt aggressive treatment and diagnostic studies . these patients during the course of adpkd have early and more severe left ventricular hypertrophy ( lvh ) . urinary tract infections are also common . a number of noncystic manifestations such as cardiovascular deficits , cardiac valve abnormalities , diverticular disease , and intracranial aneurysms are also associated with adpkd ; in fact , cardiovascular complications are the major cause of morbidity and mortality in patients with adpkd . mitral valve prolapsed ( mvp ) occurs in about 26% of affected adults compared with 2% of control subjects [ 27 , 28 ] . pericardial effusion may occur with an increased frequency in patients with adpkd ( 35% versus 9% in a control group of patients with another chronic nephropathy ) , possibly as a result of increased compliance ( or as a collagen protein dysfunction ) of the parietal pericardium . it is known that the kidney disease is strongly associated with a greater carotid intima - media thickness ( imt ) . subjects with adpkd , even with preserved renal function , have a greater carotid imt compared with healthy controls ; carotid imt is higher in hypertensive adpkd patients . it is challenging to clearly segregate those cardiovascular features due to the genetic disorder versus the secondary cardiovascular consequences of declining kidney function per se . however , recent improvement and expansion of genetically modified adpkd animal models which mimic the human form are providing additional insights into the molecular mechanisms governing these disease processes as well as the development of cardiovascular complications . several studies have provided convincing evidence that these vascular abnormalities are caused by alterations in the arterial wall linked to mutations in pkd1 or pkd2 . heterozygous mutant pkd1 or pkd2 mice appear normal but develop single cysts in the kidney or liver late in life and have a reduced overall lifespan [ 23 , 31 ] . homozygous null mutant mice are embryonically lethal and die in utero or perinatally because of systemic defects with massively enlarged cystic kidneys , pancreatic ductal cysts , and pulmonary hypoplasia and often exhibit edema , vascular leaks , and rupture of blood vessels . it suggests the role of polycystins for the structural integrity of blood vessels [ 23 , 31 , 32 ] . in addition , most of the homozygous knockout embryos display multiple cardiac abnormalities including cardiac septation defects , double outlet right ventricle , and pericardial effusions [ 31 , 33 ] . moreover , kurbegovic et al . engineered and described a pkd1 transgenic mice ( pkd1(tag ) mice ) that , in addition to the cystic phenotype , developed cardiac anomalies with severe left ventricular hypertrophy , marked aortic arch distention , and/or valvular stenosis and calcification . therefore , the cardiovascular complications seen in adpkd patients begin to be recognized not only as a consequence of declining kidney function , but also as a defect due to the loss of polycystin-1 and/or polycystin-2 function in cardiovascular organs . as previously mentioned , hypertension and its consequent left ventricular hypertrophy are common in adpkd patients , even in young adults , compared with unaffected controls . moreover , hp and lvh are associated with a faster progression to esrd and an increased cardiovascular mortality [ 35 , 36 ] . it is well known that hp and lvh are associated with an accelerated rate of renal functional deterioration . both hp and lvh are important risk factors for cardiovascular death , the most frequent cause of mortality in adpkd patients ; thus left ventricular hypertrophy may be considered a powerful indicator of mortality . hypertension in adpkd occurs before the loss of kidney function in 60% of affected individuals and increases to almost 100% in patients with ckd stage iv - v [ 39 , 40 ] . the average age of onset of hypertension is 3034 years , with men more commonly affected than women . in added , its occurrence is earlier and more common in adpkd1 than adpkd2 patients . the mechanisms leading to hypertension in adpkd are not well understood . however , it is now well known that increased activity of the intrarenal rather than the systemic renin - angiotensin system ( ras ) is responsible for many forms of hypertension . ( ang ii ) production with the inability to reduce ang ii in response to a high sodium intake will result in resetting the pressure - natriuresis relationship towards higher blood pressures leading to hypertension [ 44 , 45 ] . hypertension is associated with larger kidney size , reflecting a larger number of cysts and with the severity of kidney disease . hypertensive adpkd patients with normal kidney function show greater kidney volumes versus age - matched normotensive adpkd men and women increased proteinuria and decreased renal blood flow . renal blood flow is reduced in hypertensive adpkd patients versus matched essential hypertensive patients , and the renal resistive indices are also increased in hypertensive adpkd subjects and are correlated with a loss of kidney function . renal structural changes play an important role in the pathogenesis of the hp , and renal arteriograms from end - stage adpkd - nephrectomized specimens demonstrate marked attenuation of the vasculature due to the extrinsic compression by the presence of the cysts and their replacement by the latter . renal angiographic images of hypertensive adpkd patients ( from mild to advanced renal failure ) show a large amount of a vascular renal substance peripheral to the outermost branches of the arterial tree . hypertensive adpkd adults with normal kidney function show a greater frequency of lvh versus normotensive adpkd men ( 50% versus 30% ) and women ( 52% versus 22% ) as well as with healthy controls . they also show greater left ventricular mass index ( lvmi ) in comparison to matched essential hypertensive population [ 43 , 50 ] . the prevalence of lvh is increased even in the early stages of ckd , and the frequency increases progressively as renal function decreases . several studies have shown increased lvmi , left ventricular diastolic dysfunction , endothelial dysfunction , and increased carotid imt in young normotensive patients with adpkd with well - preserved renal function . these findings suggest the cardiovascular involvement in the early stages during the course of adpkd . in experimental studies , hypertrophy was found not only in the left , but also in the right ventricle ; these findings exclude that simple hemodynamic factors ( increased preload and afterload ) are the only explanation . it is quite important to appreciate that hypertrophic remodeling comprises not only cardiomyocyte hypertrophy , but also interstitial fibrosis and microvessel disease . a major complication of adpkd includes the intracranial aneurysms ( icas ) ; the prevalence of ica in patients with adpkd ranges from 4 to 12% , compared to a prevalence of 1% for the general population . a familial clustering of ica is found , with a 5 times greater chance of detecting an ica in a subject with a relative with a ruptured ica [ 52 , 53 ] ; it suggests that genetic factors may be associated with the development of this complication . this family clustering is in agreement with the finding that patients with mutations in the 5 region of pkd1 are more likely to have ica than patients with 3 mutations , especially in those with ica rupture before 40 years old and in families with multiple cases of ica or other vascular events . computed tomography , magnetic resonance angiography , and classical angiography are screening tests for detection of intracranial aneurysms in adpkd subjects at high risk . the routine screening for asymptomatic ica in all adpkd individuals is not indicated , but in families with a proven case of ica , a screening analysis is recommend in consultation with a neurosurgeon . in general , biomarkers can be divided into 3 subtypes based on the technical procedures used . biomarkers measured by laboratory tests are defined as laboratory or molecular biomarkers ; those related to signaling , imaging , and functional tests are defined as functional biomarkers ; those related to genetic polymorphisms and other genomic tests are defined as genetic biomarkers . in patients with adpkd , a limited number of biomarkers have been investigated . in a recent study , meijer et al . investigated urinary biomarkers for different segments of the nephron in patients with adpkd versus healthy controls . they choose urinary immunoglobulin g ( igg ) as a marker of glomerular damage ; urinary 2-microglobulin ( b2 m ) , urinary kidney injury molecule 1 ( kim-1 ) , n - acetyl--d - glucosammide ( nag ) , and neutrophil gelatinase - associated lipocalin ( ngal ) as markers for damage of the proximal tubule ; urinary heart - type fatty acid binding protein ( hfabp ) as a marker for damage of the distal tubule . urinary macrophage migration inhibitory factor ( mif ) and monocyte chemotatic protein 1 ( mcp-1 ) were chosen as markers of inflammation . the most important finding of this study is that excretion of all urinary biomarkers from all segments of the nephron was increased in patients with adpkd compared with control subjects . furthermore , ngal excretion was associated with renal blood flow and total renal volume independent of albuminuria . in addition , b2 m and h - fabp were associated inversely with measured gfr and effective renal blood flow independent of albuminuria ; kim-1 , ngal , and mcp-1 were associated positively with total renal volume independent of albuminuria . in a recent paper , investigated and reported urinary and serum ngal levels in a sample of 26 adpkd patients . they found that urinary and serum ngal levels were higher in adpkd patients than in control subjects . they found a strong correlation with the glomerular filtration rate . in addiction , they divided patients into two groups according to the cystic development and kidney dimensions ; subjects with higher cystic growth presented higher urinary and serum ngal values with respect to others . they concluded that higher levels of ngal are correlated to higher cystic growth and suggested that this protein could be also involved in the process of cystogenesis . in 92 patients with adpkd , casal et al . reported a comparative study for three biomarker tests of early kidney damage such as urinary albumin and total -n - acetylhexosaminidase ( hex ) and its isoenzymes ( hex a , hex b ) , as well as serum glutathione peroxidase , which has been considered as a marker of renal proximal tubular function . they found a frequent elevation of the urinary hex and an alteration of its isoenzymatic profile , with 31% of the normotensive adpkd subjects with normoalbuminuria already presenting an increased proportion of hex b isoenzyme . furthermore , keeping age constant , they reported a partial significant correlation between the ultrasound score ( kidney size and number of cysts ) and the proportion of hex b , but not with albuminuria or cystatin c. this confirms the hypothesis that tubular damage plays a role in the pathogenesis and progression of adpdk . wong et al . valued measure gfr and serum cystatin c ( cys c ) levels in 18 children with adpkd versus 41 children with minor renal pathological states . serum creatinine levels did not differ between the adpkd and control group , but gfr was significantly greater in the adpkd group than in controls . this study corroborates the increase of gfr in children and adolescents with adpkd and the superior diagnostic performance of cys c . in fact , in patients with ckd , cys c was proposed to perform better as a marker of gfr than serum creatinine . apart from being a good marker for renal function , cys c appears to be also a marker of cardiovascular risk in crs types 2 and 4 and offers complementary prognostic information to other cardiac biomarkers like troponin t and nt - probnp [ 58 , 59 ] . high concentrations of circulating cys c have shown to be consistently and strongly associated with the cardiovascular outcomes . another index of adpkd progression is microalbuminuria ; in fact , there are numerous reports that have established that microalbuminuria is a frequent sign of kidney impairment in the disease , associated with a major cardiovascular risk . microalbuminuria is present in patients with chronic heart failure ( chf ) and progressive renal failure [ 46 , 61 ] . in adpkd patients , microalbuminuria is associated with an increase in arterial pressure [ 61 , 62 ] and progression to renal failure , as well as with a more severe cystic involvement . the study showed a high prevalence of microalbuminuria in this group and a tendency of these patients towards a greater systolic blood pressure , plasma renin activity , and left ventricular mass . there are few information about renal alterations and vascular remodeling in adpkd patients with normal or minimally increased levels of urine albumin excretion . the natriuretic peptides ( nps ) are a well - described family of hormones with a major role in sodium and body volume homeostasis . bnp ( brain natriuretic peptide ) , and nt - probnp are correlated with the severity of heart failure ( hf ) and left ventricular ( lv ) function and are useful markers for diagnosis , management , and prognosis in patients with normal renal function . recent studies indicated that both bnp and troponin t have a diagnostic power in patients with ckd to predict cardiovascular disease [ 63 , 64 ] . the nps have shown prognostic utility in patients with various stages of renal insufficiency , demonstrating potential applications in crs types 2 and 4 . it is well established that patients with ckd have higher levels of both bnp and nt - probnp than age- and gender - matched subjects without reduced renal function , even in the absence of clinical hf . thus , the relationship between bnp , renal function , and the severity of heart failure is less clear , and the association between nps levels and renal function remains complex . cardiac troponin t ( ctnt ) is a specific marker for myocardial damage and a myocardial infarction . in hemodialysis patients , three large observational studies concordant with ctnt levels are associated strongly with the risk of incident for cardiovascular events [ 6769 ] . thus , increased ctnt levels represent a strong and independent predictor of global cardiovascular mortality in clinically stable hemodialysis patients . however , there are few studies describing the significance and the prognostic value of elevated serum ctnt levels in stable patients with moderate ckd . at this time , the relationship between renal function and serum ctnt remains still unclear , and the significance of an increased ctnt concentration in patients with renal dysfunction remains controversial . unfortunately , there are currently no published data about specific cardiac biomarkers in adpkd population . imaging techniques may enhance , extend , and refine our ability to diagnose and follow up cardiac and renal diseases . the main structural change seen for adpkd is the formation of renal cysts ; thus , it is evident that any enlargement of the cysts and the decrease in the volume of the renal parenchyma are the key factors in the progress of this disease . different imaging modalities such as ultrasonography ( us ) , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) have been used to quantify the size of the kidney in adpkd . the consortium for radiologic imaging studies of polycystic kidney disease ( crisp ) was created to develop innovative imaging techniques and analyses to follow disease progression or to evaluate treatments for adpkd . ultrasonography was the earliest method used to measure kidney volume in vivo and has the advantage of being widely available and easily performed with modest cost in comparison with ct scan and mri . unfortunately , us can not provide separate , reliable measurements of both the renal cyst volume and the renal parenchymal volume . individuals who are at risk for adpkd are often screened by ultrasound using age - graded diagnostic criteria derived from individuals with mutations in pkd1 . in families of unknown genotype , the presence of three or more ( unilateral or bilateral ) renal cysts is sufficient for establishing the diagnosis in individuals aged 15 to 39 years ; two or more cysts in each kidney are sufficient for individuals aged 40 to 59 years , and four or more cysts in each kidney are required for older individuals aged 60 years . conversely , less than two renal cysts in at - risk individuals aged 40 years are sufficient to exclude the disease . us imaging does not provide a sufficiently certain diagnosis in at - risk individuals younger than 30 years of age ; so the utility of this technique for disease exclusion is limited in younger subjects . families with mutations in pkd2 typically have less severe disease ; in adpkd2 , the mild renal cystic involvement has an adverse impact on the sensitivity of us criteria that apply to diagnose the disease . as a result of reduced test sensitivity , the diagnostic criteria in use have a suboptimal performance for individuals with mutations in pkd2 . for younger individuals in whom us might yield equivocal or indeterminate results , a negative ct scan or mri may provide further assurance that they are unaffected . ct scan and mri have greater sensitivity so that smaller cysts ( 2 mm compared with 10 mm for ultrasonography ) can be detected . both ct and mri avoid the potential pitfalls of us , that is , any operator - dependent techniques or the need for multiple image acquisitions of large kidneys . contrast media - enhanced ct or electron beam ct techniques can provide accurate measurements not only of the total kidney volume , but also of the renal cyst volumes in adpkd patients . however , ct has two significant limitations : the radiation exposure and the requirement for administering intravenous contrast media . the contrast media may be associated with a small chance of serious allergic reactions and nephrotoxicity in patients with renal insufficiency . mri has increasingly been used because it provides high - resolution 3d images with excellent tissue contrast without exposure to ionizing radiation or iodinated contrast medium . while mri is a reliable and precise method to measure renal volume , little information and data are available in the medical literature about the validity and accuracy of mri - based kidney volume measurements in adpkd patients [ 41 , 76 ] . initial preliminary reports from the crisp indicate that mri is as least as accurate as ct scan for determining the rate of increase in kidney volume . at the present time some of the limitations of mri include relatively long image - acquisition times and variability in the quality of images that can be produced from different mr scanners . echocardiography determines left / right ventricular size and function , left ventricular wall motion abnormalities , valvular function and abnormalities , diastolic function , and presence or absence of pericardial abnormalities or intracardiac masses ; it also evaluates intracardiac filling pressures . transmitral pulsed doppler is the classical noninvasive method of evaluation of diastolic dysfunction ; it is influenced by a variety of factors such as the loading condition of the left atria and heart rate . tissue doppler imaging ( tdi ) is a new technique that permits an assessment of myocardial motion , a sensitive index of ventricular relaxation , which is more independent of the hemodynamic condition and , therefore , a more reliable diastolic function index . unlike transmitral pulsed doppler , tdi directly measures the mechanical wall function by calculating the velocity of myocardial movement and has been shown to better monitoring diastolic function of the myocardium . starting as a research method little more than a decade ago , cardiovascular magnetic resonance imaging ( cmri ) has rapidly evolved to become a powerful diagnostic tool used in routine clinical cardiology . cmri provides a relatively novel method for accurate definition of cardiac dimensions and is accepted as the gold standard for the assessment of ventricular dimensions . benefits of cmri include the ability to obtain a great deal of information with one noninvasive test . cmri is used for the assessment of regional and global ventricular function and to answer questions regarding anatomy . cmri is able to assess ischemic versus nonischemic disease , infiltrative disease , valvular and congenital disorders , and hypertrophic disease , and determine viability . molecular genetic tests are helpful when imaging results are equivocal and/or when a definite diagnosis is required in a younger adult . there are 2 methods for adpkd dna testing : linkage analysis and direct mutation screening . presymptomatic testing is possible in larger families by linkage analysis using highly informative microsatellite markers flanking the pkd1 and pkd2 genes . a significant limit of linkage analysis is the need for a relatively large number of affected family members in order to establish the gene involved in the disease . in linkage analysis , the segregation of chromosomal markers is examined and compared within a family in whom the clinical status ( affected or unaffected ) of each individual is known . by examining several markers , a haplotype ( a pattern of alleles on the same chromosome that are inherited together ) that segregates with the disease can be determined . there are several limitations to linkage testing ; the most important is that no information can be obtained from testing the proband alone . furthermore , linkage analysis can not be used if a family is small , if family members refuse to participate , or if the proband is suspected to have a de novo mutation . as adpkd displays a high level of allelic heterogeneity , the complete screening of both genes is required . most mutations are private ( unique to a single family ) and scattered throughout these genes with no clear hot spots . therefore , exon - by - exon screening of these genes is required to ensure a high sensitivity in detecting disease - causing mutations , thus screening approaches are expensive . however , only approximately 65% of patients have definite mutations with approximately 26% having nondefinite changes that require further evaluation . adpkd database ( autosomal dominant polycystic kidney disease : mutation database ) collects every known variants on pkd1 and pkd2 to improve the diagnostic value of molecular screening . furthermore , the recent availability of clinical molecular genetic testing means such testing may be applied to asymptomatic at - risk relatives of subjects with adpkd . as with most renal diseases , early diagnosis with implementation of effective interventions has important implications and the best chance for preventing or slowing renal progression and cardiac complications in patients with the adpkd . for example , the early intervention of diagnosing and aggressively treating blood pressure in these patients , particularly blockade of the renin - angiotensin system , has the potential of preventing lvh , cardiovascular complications , and mortality . in that regard , schrier et al . have reported that the age of esrd in both men and women with adpkd has increased in recent years and speculate that this effect has been associated with better blood pressure control and increased therapy with ace inhibitors . other relevant indications for early identification of adpkd include the provision of more detailed and specific information regarding prognosis and risk for complications , the ability to make more informed reproductive choices , motivation for enhanced compliance and medical followup , and the evaluation of living donors form affected families . screening is also important in clearing prospective living kidney donors from affected families . screening children under 18 years old is not strongly recommended because of the potential emotional and social impact of a positive diagnosis in these younger subjects . before screening , counselling from experienced staff must be performed , in order to facilitate appropriate life - style decisions . the core of the prevention is that the reduction in the rate of progression of ckd may lead to a reduction of the incidence of chronic renocardiac syndrome . many novel therapies have been evaluated in the cardiorenal syndrome setting , including agents that may block key local factors ( e.g. , adenosine a(i ) receptor antagonists ) , improve diuresis , aquaresis , and natriuresis , and augment natural vasodilator mechanisms to improve renal perfusion . there are no disease - specific therapies for any form of adpkd , and no evidence - based guidelines on the management of adpkd have been reported perhaps due to the very slow rate of disease progression . interventions should be capable to slow down , stop , or reverse structural progression of the disease and should be able to prevent the decline of renal function improving clinical outcome . in adpkd , only blood pressure control has been shown to have a favorable impact on disease progression and cardiovascular complication rate . rigorous control of blood pressure may prevent progression of renal disease and decreases the risk of cardiovascular morbidity that characterizes all patients with ckd . the kdoqi clinical practice guidelines on hypertension indicate that goal blood pressure should be less than 130/80 mmhg . if there are no contraindications , an angiotensin - converting enzyme ( ace ) inhibitor should be the initial antihypertensive agent . increased renin - angiotensin system activity and extracellular volume expansion play an important role in the pathogenesis of hp in patients with adpkd , thus patients generally respond well to these agents . ace inhibitors or angiotensin receptor blockers ( arbs ) may have renoprotective properties increasing renal blood flow which correlates with progression of adpkd and contributes to cyst growth [ 12 , 47 , 86 ] . in a meta - analysis of 11 randomized controlled trials , jafar et al . found that ace inhibitors were more effective in lowering urine protein excretion in patients with adpkd compared to regimes without ace inhibitors , and it was more evident in patients with higher levels of proteinuria . whether salt restriction and ace inhibitors and arbs therapy fail to lower blood pressure sufficiently , it may be necessary to add a diuretic ( thiazides initially , with a switch to loop diuretics if thiazides are not effective ) . additional agents may then be added to gain an appropriate blood pressure control and ameliorate other clinical advantages , such as angina using a -blockers or calcium channel blockers . addition of -blockers to ace inhibitor / arb therapy is a very attractive choice , with documented cardiovascular protective characteristics . several studies have shown better preservation of renal function or reduction in proteinuria and lvh with ace inhibitors or arbs compared to diuretics or calcium channel blockers [ 87 , 88 ] . a controlled trial , the halt progression of polycystic kidney disease study ( nct00283686 ) , funded by the national institutes of health , is under way to determine whether treatment with ace inhibitors and arbs , administered singly or in combination , will reduce the rate of increase in kidney volume and slow the decline in gfr . significant advances in terms to understand the genetics of adpkd and molecular mechanisms responsible for cyst initiation have revealed likely targets for therapeutic intervention . at the present time , there are no therapies proving a cyst progression delay and their complications , and there is no proven antihypertensive drug of choice neither in adpkd patients nor in adpkd patients on dialysis . an effective control of hp remains one of the few modifiable factors by medical intervention and may delay the development of lvh , which is strongly related with diastolic dysfunction . a better understanding of the pathophysiology and the availability of animal models has enabled the development of preclinical trials and the identification of promising candidate drugs for clinical trials . an hopeful therapeutic strategy , to inhibit cyst development in adpkd , is modulating cyclic amp ( camp ) levels . the effect of avp , via v2 receptors , on camp levels in the collecting duct and distal nephron and the role of camp in cystogenesis provided the rationale for preclinical trials of vasopressin v2 receptor ( vpv2r ) antagonists . in particular , one of these drugs , opc-31260 , reduces the concentrations of camp and inhibits cyst development in animal models of adpkd . an antagonist with high potency and selectivity for the human vpv2r ( tolvaptan ) has also been shown to be an effective treatment in pkd2 mouse model of autosomal dominant polycystic kidney disease . the usefulness of avp - v2 inhibitors in slowing the progression of renal enlargement and insufficiency in patients with adpkd is currently evaluating in a placebo - controlled trial ( nct00428948 ) . small clinical trials ( nct00309283 , nct00426153 , and nct00565097 ) have shown that the administration of octreotide or lanreotide for a period of 6 to 12 months inhibits the growth of polycystic kidneys and livers [ 9294 ] . the absence of polycystin permits excessive kinase activity in the mammalian which is the target of rapamycin ( mtor ) pathway and the development of renal cysts . the mtor system can be blocked by rapamycin ( sirolimus , everolimus ) , so it may be another possible strategy to modulate disease progression in adpkd patients . wahl et al . found that inhibition of mtor with rapamycin slows adpkd progression and kidney enlargement in rats . in a prospective study in humans , rapamycin reduced polycystic liver volumes in adpkd renal transplant recipients . larger studies of longer duration are needed to confirm the safety and to sustain efficacy of these novel treatments . experimental and clinical studies have suggested that statins may slow the progression of chronic kidney disease in general and adpkd specifically . statins are widely used to lower cholesterol , and they have anti - inflammatory and antiproliferative qualities . however , there are some reported animal studies in han : sprd rats , an adpkd model with many of the characteristics of the disease in humans , that demonstrate that statins reduce cyst formation and improve renal function . moreover , namli et al . have shown in patients with adpkd that statins have a beneficial effect in the reversal of endothelial dysfunction , an early manifestation of vascular injury . six months of simvastatin therapy resulted in a significant improvement of endothelial dysfunction in patients with adpkd . a clinical trail is currently in progress at the university of colorado regarding adpkd and pravastatin ( nct00456365 ) . this study is designed to determine if the treatment with pravastatin can slow the progression of kidney and heart disease when initiated early in life in patients with adpkd . the endpoints of interest in this three - year study include total kidney volume and lvh index as measured mri ; urinary albumin excretion and endothelial - dependent vasodilation as assessed by brachial ultrasound ( table 3 ) . in conclusion , recent studies using different animal models of renal cystic diseases have suggested that various pharmacological interventions may modify disease progression . this clearly demonstrates that a better comprehension of the molecular and cellular defects underlying cystogenesis may lead to design novel therapeutic agents or a better use of existing ones . it is therefore likely that trials in human adpkd will be carried out in the near future , especially as methods for assessing disease progression in the short term are now available [ 41 , 101 ] . other important thing is that genetic counseling to discuss genetic risk , screening , and prenatal and predictive testing should be offered to all individuals with or at risk of inheriting adpkd .

the cardiorenal syndrome type 4 ( chronic renocardiac syndrome ) is characterized by a condition of primary chronic kidney disease ( ckd ) that leads to an impairment of the cardiac function , ventricular hypertrophy , diastolic dysfunction , and/or increased risk of adverse cardiovascular events . clinically , it is very difficult to distinguish between crs type 2 ( chronic cardiorenal syndrome ) and crs type 4 ( chronic renocardiac syndrome ) because often it is not clear whether the primary cause of the syndrome depends on the heart or the kidney . autosomal dominant polycystic kidney disease ( adpkd ) , a genetic disease that causes ckd , could be viewed as an ideal prototype of crs type 4 because it is certain that the primary cause of cardiorenal syndrome is the kidney disease . in this paper , we will briefly review the epidemiology of adpkd , conventional and novel biomarkers which may be useful in following the disease process , and prevention and treatment strategies .

1. Introduction 2. Definition, Classification, and Epidemiology 3. Biomarkers 4. Prevention, Management, and Trials 5. Conflict of Interest

in this paper , we discuss about the adpkd and its relation with cardiorenal syndrome . ( crs ) was defined as the pathophysiological disorder of the heart and kidney in which acute or chronic dysfunction in one organ may induce acute or chronic dysfunction in the other organ . for this reason , it was necessary to classify and divide the cardiorenal syndrome into different subtypes to provide a more concise and logically correct approach to this condition ( see table 1 ) . patients with ckd are at higher risk for cardiovascular events , and they have a 10- to 20-fold increased risk of cardiac death compared with age - gender - matched control subjects without ckd . clinically , it is very difficult to distinguish between crs type 2 ( chronic cardiorenal syndrome ) and crs type 4 ( chronic renocardiac syndrome ) because often it is not clear whether the primary cause of the syndrome depends on the heart or the kidney . autosomal dominant polycystic kidney disease , a genetic disease that causes ckd , could be viewed as an ideal prototype of crs type 4 because it is certain that the kidney disease is the primary process . in this paper , we will briefly review the epidemiology of adpkd , conventional and novel biomarkers which may be useful in following the disease process , and prevention and treatment strategies . it accounts for ~10% of patients on renal replacement therapy representing an important cause of end - stage renal disease ( esrd ) worldwide . prevalence of the disease is higher than that of huntington disease , hemophilia , sickle cell disease , cystic fibrosis , myotonic dystrophy , and down syndrome combined , and it occurs in approximately 1 of every 400 to 1000 live births . epidemiological data on the prevalence of adpkd have been extensively reported , mainly in the united states and europe . adpkd is the fourth leading cause of ckd in the united states accounting for approximately 3% of cases . with the advent of renal replacement therapy , adpkd is a genetically heterogeneous disease identified by two phenotypically similar forms associated with several mutations in two genes : the pkd1 gene located on chromosome 16 ( 16p13.3 ) and the pkd2 gene mapped to chromosome 4 ( 4q13q23 ) . mutations of pkd1 gene , encoding the polycystin-1 protein , result in adpkd type i ( adpkd1 ) which is responsible for approximately 85% of adpkd cases . gene pkd2 mutations , encoding the polycystin-2 protein , result in adpkd type ii ( adpkd2 ) , corresponding to 15% of adpkd cases . mutations in these genes lead to abnormalities in cell proliferation , apoptosis , tubular basement membranes , and tubular fluid secretion , ultimately resulting in slowly expanding renal cysts . several mechanisms contributing to the cyst formation have been identified , including a imbalance between epithelial cell proliferation and apoptosis , secretor defects , altered cell matrix interactions , cell polarity , ciliary dysfunction , and altered intracellular signaling . clinically , adpkd is an adult - onset disease characterized by progressive , bilateral renal cyst development and expansion of the kidneys . many patients with adpkd are completely asymptomatic and often are diagnosed because of their positive family history or the development of hypertension ( hp ) . liver cysts develop in more than 80% of patients , and the cysts are usually larger in women than in men . other locations of cysts include the spleen , arachnoid membranes , and seminal vesicles in men . the new onset of hp in a patient at risk for polycystic kidney disease should prompt aggressive treatment and diagnostic studies . these patients during the course of adpkd have early and more severe left ventricular hypertrophy ( lvh ) . a number of noncystic manifestations such as cardiovascular deficits , cardiac valve abnormalities , diverticular disease , and intracranial aneurysms are also associated with adpkd ; in fact , cardiovascular complications are the major cause of morbidity and mortality in patients with adpkd . pericardial effusion may occur with an increased frequency in patients with adpkd ( 35% versus 9% in a control group of patients with another chronic nephropathy ) , possibly as a result of increased compliance ( or as a collagen protein dysfunction ) of the parietal pericardium . it is known that the kidney disease is strongly associated with a greater carotid intima - media thickness ( imt ) . subjects with adpkd , even with preserved renal function , have a greater carotid imt compared with healthy controls ; carotid imt is higher in hypertensive adpkd patients . in addition , most of the homozygous knockout embryos display multiple cardiac abnormalities including cardiac septation defects , double outlet right ventricle , and pericardial effusions [ 31 , 33 ] . engineered and described a pkd1 transgenic mice ( pkd1(tag ) mice ) that , in addition to the cystic phenotype , developed cardiac anomalies with severe left ventricular hypertrophy , marked aortic arch distention , and/or valvular stenosis and calcification . therefore , the cardiovascular complications seen in adpkd patients begin to be recognized not only as a consequence of declining kidney function , but also as a defect due to the loss of polycystin-1 and/or polycystin-2 function in cardiovascular organs . it is well known that hp and lvh are associated with an accelerated rate of renal functional deterioration . both hp and lvh are important risk factors for cardiovascular death , the most frequent cause of mortality in adpkd patients ; thus left ventricular hypertrophy may be considered a powerful indicator of mortality . however , it is now well known that increased activity of the intrarenal rather than the systemic renin - angiotensin system ( ras ) is responsible for many forms of hypertension . hypertension is associated with larger kidney size , reflecting a larger number of cysts and with the severity of kidney disease . renal blood flow is reduced in hypertensive adpkd patients versus matched essential hypertensive patients , and the renal resistive indices are also increased in hypertensive adpkd subjects and are correlated with a loss of kidney function . renal structural changes play an important role in the pathogenesis of the hp , and renal arteriograms from end - stage adpkd - nephrectomized specimens demonstrate marked attenuation of the vasculature due to the extrinsic compression by the presence of the cysts and their replacement by the latter . hypertensive adpkd adults with normal kidney function show a greater frequency of lvh versus normotensive adpkd men ( 50% versus 30% ) and women ( 52% versus 22% ) as well as with healthy controls . the prevalence of lvh is increased even in the early stages of ckd , and the frequency increases progressively as renal function decreases . several studies have shown increased lvmi , left ventricular diastolic dysfunction , endothelial dysfunction , and increased carotid imt in young normotensive patients with adpkd with well - preserved renal function . these findings suggest the cardiovascular involvement in the early stages during the course of adpkd . it is quite important to appreciate that hypertrophic remodeling comprises not only cardiomyocyte hypertrophy , but also interstitial fibrosis and microvessel disease . a familial clustering of ica is found , with a 5 times greater chance of detecting an ica in a subject with a relative with a ruptured ica [ 52 , 53 ] ; it suggests that genetic factors may be associated with the development of this complication . the routine screening for asymptomatic ica in all adpkd individuals is not indicated , but in families with a proven case of ica , a screening analysis is recommend in consultation with a neurosurgeon . biomarkers measured by laboratory tests are defined as laboratory or molecular biomarkers ; those related to signaling , imaging , and functional tests are defined as functional biomarkers ; those related to genetic polymorphisms and other genomic tests are defined as genetic biomarkers . in patients with adpkd , a limited number of biomarkers have been investigated . investigated urinary biomarkers for different segments of the nephron in patients with adpkd versus healthy controls . they choose urinary immunoglobulin g ( igg ) as a marker of glomerular damage ; urinary 2-microglobulin ( b2 m ) , urinary kidney injury molecule 1 ( kim-1 ) , n - acetyl--d - glucosammide ( nag ) , and neutrophil gelatinase - associated lipocalin ( ngal ) as markers for damage of the proximal tubule ; urinary heart - type fatty acid binding protein ( hfabp ) as a marker for damage of the distal tubule . in addition , b2 m and h - fabp were associated inversely with measured gfr and effective renal blood flow independent of albuminuria ; kim-1 , ngal , and mcp-1 were associated positively with total renal volume independent of albuminuria . they concluded that higher levels of ngal are correlated to higher cystic growth and suggested that this protein could be also involved in the process of cystogenesis . in 92 patients with adpkd , casal et al . reported a comparative study for three biomarker tests of early kidney damage such as urinary albumin and total -n - acetylhexosaminidase ( hex ) and its isoenzymes ( hex a , hex b ) , as well as serum glutathione peroxidase , which has been considered as a marker of renal proximal tubular function . furthermore , keeping age constant , they reported a partial significant correlation between the ultrasound score ( kidney size and number of cysts ) and the proportion of hex b , but not with albuminuria or cystatin c. this confirms the hypothesis that tubular damage plays a role in the pathogenesis and progression of adpdk . in fact , in patients with ckd , cys c was proposed to perform better as a marker of gfr than serum creatinine . another index of adpkd progression is microalbuminuria ; in fact , there are numerous reports that have established that microalbuminuria is a frequent sign of kidney impairment in the disease , associated with a major cardiovascular risk . the study showed a high prevalence of microalbuminuria in this group and a tendency of these patients towards a greater systolic blood pressure , plasma renin activity , and left ventricular mass . bnp ( brain natriuretic peptide ) , and nt - probnp are correlated with the severity of heart failure ( hf ) and left ventricular ( lv ) function and are useful markers for diagnosis , management , and prognosis in patients with normal renal function . it is well established that patients with ckd have higher levels of both bnp and nt - probnp than age- and gender - matched subjects without reduced renal function , even in the absence of clinical hf . thus , the relationship between bnp , renal function , and the severity of heart failure is less clear , and the association between nps levels and renal function remains complex . in hemodialysis patients , three large observational studies concordant with ctnt levels are associated strongly with the risk of incident for cardiovascular events [ 6769 ] . the main structural change seen for adpkd is the formation of renal cysts ; thus , it is evident that any enlargement of the cysts and the decrease in the volume of the renal parenchyma are the key factors in the progress of this disease . different imaging modalities such as ultrasonography ( us ) , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) have been used to quantify the size of the kidney in adpkd . the consortium for radiologic imaging studies of polycystic kidney disease ( crisp ) was created to develop innovative imaging techniques and analyses to follow disease progression or to evaluate treatments for adpkd . in families of unknown genotype , the presence of three or more ( unilateral or bilateral ) renal cysts is sufficient for establishing the diagnosis in individuals aged 15 to 39 years ; two or more cysts in each kidney are sufficient for individuals aged 40 to 59 years , and four or more cysts in each kidney are required for older individuals aged 60 years . conversely , less than two renal cysts in at - risk individuals aged 40 years are sufficient to exclude the disease . families with mutations in pkd2 typically have less severe disease ; in adpkd2 , the mild renal cystic involvement has an adverse impact on the sensitivity of us criteria that apply to diagnose the disease . mri has increasingly been used because it provides high - resolution 3d images with excellent tissue contrast without exposure to ionizing radiation or iodinated contrast medium . at the present time some of the limitations of mri include relatively long image - acquisition times and variability in the quality of images that can be produced from different mr scanners . echocardiography determines left / right ventricular size and function , left ventricular wall motion abnormalities , valvular function and abnormalities , diastolic function , and presence or absence of pericardial abnormalities or intracardiac masses ; it also evaluates intracardiac filling pressures . transmitral pulsed doppler is the classical noninvasive method of evaluation of diastolic dysfunction ; it is influenced by a variety of factors such as the loading condition of the left atria and heart rate . tissue doppler imaging ( tdi ) is a new technique that permits an assessment of myocardial motion , a sensitive index of ventricular relaxation , which is more independent of the hemodynamic condition and , therefore , a more reliable diastolic function index . unlike transmitral pulsed doppler , tdi directly measures the mechanical wall function by calculating the velocity of myocardial movement and has been shown to better monitoring diastolic function of the myocardium . a significant limit of linkage analysis is the need for a relatively large number of affected family members in order to establish the gene involved in the disease . by examining several markers , a haplotype ( a pattern of alleles on the same chromosome that are inherited together ) that segregates with the disease can be determined . adpkd database ( autosomal dominant polycystic kidney disease : mutation database ) collects every known variants on pkd1 and pkd2 to improve the diagnostic value of molecular screening . furthermore , the recent availability of clinical molecular genetic testing means such testing may be applied to asymptomatic at - risk relatives of subjects with adpkd . for example , the early intervention of diagnosing and aggressively treating blood pressure in these patients , particularly blockade of the renin - angiotensin system , has the potential of preventing lvh , cardiovascular complications , and mortality . have reported that the age of esrd in both men and women with adpkd has increased in recent years and speculate that this effect has been associated with better blood pressure control and increased therapy with ace inhibitors . other relevant indications for early identification of adpkd include the provision of more detailed and specific information regarding prognosis and risk for complications , the ability to make more informed reproductive choices , motivation for enhanced compliance and medical followup , and the evaluation of living donors form affected families . screening children under 18 years old is not strongly recommended because of the potential emotional and social impact of a positive diagnosis in these younger subjects . the core of the prevention is that the reduction in the rate of progression of ckd may lead to a reduction of the incidence of chronic renocardiac syndrome . many novel therapies have been evaluated in the cardiorenal syndrome setting , including agents that may block key local factors ( e.g. , adenosine a(i ) receptor antagonists ) , improve diuresis , aquaresis , and natriuresis , and augment natural vasodilator mechanisms to improve renal perfusion . there are no disease - specific therapies for any form of adpkd , and no evidence - based guidelines on the management of adpkd have been reported perhaps due to the very slow rate of disease progression . interventions should be capable to slow down , stop , or reverse structural progression of the disease and should be able to prevent the decline of renal function improving clinical outcome . rigorous control of blood pressure may prevent progression of renal disease and decreases the risk of cardiovascular morbidity that characterizes all patients with ckd . ace inhibitors or angiotensin receptor blockers ( arbs ) may have renoprotective properties increasing renal blood flow which correlates with progression of adpkd and contributes to cyst growth [ 12 , 47 , 86 ] . whether salt restriction and ace inhibitors and arbs therapy fail to lower blood pressure sufficiently , it may be necessary to add a diuretic ( thiazides initially , with a switch to loop diuretics if thiazides are not effective ) . a controlled trial , the halt progression of polycystic kidney disease study ( nct00283686 ) , funded by the national institutes of health , is under way to determine whether treatment with ace inhibitors and arbs , administered singly or in combination , will reduce the rate of increase in kidney volume and slow the decline in gfr . significant advances in terms to understand the genetics of adpkd and molecular mechanisms responsible for cyst initiation have revealed likely targets for therapeutic intervention . an effective control of hp remains one of the few modifiable factors by medical intervention and may delay the development of lvh , which is strongly related with diastolic dysfunction . in particular , one of these drugs , opc-31260 , reduces the concentrations of camp and inhibits cyst development in animal models of adpkd . an antagonist with high potency and selectivity for the human vpv2r ( tolvaptan ) has also been shown to be an effective treatment in pkd2 mouse model of autosomal dominant polycystic kidney disease . small clinical trials ( nct00309283 , nct00426153 , and nct00565097 ) have shown that the administration of octreotide or lanreotide for a period of 6 to 12 months inhibits the growth of polycystic kidneys and livers [ 9294 ] . the mtor system can be blocked by rapamycin ( sirolimus , everolimus ) , so it may be another possible strategy to modulate disease progression in adpkd patients . experimental and clinical studies have suggested that statins may slow the progression of chronic kidney disease in general and adpkd specifically . however , there are some reported animal studies in han : sprd rats , an adpkd model with many of the characteristics of the disease in humans , that demonstrate that statins reduce cyst formation and improve renal function . this clearly demonstrates that a better comprehension of the molecular and cellular defects underlying cystogenesis may lead to design novel therapeutic agents or a better use of existing ones . it is therefore likely that trials in human adpkd will be carried out in the near future , especially as methods for assessing disease progression in the short term are now available [ 41 , 101 ] . other important thing is that genetic counseling to discuss genetic risk , screening , and prenatal and predictive testing should be offered to all individuals with or at risk of inheriting adpkd .

iron overload contributes significantly to morbidity and mortality in patients receiving regular red blood - cell transfusions as supportive therapy for chronic anemias such as -thalassemia , sickle - cell disease ( scd ) , and myelodysplastic syndromes ( mdss ) . in the absence of physiological mechanisms to excrete this excess iron resulting from transfusions , administration of effective iron - chelation therapy that results in net negative iron balance is the only way to prevent end - organ damage from iron deposition in various tissues . while deferoxamine ( dfo ) is an effective iron chelator that has been shown to improve survival in iron - overloaded -thalassemia patients , its delivery and compliance with therapy are less than ideal.1 dfo is typically administered as a subcutaneous infusion over 824 hours , 57 days per week , delivered overnight by a small portable pump.2 this arduous schedule interferes with patients quality of life and results in poor patient compliance.3 the urgent need for an effective and safe oral iron chelator led to the expedited approval of deferasirox ( exjade , novartis pharmaceuticals corporation , basel , switzerland ) by the us food and drug administration ( fda ) in november 2005 for the treatment of transfusional iron overload associated with thalassemia and other iron - loading conditions with an orphan - drug designation.4 deferiprone , another orally bioavailable iron chelator , has been shown to be safe and efficacious in iron - overloaded -thalassemia patients . although it was being used in other countries , deferiprone received fda approval only in october 2011 for treatment of patients with transfusional iron overload due to thalassemia syndromes who were not adequately chelated with other agents . however , the use of deferiprone has been limited , due to the occurrence of rare but serious adverse events during treatment , such as neutropenia and agranulocytosis.5,6 this review will focus on the long - term safety and efficacy of deferasirox in trials conducted for transfusional hemosiderosis in hemoglobinopathies and bone marrow - failure syndromes . deferasirox is a tridentate chelator with a long half - life of 816 hours , allowing for convenient once - daily administration.7 it is rapidly absorbed , and has a bioavailability of about 70% . it is mainly excreted in the feces ( 84% of the dose ) after it is metabolized by glucuronidation and subsequent biliary excretion.8 the highest dose of deferasirox approved by most health authorities is 30 mg / kg / day . however , most recently the fda and european medicines agency have approved doses up to 40 mg / kg / day in patients not adequately chelated with doses of 30 mg / kg / day ; doses above 40 mg / kg / day are generally not recommended.4,9 the efficacy and safety of deferasirox were initially studied in a series of 1-year phase ii and iii clinical trials involving more than 1,000 patients . in order to assess its long - term efficacy and safety , 4-year extension phases of the 1-year core trials were conducted ( table 1 ) . results from the extension phases , with a median follow - up of 5 years now , have confirmed the efficacy of deferasirox to be dose- and transfusion - dependent.10 across these clinical trials , deferasirox was well tolerated , with the most common drug - related adverse events being mild to moderate in severity and often self - limiting ( table 1 ) . serious albeit rare adverse effects reported with long - term deferasirox use include renal failure , agranulocytosis , hepatic toxicity , and gastrointestinal bleeding , making close toxicity monitoring mandatory.11 thalassemia , first described by cooley and lee in 1925 , is a hereditary form of anemia resulting from an imbalance in the synthesis of the - and -chains of hemoglobin leading to hemolysis and ineffective erythropoiesis.12 iron overload in patients with thalassemia is mainly the result of regular red blood - cell transfusions and can result in an iron load of up to 10 g per year.13 iron overload can also result from increased intestinal iron absorption , particularly in patients with thalassemia intermedia.14 patients who receive regular blood transfusions without adequate chelation therapy develop evidence of iron excess ( elevated liver iron concentration [ lic ] and serum ferritin ) within 12 years of transfusion therapy . adequate iron chelation with dfo from early on in life in these iron - overloaded -thalassemia patients has significant benefits on both morbidity and early mortality.15,16 unfortunately , effective administration of iron - chelation therapy with dfo is often compromised secondary to the arduous schedule of overnight subcutaneous infusions leading to reduced compliance.17 dfo is also associated with local skin reactions , ophthalmological adverse effects ( optic neuropathy , retinal pigmentation ) , ototoxicity , anaphylactic reactions , pulmonary fibrosis , and rarely renal impairment.18,19 in various trials done to date , deferasirox has proved to be an effective , safe oral iron - chelating agent in patients with thalassemia . the results of two initial short - term studies investigating mainly the safety and pharmacokinetics of deferasirox were reported by galenello et al20 and nisbet - brown et al.21 following their results , further clinical testing in phase ii and iii trials was performed . piga et al conducted a randomized open - label phase ii trial in 71 -thalassemia patients focusing mainly on safety of deferasirox . efficacy was judged to be comparable for 20 mg / kg / day of deferasirox and 40 mg / kg / day of dfo , supporting the assumed 1:2 equivalence ratio.22 in a subsequent large phase iii comparative trial , patients with -thalassaemia major were randomized to receive either deferasirox ( 296 patients ) or dfo ( 290 patients ) , dosed according to their baseline lic . this was a noninferiority trial that led to the approval of deferasirox in the us . over 1 year , a deferasirox dose of 20 mg / kg / day stabilized the mean serum ferritin level close to 2,000 ng / ml , achieved negative iron balance , and maintained lic . maintenance of lic at 7 mg iron / g dry weight was achieved in 59.7% of patients in the deferasirox arm versus 58.7% of patients in the dfo arm . for both drugs , a clear dose a deferasirox dose of 30 mg / kg / day reduced serum ferritin by about 1,200 ng / ml and achieved negative iron balance , with a fall in lic of 8.9 mg iron / g dry weight over 1 year , whereas lower doses of deferasirox were not as effective and led to a positive iron balance in most patients.23 transient gastrointestinal disturbances , including abdominal pain , nausea , vomiting , diarrhea , and constipation , were reported in 15.2% of patients . mild , dose - dependent increases in serum creatinine were noted in 38% of patients on deferasirox ; however , only 13% of patients required dose reduction , as creatinine spontaneously normalized in a number of patients . among patients who required dose reduction , creatinine returned to baseline in 25% of patients , and in the rest either remained stable or fluctuated between baseline and the maximum increase observed prior to dose adjustment . despite fluctuations in serum copper and zinc levels , no patient developed trace - element deficiency in the trial . as expected , satisfaction with and convenience of deferasirox treatment was significantly higher , although this was not reflected in the rate of discontinuation of deferasirox when compared to dfo . in order to evaluate the long - term efficacy and safety of deferasirox in patients with -thalassemia who completed the 1-year trial , a 4-year extension study was conducted.24 in this extension trial , patients either continued receiving deferasirox ( deferasirox cohort ) or crossed over from dfo to deferasirox ( crossover cohort ) . the results were reassuring , as irrespective of whether patients switched from dfo or continued on deferasirox , significant decreases in both lic and serum ferritin levels were observed . median serum ferritin significantly decreased by 706 ng / ml in the deferasirox arm and by 371 ng / ml in the crossover arm . mean lic significantly decreased by 7.8 11.2 mg iron / g dry weight and 3.1 7.9 mg iron / g dry weight in the deferasirox and crossover cohorts , respectively . deferasirox was generally well tolerated over the long term in both pediatric and adult patients . no adverse effects on growth and development were noted in younger patients on deferasirox treatment . gastrointestinal adverse effects ( 9% ) and increased serum creatinine ( 11% ) were transient and mild to moderate in severity . renal and liver functions were closely monitored during the trial , and no progressive increases in serum creatinine or liver transaminase levels were observed during long - term deferasirox treatment . long - term safety of iron chelators is critical in -thalassemia patients , since most require lifelong red cell transfusions and therefore iron - chelation therapy . this is particularly relevant in pediatric populations , as intensive chelation therapy initiated to prevent iron toxicity can cause growth deceleration and skeletal changes.25 long - term consequences of deferasirox on growth and development in chelation - nave children below 5 years of age were explored in an observational study . deferasirox was found to be an effective first - line therapy to maintain negative iron balance without adverse effects on growth and development in this age - group.26 the efficacy of deferasirox in reducing cardiac iron and thereby preventing cardiac iron loading was assessed using cardiovascular magnetic resonance by measuring myocardial t2 * in a subgroup of patients with -thalassemia ( n = 192 ) who were enrolled in the large evaluation of patients iron chelation with exjade ( epic ) trial.27 a cardiac t2 * value of < 20 ms is considered to indicate cardiac iron levels below the normal range , whereas a value < 10 ms is associated with reduction in left and right ventricular ejection fraction and an increased risk of cardiac failure and arrhythmia . results of this study were significant for marginal but statistically significant improvement in myocardial t2 * in the treatment arm , improved from a baseline of 11.2 ms ( 40.5% ) to 12.9 ms ( 49.5% ) with maintained left ventricular function , while in patients without cardiac iron overload ( prevention arm ) , the ejection fraction improved significantly . seventy - one patients from this study elected to continue deferasirox and entered the 3-year extension phase . over the 3 years of follow - up , myocardial t2 * increased significantly from 12.0 ms 39.1% at baseline to 17.1 ms 62.0% at the end of the study , corresponding to a continued reduction in cardiac iron concentration from 2.43 1.2 mg iron / g dry weight at baseline to 1.80 1.4 mg iron / g dry weight at the end of the study . mean deferasirox doses were high ( 33.6 9.8 mg / kg / day ) , with no increase in adverse effects noted.28 in another study of 19 patients , significant improvements of myocardial t2 * were demonstrated in heavily transfused iron - overloaded patients who demonstrated greater than 95% compliance to therapy with deferasirox , reiterating the fact that compliance is essential for success of treatment in myocardial siderosis.29 deugnier et al reported long - term effects of deferasirox treatment on liver fibrosis , and found that deferasirox treatment for 3 or more years reversed or stabilized liver fibrosis and necroinflammation in 83% of patients with iron - overloaded -thalassemia . this therapeutic effect was independent of reduction in lic.30 another prospective , open - label , 1-year study , the escalator ( extension study of the efficacy and safety of deferasirox treatment in beta - thalassemia patients with transfusional hemosiderosis ) trial was conducted in the middle east to evaluate the efficacy and safety of deferasirox in patients who had been previously treated with dfo and/or deferiprone . mean baseline lic in the study was 18.0 9.1 mg iron / g dry weight , while median serum ferritin was 3356 ng / ml . after 1 year of deferasirox treatment , the intent - to - treat population experienced a significant mean reduction in lic of 3.4 mg iron / g dry weight and a decrease in median serum ferritin of 341 ng / ml at 52 weeks . most patients ( 78.1% ) underwent dose increases above 20 mg / kg / day , mostly to 30 mg / kg / day . for reasons that are not so clear , reductions in serum ferritin and lic were noted to be more pronounced in the adult population when compared to the pediatric population . deferasirox was well tolerated overall , with manageable side effects and good safety profile , and there were no reported discontinuations due to adverse effects . there were no negative effects noted on physical or gonadal development in children ; however , longer follow - up studies are needed to make such claims . another important result of the study was a statistically significant improvement in left ventricular ejection fraction observed by week 52 , suggesting a positive effect of deferasirox on cardiac function.31 red cell - transfusion therapy has been shown effectively to prevent strokes and other complications , mainly acute chest syndrome , pain crisis , and growth retardation in scd patients.3234 many patients with scd have received multiple blood transfusions by adulthood . unlike -thalassemia , iron overload can be easily overlooked in scd patients due to episodic requirements for red cell transfusions , even though these patients often develop iron - induced complications later in life similar to other chronically transfused populations.35 therefore , guidelines from consensus panels have recommended iron - chelation therapy in scd patients with evidence of iron overload.36 current recommendations are to initiate iron - chelation therapy in patients with scd once lic increases to 7 mg iron / g dry weight , if serum ferritin steady - state levels are > 1000 ng / ml , or if patients have received cumulative transfusions of 120 ml packed red blood cells / kg ( usa ) or at least 20 top - up transfusions ( uk ) ( national institutes of health nhlbi , 2002 ; sickle cell society , 2008 ) . a major issue with the use of deferasirox in scd patients who may have baseline compromised renal function was the concern for renal toxicity , which was seen in some preclinical studies done with the drug in animals . another concern was the lack of knowledge on whether treatment with deferasirox would provoke or exacerbate sickle - cell crisis . vichinsky et al compared the safety and efficacy of deferasirox versus dfo in a 1-year , multicenter , open - label , randomized phase ii trial . in this trial , 203 eligible patients were randomized in a 2:1 ratio to receive either deferasirox or dfo . deferasirox treatment resulted in statistically significant and similar reductions in lic compared to dfo ( 3.0 6.2 vs 2.8 10.4 mg iron / g dry weight for deferasirox vs dfo , respectively).37 the most notable adverse effects irrespective of the relationship to study medication were transient gastrointestinal disturbances and skin rash . in the deferasirox group , mild , stable increase in serum creatinine of unknown clinical relevance was noted in 36.4% of patients . despite greater satisfaction with deferasirox treatment , the rate of discontinuation of therapy was similar in both arms . in order to assess the long - term efficacy and safety of deferasirox in patients with scd , patients who completed the 1-year study entered a 4-year extension phase where they continued to receive deferasirox , or switched from dfo to deferasirox.38 of the patients who received at least one dose of deferasirox , only 62 patients ( 33.5% ) completed the extension study ; the reasons for such a high rate of discontinuation in scd patients remain unclear . another drawback of the study was the inability to assess the impact of deferasirox therapy on the frequency of sickle - cell crisis secondary to the lack of control group in the study design . however , serum ferritin levels in patients with 4 years of deferasirox exposure significantly decreased by 591 ng / ml , and the tolerability of the drug improved with long - term follow - up , as a decrease in the most common adverse effect was noted with continued therapy . cancado et al investigated the efficacy and safety of a 2-year treatment program with deferasirox in 31 patients with scd and transfusional iron overload . their results were similar to those reported in prior studies of -thalassemia and scd . at 24 months , there were significant decreases from baseline in mean serum ferritin ( from 2,344.6 to 1,986.3 ng / ml ) and in mean lic ( from 13.0 5.4 to 9.3 5.7 mg iron / g dry weight ) . the authors also noted significantly improved left ventricular function at 24 months when compared to baseline , despite a statistically nonsignificant decline in myocardial mean t2 * from baseline . the most common adverse effects in the study were transient and mild , with diarrhea and headaches reported in 22.6% patients each . three patients ( 9.5% ) were noted to have nonprogressive increase in serum creatinine.39 taken together , these studies support the efficacy of deferasirox in iron - overloaded scd patients and demonstrate its safety over a reasonably long time period . the mdss comprise a heterogeneous group of malignant hematopoietic stem cell disorders characterized by dysplastic and ineffective blood - cell production and a variable risk of transformation to acute leukemia.40 the major cause of iron overload in these patients is red cell transfusions for anemia . with every unit of transfused blood , the patients receive approximately 200250 mg iron.41 another less recognized cause of iron excess in mds patients is increased absorption of iron from the gut due to ineffective erythropoiesis.42 overall survival and leukemia - free survival are significantly shorter in transfusion - dependent mds patients.43 this may simply be related to disease severity or be an effect of iron overload itself . gattermann and rachmilewitz noted shortened overall survival with increasing ferritin level , with a hazard ratio of 1.42 for every 500 ng / ml increase in ferritin over 1,000 ng / ml.44 even in patients undergoing hematopoietic stem cell transplantation ( hsct ) , elevated pre - transplant serum ferritin level adversely affects survival.45 excess iron has also been shown to suppress erythropoiesis , as evidenced by decreased burst - forming units erythroid in patients with elevated ferritin compared to patients with normal ferritin.46 thus , iron overload has many deleterious effects , especially in the usually older mds population , with significant comorbidities that make them more sensitive to the effects of iron overload.47 as phlebotomy is not a feasible option in mds patients , an effective way to prevent adverse consequences of iron overload is iron - chelation therapy . there are several published guidelines recommending chelation in chronically transfused mds patients , mostly based on retrospective case - control studies and some prospective randomized supportive data extrapolated from other iron - overload disease populations . in this regard , the results of the ongoing telesto ( myelodysplastic syndromes event - free survival with iron chelation therapy study ) trial , a placebo - controlled randomized trial in low- and intermediate-1-risk mds patients , are much awaited . currently , the main factors taken into account to make decisions about initiating iron chelation in mds patients include life expectancy , transfusion burden , evidence of iron excess ( elevated ferritin ) , and related comorbidities . recommendation are that low - risk disease ( low- or intermediate-1-risk international prognostic scoring system [ ipss ] ) with a life expectancy of 5 years , and either a serum ferritin level over 2000 mg / l or a transfusion intake of at least 20 units of packed red blood cells , or clinical , biopsy , or imaging evidence of iron overload and those who are eligible for hematopoietic stem cell transplantation should receive iron - chelation therapy.4851 porter et al have reported the results of a prospective phase ii trial that evaluated the efficacy of deferasirox in 184 regularly transfused patients , including 47 mds patients.52 overall , the results of the trial showed that there were no significant statistical differences between disease groups in terms of response to iron - chelation therapy , and that liver lic changes were dependent on dose and transfusion iron intake . change in serum ferritin in relation to the change in lic followed a similar trend for all disease groups treated with the drug . in this trial , changes in lic were more pronounced in the mds subgroup of patients , even though these patients had the lowest transfusion iron intake . in another small study of 20 mds patients by metzegeroth et al , deferasirox was effective in reducing ferritin concentration ; however , the authors noted that 70% of the patients had an increase in ferritin levels during the first 4 weeks of deferasirox therapy , and hence concluded that serum ferritin levels may not be very reliable for dose escalation earlier in the treatment course.53 wimazal et al reported efficacy of deferasirox in 14 mds patients treated for up to 24 months . all patients except one demonstrated decreased ferritin levels during therapy , with four patients achieving a complete response.54 in a recent multicenter trial , nolte et al evaluated the efficacy and safety of deferasirox ( mean daily trial dose 19 mg / kg ) in 50 low- or intermediate-1-risk ( ipss ) mds patients . a significant reduction of ferritin and hematological improvement was reported in six patients ( 11%).55 the largest prospective trial to date ( epic ) enrolled 341 mds patients among a total of 1,744 patients . serum ferritin levels decreased significantly from baseline after 1 year of treatment , with a median reduction of 606 ng / ml . responses were seen in both pretreated and chelation - nave patients . however , only about 52% of the mds patients were able to complete 1 year of treatment , with about 13% of the patients discontinuing due to adverse effects.56 the second - largest trial ( us03 ) was an open - label , single - arm , phase iii trial that included 176 mds patients . in this trial , median serum ferritin levels decreased by 23% in the 53% of patients who completed 12 months of treatment , by 36.7% in patients who completed 2 years , and by 36.5% in patients who completed 3 years , despite continued transfusion requirements . over 1 year of deferasirox treatment , lineage - specific improvements in hematologic parameters were noted in 15%22% of patients , suggesting that hematologic improvement may occur with chelation therapy in mds patients . reduction in serum ferritin significantly correlated with alkaline phosphate improvement , a finding also noted in the epic trial.56,57 across these clinical trials , the most common toxicities reported in up to 45% of mds patients were gastrointestinal symptoms , including diarrhea , nausea , vomiting , and abdominal pain , which may be partly related to the presence of lactose in the formulation of deferasirox.52,53 skin rash was reported in about 9% of patients and nonprogressive transient increase in serum creatinine has also been commonly reported . in the epic trial , apart from the usual gastrointestinal and skin toxicities , hearing loss and lens opacities were uncommonly reported.56 in the extend and exjange studies , among the 335 treatment - nave and prechelated mds patients enrolled , seven of them ( five nave and two prechelated ) experienced serious adverse effects , including gastrointestinal bleeding , myocardial infarction , neutropenia , lens opacity , and acute renal failure.58 in the us03 trial , the rate of drug discontinuation due to adverse effects was reported as 24.8% , most commonly related to gastrointestinal disturbances and creatinine increases.57 iron chelation with deferasirox in mds patients seems to have favorable effects on hematopoiesis in a subset of patients with about 28% low- and intermediate-1-grade mds patients having hematological improvement by international working group criteria in the us03 trial . in the post hoc analysis evaluating hematological response to deferasirox therapy in the iron - overloaded mds patient cohort enrolled in the epic trial , improvement in hematologic parameters with an overall erythroid response was noted in 21.5% of the patients , platelet response in 13.0% of the patients , and neutrophil response in 22.0% of the patients.59 with its confirmed efficacy and acceptable safety profile , deferasirox is a reasonable option for selected iron - overloaded mds patients . however , particular attention should be paid to toxicity monitoring in this group of patients , most of whom are elderly with significant comorbidities and side effects from other concomitant therapies for mds . there is evidence from registry data as well as retrospective analyses that iron chelation improves overall survival and delays time to development of acute myeloid leukemia in patients with mds.60,61 if these findings are confirmed in prospective studies like the telesto trial mentioned previously , iron chelation may prove to be a disease - modifying therapy for mds . the epic trial also enabled evaluation of efficacy of chelation therapy in patients with aplastic anemia ( n = 116 ) . at a mean deferasirox dose of 17.6 mg / kg / day , there was a significant reduction in serum ferritin by 964 ng / ml . the most common toxicities reported in this patient population were nausea , diarrhea , and skin rash . there were no reported progressive increases in serum creatinine or hepatic transaminases.62 in addition , a post hoc analysis of the epic trial has shown hematologic responses in eleven of 24 patients who received deferasirox as sole therapy.63 the efficacy of deferasirox has also been demonstrated in diamond blackfan anemia and fanconi anemia.52,64 iron overload in hereditary hemochromatosis ( hh ) results from increased iron absorption from the gut . although phlebotomy is effective and remains the preferred treatment for hh , oral iron chelation may be required in some patients due to factors like poor venous access , concurrent anemia , or patient preference . deferasirox is not fda - approved for treatment of iron overload in hh . in a phase i / ii trial in which a total of 49 patients with hfe - related hh were treated with 5 , 10 , or 15 mg / kg of deferasirox daily , serum ferritin levels declined by 63.5% , 74.8% , and 74.1% in the 5 , 10 , and 15 mg / kg cohorts , respectively , at 48 weeks . seven patients discontinued treatment due to adverse effects . in this study , 10 mg / kg / day was the best - tolerated and most effective dose.65 localized iron overload in the brain is implicated in the pathogenesis of some neurodegenerative disorders.66 however , no systematic trials of deferasirox or other iron chelators have been performed for these conditions . in order to achieve effective iron chelation with minimal toxicity in individual patients , regular monitoring to assess iron overload and adverse effects of deferasirox treatment is essential . despite marked day - to - day variations , serum ferritin levels correlate well with lic . based on monthly measurements of serum ferritin , doses of deferasirox can be adjusted every 36 months if required , with accurate and careful assessment of toxicity profiles . in clinical evaluation of deferasirox , the most commonly reported adverse effect was transient , mild to moderate gastrointestinal disturbance , which tended to occur early in the course of the treatment . abdominal pain has been reported in about 5% of patients.23 it is recommended to avoid concomitant use of aluminum - containing antacids , nonsteroidal anti - inflammatory drugs , and narcotics with deferasirox . it is also helpful to advise patients to take the medication at bedtime with sips of water . in cases with persistent gastrointestinal side effects , splitting the total daily dose into two doses or changing the medium in which tablets are dissolved ( water , apple juice , or orange juice ) may be helpful . adequate hydration also helps with nausea associated with the drug . in the majority of patients , diarrhea is self - limiting or can be easily managed with antidiarrheal treatments . drug interruption may be required in patients in whom diarrhea persists for more than 2 weeks . deferasirox can be continued without dose adjustment in cases of mild rashes , as the majority resolve spontaneously . in cases of severe rash some investigators have utilized low - dose prednisone in the management of rash ; doses of 5 mg in children and 10 mg in adults for a maximum of 1 month can be considered . serum creatinine levels should be regularly monitored with special attention in patients with scd who may have compromised baseline renal function . increases in serum creatinine may be related to overchelation . about 2% of patients developed elevations in serum transaminases , unrelated to dose of deferasirox . liver - function tests should be obtained prior to initiation of therapy and at regular intervals thereafter . auditory and ocular adverse events with deferasirox are rare ; however , it is recommended that patients be monitored at baseline and annually during treatment.26 serious adverse effects may require termination of deferasirox in about 10% of patients.67 chronic iron overload is a serious condition that necessitates close monitoring and effective chelation therapy in order to prevent long - term complications . the availability of deferasirox is a major milestone in iron - chelation therapy , and has made such therapy convenient for a large number of patients . there is enough evidence from various pivotal clinical trials and subsequent extension studies validating the efficacy and safety of deferasirox for the treatment of iron - overloaded , regularly transfused patient populations , including those with thalassemia , scd , mds , and other rare anemias , including aplastic anemia . however , even longer follow - up is needed to establish fully the long - term efficacy and toxic side effects of deferasirox , particularly in patients with hemoglobinopathies , who are likely to be receiving deferasirox therapy for decades .

deferasirox is a once - daily , oral iron chelator that is widely used in the management of patients with transfusional hemosiderosis . several phase ii trials along with their respective extension studies as well as a phase iii trial have established the efficacy and safety of this novel agent in transfusion - dependent patients with -thalassemia , sickle - cell disease and bone marrow - failure syndromes , including myelodysplastic syndrome and aplastic anemia . data from various clinical trials show that a deferasirox dose of 20 mg / kg / day stabilizes serum ferritin levels and liver iron concentration , while a dose of 3040 mg / kg / day reduces these parameters and achieves negative iron balance in red cell transfusion - dependent patients with iron overload . across various pivotal clinical trials , deferasirox was well tolerated , with the most common adverse events being gastrointestinal disturbances , skin rash , nonprogressive increases in serum creatinine , and elevations in liver enzyme levels . longer - term extension studies have also confirmed the efficacy and safety of deferasirox . however , it is essential that patients on deferasirox therapy are monitored regularly to ensure timely management for any adverse events that may occur with long - term therapy .

Introduction Pharmacology of deferasirox Thalassemia Sickle-cell disease Myelodysplastic syndromes Nontransfusional iron overload Management of adverse effects of deferasirox Conclusion

iron overload contributes significantly to morbidity and mortality in patients receiving regular red blood - cell transfusions as supportive therapy for chronic anemias such as -thalassemia , sickle - cell disease ( scd ) , and myelodysplastic syndromes ( mdss ) . while deferoxamine ( dfo ) is an effective iron chelator that has been shown to improve survival in iron - overloaded -thalassemia patients , its delivery and compliance with therapy are less than ideal.1 dfo is typically administered as a subcutaneous infusion over 824 hours , 57 days per week , delivered overnight by a small portable pump.2 this arduous schedule interferes with patients quality of life and results in poor patient compliance.3 the urgent need for an effective and safe oral iron chelator led to the expedited approval of deferasirox ( exjade , novartis pharmaceuticals corporation , basel , switzerland ) by the us food and drug administration ( fda ) in november 2005 for the treatment of transfusional iron overload associated with thalassemia and other iron - loading conditions with an orphan - drug designation.4 deferiprone , another orally bioavailable iron chelator , has been shown to be safe and efficacious in iron - overloaded -thalassemia patients . although it was being used in other countries , deferiprone received fda approval only in october 2011 for treatment of patients with transfusional iron overload due to thalassemia syndromes who were not adequately chelated with other agents . however , the use of deferiprone has been limited , due to the occurrence of rare but serious adverse events during treatment , such as neutropenia and agranulocytosis.5,6 this review will focus on the long - term safety and efficacy of deferasirox in trials conducted for transfusional hemosiderosis in hemoglobinopathies and bone marrow - failure syndromes . deferasirox is a tridentate chelator with a long half - life of 816 hours , allowing for convenient once - daily administration.7 it is rapidly absorbed , and has a bioavailability of about 70% . it is mainly excreted in the feces ( 84% of the dose ) after it is metabolized by glucuronidation and subsequent biliary excretion.8 the highest dose of deferasirox approved by most health authorities is 30 mg / kg / day . however , most recently the fda and european medicines agency have approved doses up to 40 mg / kg / day in patients not adequately chelated with doses of 30 mg / kg / day ; doses above 40 mg / kg / day are generally not recommended.4,9 the efficacy and safety of deferasirox were initially studied in a series of 1-year phase ii and iii clinical trials involving more than 1,000 patients . results from the extension phases , with a median follow - up of 5 years now , have confirmed the efficacy of deferasirox to be dose- and transfusion - dependent.10 across these clinical trials , deferasirox was well tolerated , with the most common drug - related adverse events being mild to moderate in severity and often self - limiting ( table 1 ) . serious albeit rare adverse effects reported with long - term deferasirox use include renal failure , agranulocytosis , hepatic toxicity , and gastrointestinal bleeding , making close toxicity monitoring mandatory.11 thalassemia , first described by cooley and lee in 1925 , is a hereditary form of anemia resulting from an imbalance in the synthesis of the - and -chains of hemoglobin leading to hemolysis and ineffective erythropoiesis.12 iron overload in patients with thalassemia is mainly the result of regular red blood - cell transfusions and can result in an iron load of up to 10 g per year.13 iron overload can also result from increased intestinal iron absorption , particularly in patients with thalassemia intermedia.14 patients who receive regular blood transfusions without adequate chelation therapy develop evidence of iron excess ( elevated liver iron concentration [ lic ] and serum ferritin ) within 12 years of transfusion therapy . adequate iron chelation with dfo from early on in life in these iron - overloaded -thalassemia patients has significant benefits on both morbidity and early mortality.15,16 unfortunately , effective administration of iron - chelation therapy with dfo is often compromised secondary to the arduous schedule of overnight subcutaneous infusions leading to reduced compliance.17 dfo is also associated with local skin reactions , ophthalmological adverse effects ( optic neuropathy , retinal pigmentation ) , ototoxicity , anaphylactic reactions , pulmonary fibrosis , and rarely renal impairment.18,19 in various trials done to date , deferasirox has proved to be an effective , safe oral iron - chelating agent in patients with thalassemia . efficacy was judged to be comparable for 20 mg / kg / day of deferasirox and 40 mg / kg / day of dfo , supporting the assumed 1:2 equivalence ratio.22 in a subsequent large phase iii comparative trial , patients with -thalassaemia major were randomized to receive either deferasirox ( 296 patients ) or dfo ( 290 patients ) , dosed according to their baseline lic . over 1 year , a deferasirox dose of 20 mg / kg / day stabilized the mean serum ferritin level close to 2,000 ng / ml , achieved negative iron balance , and maintained lic . for both drugs , a clear dose a deferasirox dose of 30 mg / kg / day reduced serum ferritin by about 1,200 ng / ml and achieved negative iron balance , with a fall in lic of 8.9 mg iron / g dry weight over 1 year , whereas lower doses of deferasirox were not as effective and led to a positive iron balance in most patients.23 transient gastrointestinal disturbances , including abdominal pain , nausea , vomiting , diarrhea , and constipation , were reported in 15.2% of patients . mild , dose - dependent increases in serum creatinine were noted in 38% of patients on deferasirox ; however , only 13% of patients required dose reduction , as creatinine spontaneously normalized in a number of patients . in order to evaluate the long - term efficacy and safety of deferasirox in patients with -thalassemia who completed the 1-year trial , a 4-year extension study was conducted.24 in this extension trial , patients either continued receiving deferasirox ( deferasirox cohort ) or crossed over from dfo to deferasirox ( crossover cohort ) . renal and liver functions were closely monitored during the trial , and no progressive increases in serum creatinine or liver transaminase levels were observed during long - term deferasirox treatment . long - term safety of iron chelators is critical in -thalassemia patients , since most require lifelong red cell transfusions and therefore iron - chelation therapy . deferasirox was found to be an effective first - line therapy to maintain negative iron balance without adverse effects on growth and development in this age - group.26 the efficacy of deferasirox in reducing cardiac iron and thereby preventing cardiac iron loading was assessed using cardiovascular magnetic resonance by measuring myocardial t2 * in a subgroup of patients with -thalassemia ( n = 192 ) who were enrolled in the large evaluation of patients iron chelation with exjade ( epic ) trial.27 a cardiac t2 * value of < 20 ms is considered to indicate cardiac iron levels below the normal range , whereas a value < 10 ms is associated with reduction in left and right ventricular ejection fraction and an increased risk of cardiac failure and arrhythmia . results of this study were significant for marginal but statistically significant improvement in myocardial t2 * in the treatment arm , improved from a baseline of 11.2 ms ( 40.5% ) to 12.9 ms ( 49.5% ) with maintained left ventricular function , while in patients without cardiac iron overload ( prevention arm ) , the ejection fraction improved significantly . mean deferasirox doses were high ( 33.6 9.8 mg / kg / day ) , with no increase in adverse effects noted.28 in another study of 19 patients , significant improvements of myocardial t2 * were demonstrated in heavily transfused iron - overloaded patients who demonstrated greater than 95% compliance to therapy with deferasirox , reiterating the fact that compliance is essential for success of treatment in myocardial siderosis.29 deugnier et al reported long - term effects of deferasirox treatment on liver fibrosis , and found that deferasirox treatment for 3 or more years reversed or stabilized liver fibrosis and necroinflammation in 83% of patients with iron - overloaded -thalassemia . this therapeutic effect was independent of reduction in lic.30 another prospective , open - label , 1-year study , the escalator ( extension study of the efficacy and safety of deferasirox treatment in beta - thalassemia patients with transfusional hemosiderosis ) trial was conducted in the middle east to evaluate the efficacy and safety of deferasirox in patients who had been previously treated with dfo and/or deferiprone . most patients ( 78.1% ) underwent dose increases above 20 mg / kg / day , mostly to 30 mg / kg / day . deferasirox was well tolerated overall , with manageable side effects and good safety profile , and there were no reported discontinuations due to adverse effects . unlike -thalassemia , iron overload can be easily overlooked in scd patients due to episodic requirements for red cell transfusions , even though these patients often develop iron - induced complications later in life similar to other chronically transfused populations.35 therefore , guidelines from consensus panels have recommended iron - chelation therapy in scd patients with evidence of iron overload.36 current recommendations are to initiate iron - chelation therapy in patients with scd once lic increases to 7 mg iron / g dry weight , if serum ferritin steady - state levels are > 1000 ng / ml , or if patients have received cumulative transfusions of 120 ml packed red blood cells / kg ( usa ) or at least 20 top - up transfusions ( uk ) ( national institutes of health nhlbi , 2002 ; sickle cell society , 2008 ) . in order to assess the long - term efficacy and safety of deferasirox in patients with scd , patients who completed the 1-year study entered a 4-year extension phase where they continued to receive deferasirox , or switched from dfo to deferasirox.38 of the patients who received at least one dose of deferasirox , only 62 patients ( 33.5% ) completed the extension study ; the reasons for such a high rate of discontinuation in scd patients remain unclear . another drawback of the study was the inability to assess the impact of deferasirox therapy on the frequency of sickle - cell crisis secondary to the lack of control group in the study design . however , serum ferritin levels in patients with 4 years of deferasirox exposure significantly decreased by 591 ng / ml , and the tolerability of the drug improved with long - term follow - up , as a decrease in the most common adverse effect was noted with continued therapy . cancado et al investigated the efficacy and safety of a 2-year treatment program with deferasirox in 31 patients with scd and transfusional iron overload . the most common adverse effects in the study were transient and mild , with diarrhea and headaches reported in 22.6% patients each . with every unit of transfused blood , the patients receive approximately 200250 mg iron.41 another less recognized cause of iron excess in mds patients is increased absorption of iron from the gut due to ineffective erythropoiesis.42 overall survival and leukemia - free survival are significantly shorter in transfusion - dependent mds patients.43 this may simply be related to disease severity or be an effect of iron overload itself . gattermann and rachmilewitz noted shortened overall survival with increasing ferritin level , with a hazard ratio of 1.42 for every 500 ng / ml increase in ferritin over 1,000 ng / ml.44 even in patients undergoing hematopoietic stem cell transplantation ( hsct ) , elevated pre - transplant serum ferritin level adversely affects survival.45 excess iron has also been shown to suppress erythropoiesis , as evidenced by decreased burst - forming units erythroid in patients with elevated ferritin compared to patients with normal ferritin.46 thus , iron overload has many deleterious effects , especially in the usually older mds population , with significant comorbidities that make them more sensitive to the effects of iron overload.47 as phlebotomy is not a feasible option in mds patients , an effective way to prevent adverse consequences of iron overload is iron - chelation therapy . recommendation are that low - risk disease ( low- or intermediate-1-risk international prognostic scoring system [ ipss ] ) with a life expectancy of 5 years , and either a serum ferritin level over 2000 mg / l or a transfusion intake of at least 20 units of packed red blood cells , or clinical , biopsy , or imaging evidence of iron overload and those who are eligible for hematopoietic stem cell transplantation should receive iron - chelation therapy.4851 porter et al have reported the results of a prospective phase ii trial that evaluated the efficacy of deferasirox in 184 regularly transfused patients , including 47 mds patients.52 overall , the results of the trial showed that there were no significant statistical differences between disease groups in terms of response to iron - chelation therapy , and that liver lic changes were dependent on dose and transfusion iron intake . in another small study of 20 mds patients by metzegeroth et al , deferasirox was effective in reducing ferritin concentration ; however , the authors noted that 70% of the patients had an increase in ferritin levels during the first 4 weeks of deferasirox therapy , and hence concluded that serum ferritin levels may not be very reliable for dose escalation earlier in the treatment course.53 wimazal et al reported efficacy of deferasirox in 14 mds patients treated for up to 24 months . all patients except one demonstrated decreased ferritin levels during therapy , with four patients achieving a complete response.54 in a recent multicenter trial , nolte et al evaluated the efficacy and safety of deferasirox ( mean daily trial dose 19 mg / kg ) in 50 low- or intermediate-1-risk ( ipss ) mds patients . however , only about 52% of the mds patients were able to complete 1 year of treatment , with about 13% of the patients discontinuing due to adverse effects.56 the second - largest trial ( us03 ) was an open - label , single - arm , phase iii trial that included 176 mds patients . in this trial , median serum ferritin levels decreased by 23% in the 53% of patients who completed 12 months of treatment , by 36.7% in patients who completed 2 years , and by 36.5% in patients who completed 3 years , despite continued transfusion requirements . reduction in serum ferritin significantly correlated with alkaline phosphate improvement , a finding also noted in the epic trial.56,57 across these clinical trials , the most common toxicities reported in up to 45% of mds patients were gastrointestinal symptoms , including diarrhea , nausea , vomiting , and abdominal pain , which may be partly related to the presence of lactose in the formulation of deferasirox.52,53 skin rash was reported in about 9% of patients and nonprogressive transient increase in serum creatinine has also been commonly reported . in the epic trial , apart from the usual gastrointestinal and skin toxicities , hearing loss and lens opacities were uncommonly reported.56 in the extend and exjange studies , among the 335 treatment - nave and prechelated mds patients enrolled , seven of them ( five nave and two prechelated ) experienced serious adverse effects , including gastrointestinal bleeding , myocardial infarction , neutropenia , lens opacity , and acute renal failure.58 in the us03 trial , the rate of drug discontinuation due to adverse effects was reported as 24.8% , most commonly related to gastrointestinal disturbances and creatinine increases.57 iron chelation with deferasirox in mds patients seems to have favorable effects on hematopoiesis in a subset of patients with about 28% low- and intermediate-1-grade mds patients having hematological improvement by international working group criteria in the us03 trial . in the post hoc analysis evaluating hematological response to deferasirox therapy in the iron - overloaded mds patient cohort enrolled in the epic trial , improvement in hematologic parameters with an overall erythroid response was noted in 21.5% of the patients , platelet response in 13.0% of the patients , and neutrophil response in 22.0% of the patients.59 with its confirmed efficacy and acceptable safety profile , deferasirox is a reasonable option for selected iron - overloaded mds patients . at a mean deferasirox dose of 17.6 mg / kg / day , there was a significant reduction in serum ferritin by 964 ng / ml . there were no reported progressive increases in serum creatinine or hepatic transaminases.62 in addition , a post hoc analysis of the epic trial has shown hematologic responses in eleven of 24 patients who received deferasirox as sole therapy.63 the efficacy of deferasirox has also been demonstrated in diamond blackfan anemia and fanconi anemia.52,64 iron overload in hereditary hemochromatosis ( hh ) results from increased iron absorption from the gut . in a phase i / ii trial in which a total of 49 patients with hfe - related hh were treated with 5 , 10 , or 15 mg / kg of deferasirox daily , serum ferritin levels declined by 63.5% , 74.8% , and 74.1% in the 5 , 10 , and 15 mg / kg cohorts , respectively , at 48 weeks . in this study , 10 mg / kg / day was the best - tolerated and most effective dose.65 localized iron overload in the brain is implicated in the pathogenesis of some neurodegenerative disorders.66 however , no systematic trials of deferasirox or other iron chelators have been performed for these conditions . about 2% of patients developed elevations in serum transaminases , unrelated to dose of deferasirox . auditory and ocular adverse events with deferasirox are rare ; however , it is recommended that patients be monitored at baseline and annually during treatment.26 serious adverse effects may require termination of deferasirox in about 10% of patients.67 chronic iron overload is a serious condition that necessitates close monitoring and effective chelation therapy in order to prevent long - term complications . the availability of deferasirox is a major milestone in iron - chelation therapy , and has made such therapy convenient for a large number of patients . there is enough evidence from various pivotal clinical trials and subsequent extension studies validating the efficacy and safety of deferasirox for the treatment of iron - overloaded , regularly transfused patient populations , including those with thalassemia , scd , mds , and other rare anemias , including aplastic anemia . however , even longer follow - up is needed to establish fully the long - term efficacy and toxic side effects of deferasirox , particularly in patients with hemoglobinopathies , who are likely to be receiving deferasirox therapy for decades .

dna - protein interactions are involved in several biological processes like transcription , replication , dna repair , or recombination . the variety of these mechanisms involves variations of the electrostatic potential due to groove narrowing or specific hydrogen bond donors and acceptors of the dna helix that are recognized by a complementary set of amino acids . label - based methods require the ligation of the analyte and/or ligand with reporters like enzymes , fluorescent dyes , or radioisotopes . these labels possess the disadvantage that they can adulterate the results by interfering with the molecular interaction . blocking the active binding site or affecting the conformation of the analyte can lead to false negatives . moreover , unspecific background binding leading to false positives is another issue in these assays [ 2 , 3 ] . in label - free approaches like atomic force microscopy - dynamic force spectroscopy experiments [ 4 , 5 ] , acoustic biosensors based on quartz crystal resonators , calorimetric biosensors , and surface plasmon resonance ( spr ) inherently properties ( e.g. , mass ) of the interacting molecules are measured . this article will focus on the most widely used label - free detection method : surface plasmon resonance . offer spr - based instruments , biacore ( ge healthcare ) is by far the main supplier on the spr market . in 2007 , 89% of all publications dealing with a beam of polarized light that propagates in a medium of high refractive index ( e.g. , a prism ) is totally reflected , if it encounters an interface at a medium of low refractive index ( n2 ) at a specific angle ( ) . although a total reflection occurs , the electromagnetic field component penetrates over a short distance into the medium of low refractive index . if the interface is coated with a thin layer of metal ( e.g. , gold ) , a dip in the intensity of the polarized light will be visible ( kretschmann - reather atr configuration ) . the surface plasmons can couple with the photons of the polarized light , if the wavevector of the photon ( kx ) equals the wavevector of the surface plasmons ( ksp ) . coupling of both quasiparticles leads to an enhancement of the evanescent field amplitude ( e ) . this phenomenon , called surface plasmon resonance , results in the observed dip of the light intensity . the wavevector of the plasmons depends on the refractive index of the conductor and the neighboring medium of low refractive index ( n2 ) . the wavevector of the photon depends on the wavelength of the polarized light and the angle of incidence ( ) . in conclusion , the refractive index n2 can be determined by measuring the intensity of the reflected light at different angles of incidence ( ) , if the wavelength of the polarized light and the refractive index of the conductor are both known [ 9 , 10 ] . in most practical applications of spr , the kretschmann - reather atr method that was already described in the last section is used . in this setup , a thin metal film ( typically around 50 nm thick gold layer ) is evaporated onto the glass prism and kept in direct contact with the medium of lower refractive index ( n2 ) [ 10 , 11 ] . in order to evaluate the interactions of a protein with a nucleic acid that results in the formation of a protein - dna complex , one of the two interaction partners has to be immobilized on the surface of the conductor ( ligand in figure 1 ) . in most cases , a sensor chip with preimmobilized streptavidin is used to immobilize biotinylated oligonucleotides ( more details concerning immobilization will be discussed below ) . the other interaction partner ( e.g. , the protein = analyte in figure 1 ) is injected into the running buffer that passes the surface at a constant flow . in biacore instruments , the resulting change in concentration of molecules at the gold surface due to the formation of the protein - dna complex is measured in resonance units ( rus ) and can be described according to ( 1 ) : ( 1)ru = nx=[riic]x=[(nc)ligandc]x . in this equation , n is the changing refractive index at the surface , x is a multiplier to convert n to ru , rii is the refractive index increment of the protein that is binding to the immobilized oligonucleotide , and c is the concentration of the protein . in general , ru correspond to a change in angle of 0.1 , or a protein concentration of 1 ngmm ( alternatively 10 mgml ) [ 13 , 14 ] . one set of problems that is connected to the rii has to be mentioned when using the correlation of ru and protein concentration . the rii value of the molecules used is presumed to be in a range of ~0.180.19 mlg . however , nonprotein molecules exhibit rii values beyond this range . in order to accurately perform an affinity ranking and correct stoichiometric measurements of small molecules the ru value has to be normalized for each measured compound . fortunately , the rii value is not important to get correct kinetic and thermodynamic results in simple protein - protein or protein - oligonucleotide interactions . the typical shape of a sensogram that displays the change of the response units during the course of the experiment is shown in figure 2 . it can be divided into four different phases : association phase , steady - state or equilibrium phase , dissociation phase , and regeneration phase . the association phase starts with the injection of the analyte ( e.g. , protein ) . due to the formation of a protein - dna complex , the refractive index changes , resulting in a variation of the specific angle ( ) where the dip in intensity of the reflected light is at its minimum . during the following equilibrium phase , association and dissociation of the complex occur at equal rates . shortly , after the injection is terminated , dissociation of the analyte ( e.g. , protein ) from the ligand ( e.g. , oligonucleotide ) leads to a decrease in the response units . at any point of the dissociation phase it either contains a high salt concentration or detergents like sds that release all remaining analyte molecules from the surface [ 12 , 14 ] . after having finished the described cycle , another concentration of the analyte can be injected . every standard biacore instrument is equipped with an integrated microfluidic cartridge ( ifc ) that forms four flow cells on the sensor chip and thereby enables the measurement of four different ligands at a time . in most applications , the first flow cell is used to substract response units resulting from unspecific interactions between the analyte ( e.g. , protein ) and the chip surface or the analyte and flanking regions of the oligonucleotides ' recognition sequence . this on - line referencing can be achieved by either keeping the first flow cell blank or by immobilizing an oligonucleotide that exhibits a random sequence [ 14 , 16 ] . after having performed the experiment and referenced the results , the data is evaluated for example , using the bia - evaluation software or scrubber-2 ( more aspects concerning data evaluation will be discussed below ) . the guidance of light inside an optical fiber is also based on total internal reflection ( tir ) . therefore , the prism in the kretschmann - reather atr setup can be replaced by the core of a fiber ( cf . figure 3 ) . in order to assemble a fiber - optic - based spr ( fo - spr ) , the silicon cladding has to be removed in a small area of the optical fiber . the cleared core is surrounded with a thin metal coating and a dielectric sensing layer ( cf . figure 3 ) . unlike prism - based spr instruments , sensing is accomplished by changing the wavelength instead of the angle of incidence . therefore , the resonance wavelength is measured instead of the resonance angle ( ) . moreover , there is more than one reflection event . due to the fact that the width of the spr curve that affects the detection accuracy of the spr instrument depends on the number of reflections , fiber parameters like length , sensing region , and fiber core diameter are crucial for the performance . to further change the sensitivity , detection accuracy and operating range of fiber optic - based sprs , several modifications including a bimetallic coating , a tapered or u - shaped probe or the addition of dopants like geo2 have been used . although fiber - optic - based spr instruments have some advantages like the simple and miniaturized setup that lacks moving parts or the possibility to assemble an inexpensive disposable sensor for medical or sterile tasks , the sensitivity is limited in comparison to the kretschmann - reather setup [ 10 , 11 ] . a combination of protein arrays and techniques like spr would result in label - free alternatives to existing high - throughput methods that access kinetic data . however , the restriction that standard biacore instruments are equipped with an ifc that forms four flow cells on the sensor chip complicates the expansion of spr to high - throughput screening applications . therefore , the sensitivity of spr was combined with the features of imaging methods resulting in spr imaging ( spri ) . in spri , a coherent polarized light beam is expanded in order to cover a larger area of the sensing surface . the intensity of the reflected light is detected by a ccd ( charge - coupled device ) camera as image . in contrast to conventional spr , the measurement is performed at a fixed wavelength and a fixed angle within a linear part of the spr curve ( cf . due to these confinements , changes in light intensity are proportional to alterations in the refractive index near the surface . in an spri experiment , it is either possible to substract an image that was taken before the injection start or to do on - line referencing by keeping one spot of the array blank . finally , a difference image of the array and a chart that displays the change in reflectivity for each probe during the course of time is obtained and can be evaluated further . one problematic issue of spri is the intensity of the light source . due to the proportional dependency of the signal strength on the incoming light intensity , laser beams are the preferred source . however , expanding the laser beam using optical elements results in an inhomogeneous illumination of the surface . new instruments with a scalable light source overcome the mentioned disadvantage by providing a more flexible illumination area . one advantage of label - free screens is that reporter groups that might interfere with the molecular interactions are unnecessary . however , in nearly all of the corresponding screening methods , also in the case of surface plasmon resonance , one of the interaction partners has to be immobilized . it is the most convenient and cost - effective way to immobilize short oligonucleotides as ligands on the surface to study protein - dna interactions . if the unspecific interactions between the sensor chip and the analyte are too big or if the interaction between a single protein and several oligonucleotides has to be analyzed , it might be necessary to reverse the alignment of the experimental setup by immobilizing the protein on the sensor chip . in general , it is essential for the quality , validity , and reproducibility of the results to test and select the optimum immobilization method and assembly . due to the formation of stable sulfur - gold bonds , direct immobilization of thiol - containing ligands on a gold surface is possible . proteins that lack cysteine residues can easily be modified using intein - mediated protein splicing combined with native chemical ligation , thus connecting a purification method with the ligation of a c - terminal cysteine . however , proteins coming into contact with the metal can denature and undesired interactions with the surface are possible leading to inconsistent results [ 19 , 20 ] . therefore , in most spr implementations , the gold surface is covered with hydroxyalkyl - thiols like 16-mercapto - hexadecan-1-ol thus creating a hydrophobic self - assembled monolayer ( sam ) that prevents analytes and ligands to interact with the metal . moreover , this layer serves as a functionalized structure that enables a further modification with carboxymethyl - modified dextran ( e.g. , biacore 's cm5-chip ) . this hydrogel facilitates the application of several immobilization strategies through linker molecules that can be attached covalently . biomolecules that exhibit free primary amines can be attached covalently to amine - reactive surfaces . in the case of sensor chips functionalized with a carboxymethyl - modified dextran layer ( e.g. , biacore 's cm5 ) , the carboxyl groups can be activated by n - ethyl - n-(dimethylaminopropyl)-carbodiimide ( edc ) , and n - hydroxyl succinimide ( nhs ) . sensor chips functionalized with aldehyde - terminated sams can be reacted with amine - modified oligonucleotides and proteins in aqueous buffer at basic ph , respectively . in both cases , a similar approach starts with coupling of hydrazine to the carboxymethyl - modified dextran layer using edc / nhs coupling followed by the addition of ligands with aldehyde substituents ( cf . one disadvantage of these methods is the abundance of reactive amines in proteins . due to the fact that several proteins contain more than one lysine residue , coupling of amines will lead to a heterogeneous population of ligands with random orientation and eventually random accessibility of the interaction site . due to the lower abundance of cysteine residues in proteins , thiol - reactive surfaces combine the advantages of sensor chips equipped with sams with a more specific immobilization mechanism in comparison to the amine - based coupling reactions . gold surfaces coated with carboxymethyl - modified dextran can be derivatized with sulfhydryl - reactive reagents like pyridinyldithioethanamine ( pdea ) using the edc / nhs coupling described above . addition of a cysteine - containing protein results in the formation of a reversible disulphide linkage between the ethanamine and the ligand ( cf . residual free thiol groups are subsequently saturated using pdea . a second method based on thiol - reactive sensor chips therefore , n-[-maleimidocaproic acid]-hydrazide ( emch ) is coupled to a chip coated with carboxymethyl dextran ( e.g. , cm5 ) using edc / nhs ( cf . another similar approach involves coupling of ethylenediamine using edc / nhs followed by the addition of n-[-maleimidobutyryloxy]sulfo - succinimide ester ( sulfo - gmbs ) . direct immobilization of maleimide derivatives on a gold surface without using a carboxymethyl dextran - coated sensor chip can be achieved by addition of maleimide - ethylene glycol - terminated disulfide ( meg ) to a bare gold surface or sulfosuccinimidyl 4-(n - maleimidomethyl)-cyclohexane-1-carboxylate ( ssmcc ) to a gold surface coated with fmoc - protected 11-mercaptoundecylamine ( muam ) . in both cases , thiolated oligonucleotides ( sh - dna ) coupling of thiazolidine-4-carboxylic acid ( 2-aminoethyl ) amide to a cm5 sensor chip using edc / nhs followed by deprotection of the thiazolidine ring with methoxyamine hydrochloride results in a free immobilized cysteine residue that readily undergoes native chemical ligation with peptide thioesters or expressed protein thioesters ( cf . figure 5(e ) ) . if commercially obtained thiol - functionalized dna is used without further purification in any of the immobilization procedures described above , it has to be taken into consideration that the diversity of techniques for synthesis and purification can lead to a variety of impurities still present in the sample . compounds like dithiothreitol ( dtt ) used to cleave the dimethoxytrityl protection group ( dmt ) can lead to a reduced amount of surface bound dna . in order to increase the specificity of the immobilization process o - alkylguanine - dna - alkyltransferase ( hagt ) is an important dna repair protein that removes alkyl groups from the o - position of guanine . one pseudosubstrate of the hagt , o - benzylguanine is known to inhibit the transferase irreversibly . hence , immobilizing an o - benzylguanine peg - amino derivative via edc / nhs chemistry followed by the addition of hagt fused to a ligand of interest results in a covalent attachment of the fusion protein to the carboxymethyl dextran coated surface . a second approach is based on a transpeptidase reaction catalyzed by the sortase a ( srta ) from staphylococcus aureus . srta cleaves between the threonin and the terminal glycine residues within the amino acid sequence leu - pro - x - thr - gly and links it to nucleophiles that exhibit an n - terminal glycine . therefore , immobilizing a peptide with the amino acid sequence h - gly - gly - ser - ser - cys - oh on a sensor chip surface using one of the thiol - coupling methods described above enables to attach proteins that contain the srta recognition sequence to the sensor chip surface by injecting the ligand of interest with the enzyme ( cf . 5-phosphate - terminated single - stranded dna molecules are chemically immobilized on the sensor surface . t4 rna ligase is then able to ligate rna strands to the 5-phosphate of the dna . in order to eliminate the shielding effect of hairpin formation , the rna has to start with eight adenosine bases at the 3 end ( cf . moreover , they are easily regenerated at low ph , can be immobilized using most of the procedures presented here , and are commercially available for most antigens ( cf . the variable region ( f(ab)2 ) of the antibody has to be exposed to the analyte . if physical adsorption or chemical coupling methods are used , only 20% of the antibodies have the right orientation to bind analytes properly . therefore , self - oriented immobilization methods involving proteins like protein a or g that specifically recognize the tail region ( fc ) of the antibody have been developed . hence , sensor chips coated with neutravidin - protein a complexes or protein g - dna conjugates lead to an enhancement of the antibody / antigen binding ability [ 34 , 35 ] . the biotin - streptavidin system exhibits the strongest noncovalent biological interaction known ( ka = 10 m ) . therefore , biotinylated ligands can be tethered to the surfaces of sensor chips functionalized with streptavidin ( cf . figure 6(d ) ) . the tetrameric protein can be immobilized on a standard sensor chip coated with carboxymethyl dextran ( e.g. , cm5 ) using edc / nhs coupling . ready - to - use chips can also be purchased for example , from ge healthcare ( sa - chip ) . the biotin - streptavidin system is the method of choice for immobilizing nucleic acids . in order to be applicable for immobilization , proteins can be biotinylated using nhs - active esters of biotin analogs that react with the -amino function of lysines . further chemical biotinylation methods are based on p - diazobenzoylbiocytin that specifically labels tyrosine and histidine residues , or 3-(n - maleimidopropionyl)-biotin and iodoacetyl - lc - biotin that exclusively react with free thiols . although a substitution level of one biotin per ligand is recommended , the chemical methods described often result in multilabeled compounds , thus impairing the validity and significance of the spr results . intein - mediated protein splicing combined with native chemical ligation using a cysteine biotin derivative is a more specific approach that overcomes this set of problems . moreover , the escherichia coli ( e. coli ) biotin ligase ( bira ) can be used to biotinylate site specifically a ligand fused to the recognition sequence of bira . short biotinylated oligonucleotides , the most frequently used ligands for the analysis of protein - dna interactions , can be readily purchased at every oligonucleotide supplier . the nucleotide can be incorporated into an oligonucleotide using nick translation or added as nontemplated nucleotide to the 3-end of single and duplex dna by the enzyme terminal deoxynucleotidyl transferase ( tdt ) [ 41 , 42 ] . although biotin interacts noncovalently with streptavidin , a reuse of the sensor chip is almost impossible due to the high affinity of the complex . streptavidin binding peptide ( sbp ) is another interaction partner of streptavidin that possesses nanomolar affinity . preparing a fusion protein consisting of the desired ligand and sbp facilitates moderate binding of the ligand and a complete removal from the surface using 1-min injections of 50 mm naoh . an amino acid repeat of 515 glycine and serine residues between the ligand and the sbp enhances the flexibility and assures that the fusion protein is correctly folded . one problem connected with streptavidin - coated chips is the occurrence of unspecific interactions with the surface . electrostatic interactions between the negatively charged carboxymethyl dextran layer of the sensor chip and the protein used as analyte can significantly influence the spr results . first of all , changing the composition of the running buffer might help to reduce nonspecific binding ( see below ) . moreover , alternative usage of neutravidin which does not contain carbohydrate moieties minimizes nonspecific interactions . a third method involves the usage of the commercial available hydrophobic sensor chip ( hpa ) . the hpa - chip consists of a gold surface coated with an alkane - thiol layer . addition of oligonucleotides tagged with a 3-cholesterol group results in double - stranded dna immobilized in a supported lipid monolayer that chemically and physically resembles a cell membrane surface and extensively reduces background interactions . another affinity immobilization method is based on nitrilotriacetic acid- ( nta- ) modified sensor chips . proteins labeled at the n- or c - terminus with oligo histidine ( his ) can be captured via ni nta chelation . the choice of the utilized histidin - tag ( e.g. , hexa - his , deca - his , double - his tags ) depends on the application of the spr experiment . the surface can be regenerated conveniently by stripping the nickel using ethylenediaminetetraacetic acid ( edta ) solutions [ 47 , 48 ] . site - directed immobilization of a fusion protein consisting of a ligand of interest and the dna binding domain of yeast gal4 or the bacterial lexa is an affinity immobilization method based on protein - dna interactions . therefore , double - stranded oligonucleotides containing the recognition sequence of the dna binding domain are spotted on poly(l - lysine- ) coated gold chips . in general , the analyte and the ligand have to be chemically and conformationally homogenous to assure that the data is not corrupted by artifacts based on contaminations . however , even crude samples like nuclear extracts that contain several different dna binding proteins were successfully employed as analytes for oligonucleotides immobilized on a streptavidin - coated sensor chip . in order to specifically recognize the desired transcriptional activator and to amplify the corresponding signals , a primary antibody against the protein and a secondary antibody concentration of the analyte is another very important aspect for reliable and high - quality results . moreover , at least five different concentrations should be used , including zero - concentration injections ( blank injections , see below ) . proteins used as ligands might lose their functionality in the course of time resulting in a signal drift . this phenomenon is , for example , known for proteases [ 54 , 55 ] . by using stabilized mutants or by chemically crosslinking the protein , the stability for the hiv-1 protease was increased . although , the kinetic property of the crosslinked enzyme was similar to those of the native variant , the latter method has to be handled with precaution . in the case of oligonucleotide ligands , the size of the molecule should correspond to the length of the dna footprint , elongated by 36 extra base pairs as spacers on both sides . due to the fact that the surface plasmon wave decays evanescently approximately 200 nm into the solution , even oligonucleotides of considerable length can be used as ligands . the interactions of the transcription factor leafy from arabidopsis thaliana to oligonucleotides that exhibit the sequences of the entire gene promoters apetala3 ( 2386 bp ) and apetala3 ( 3050 bp ) were successfully analyzed and the data quantitatively evaluated . also , oligonucleotides that possess hairpin conformation and contain a nick were directly immobilized on a gold surface . ligation of the nick by dna ligase of e. coli caused a change in the conformation from the hairpin structure to a rigid , linear double helix . the resulting change in the spr dip shift was recorded using a noncommercial high - resolution spr instrument . as already mentioned above , therefore , the flow cell is either left blank or a reference compound is immobilized . if the cell is left blank , only unspecific interactions resulting from bulk refractive index changes , injection noise , baseline drift , or unspecific binding of the analyte to the surface are detected [ 50 , 58 ] . in order to account for the refractive index changes caused by unspecific interactions between protein and dna an oligonucleotide that exhibits a randomized sequence of the same length as the analyzed ligand should be immobilized . if a protein is used as ligand , a compound with similar molecular weight and charge characteristics ( e.g. , a point mutant or denaturated sample that exhibits no affinity for the oligonucleotide ) should be used as reference . in both cases , the density of the ligand on the reference cell should approximate the density of the analyzed ligand [ 14 , 59 ] . one referencing method that significantly improves the quality of the results is called double referencing . in doing so , afterwards , the average of the response units from injections of pure buffer is substracted from all obtained data sets [ 50 , 53 , 60 ] . interactions of proteins with oligonucleotides can be very fast . if the kinetic rate constant kon is above 110 ms , it will be limited by the diffusion of the analyte to the immobilized ligand . due to the heterogeneity of the sensor chip surface , the transport of analyte through the microfluidic system and the nonstirred layer over the surface and the diffusion within the dextran matrix must be considered [ 3 , 62 ] . this phenomenon , called mass transport limitation , can be reduced by optimizing the experimental system . for kinetic measurements , the maximal response unit difference after injection of the analyte should not exceed rmax = 100 ru . the corresponding amount of ligand immobilized on the surface can be calculated using ( 2 ) , where mligand / analyte is the corresponding molecular weight , ligand the valency of the ligand , and rligand the amount of immobilized ligand in ru : ( 2)rligand = rmaxmligandligandmanalyte . if the association and dissociation rate values for a given system are identical at different flow rates , no mass transfer limitation is to be expected . new biacore systems like the biacore 3000 reduce these effects due to an optimized geometry . if , however , mass transport still affects the kinetics after experimental optimization , a mass transport rate constant ( km ) can be incorporated into the binding model . the corresponding value can be determined using a modified kinetic model of the biacore evaluation software . typical values for 50100 kda proteins are of the order of 10 rums . typical buffers for dna protein interaction analysis using surface plasmon resonance involve hbs - ep buffer ( 10 mm hepes , 150 mm nacl , 3 mm edta , 0.005% v / v polysorbate 20 , ph 7.4 ) , mes10 buffer ( 10 mm 2-(n - morpholino ) ethanesulfonic acid , 100 mm nacl , 1 mm edta , 0.005% v / v polysorbate 20 , ph 6.25 ) , or tris10 buffer ( 10 mm tris , 100 mm nacl , 1 mm edta , 0.005% v / v polysorbate 20 , ph 7.4 ) . as already mentioned in section 3.1.4 , the negatively charged dextran matrix can lead to strong nonspecific interactions and reduce the data quality drastically . changing the composition of the salt concentration influences the protein - dna interactions . a slight increase of the salt concentration decreases the overall binding affinity and reduces nonspecific recognition of oligonucleotides to an undetectable level . however , solutions with high ionic strength can be used to remove the protein from the oligonucleotide in order to regenerate the sensor chip for an additional experiment [ 58 , 66 , 67 ] . the nonionic polyoxyethylene surfactant polysorbate 20 ( trade name : tween 20 ) is widely employed in immunoassays , afm , and spr to reduce nonspecific adsorption of proteins due to hydrophobic interactions . it should be taken into consideration that a small increase in binding affinity might be observed when the amount of surfactant is increased in the running buffer . adding 0.05% of the polyanionic carboxymethyl dextran is known to improve the signal - to - noise ratio in the case of protein - protein interactions by competing with the surface dextran [ 69 , 70 ] . regeneration of the surface by injecting a solution that disrupts the analyzed complex might be necessary in the case of very slow dissociation . for the optimization of the regeneration protocol , it has to be kept in mind that enough data points of the dissociation should be recorded to ensure an accurate fit of the dissociation part of the sensogram , afterwards . moreover , the immobilized oligonucleotide must persist undamaged to facilitate additional measurements . a few potential solutions should be scouted to determine the most applicable regeneration buffer , by applying the corresponding conditions to approximately five cycles of analyte binding and regeneration . an overlay of the responses of the analyte binding steps indicates if the immobilized ligand is stable during the regeneration procedure . typical regeneration conditions normally involve low ( 10 mm glycine - hcl ) or high ( 1100 mm naoh ) ph , high ionic strength ( up to 5 m nacl ) , or low concentrations of sds ( up to 0.5% ) . if oligonucleotides are used as ligands , an intense change of the ph results in an unfolding and denaturation of the dna . washing the surface with buffer that exhibits ph 7 and hybridization of the oligonucleotide by readdition of the complementary strand is necessary before initiating the next cycle . superior regeneration methods for dna ligands involve the injection of 1 mm hcl , a mixture of 50 mm naoh with 1 m nacl , or low amounts of sds [ 14 , 26 ] . a considerate evaluation of the data is as essential as performing an optimally planned experiment . moreover , publishing the results according to several high - quality norms is another issue every biosensor user should be capable of and perform . in 2010 , rich and myszka published a biosensor literature review that provides rules and guidelines concerning the preparation of publishable high - quality data . although the responses of the biosensor community varied , we definitely recommend every user to read the mighty binders and to reconsider ones ' own way of dealing with spr results critically [ 72 , 73 ] . we will , therefore , recapitulate the main guidelines of rich and myszka and clarify them with basic knowledge about data evaluation in the following section . as already mentioned in section 3.2.1 , the analyte concentration used should cover a range from 0.110kd . different sample concentrations should be analyzed in a randomized fashion or high concentrations are analyzed at the beginning and additionally at the end of the experiment . taking into consideration that several factors like running buffer composition , regeneration conditions , immobilization procedures , and chemistry , potential impurities or even the analyte on its own might cause a degradation of the ligand or the sensor surface during the course of time , it is obvious , that the provisions mentioned above facilitate that the experimental setup is consistent . in order to prove this consistency and the reliability of the developed biosensor method , replicates of at least one series of measurements must be undertaken and an overlay of the results should be published . by mischance , this fundamental scientific principle is neglected in biosensor publications very often [ 50 , 71 ] . even during the preparation of this review , most of the literature found did not contain replicates or included sensograms at all . one nice feature of ge healthcares ' instruments is that the biacore wizard included in the control software provides the programming of flexible applications . an automated routine of the developed cycle conveniently enables measurements over night or over the weekend and facilitates an accurate and consistent accomplishment of the planned steps for every analyte concentration . before extracting the kinetic and thermodynamic parameters , the responses measured in the reference cell are subtracted , unwanted parts of the sensogram ( e.g. , regeneration ) are removed , the baseline of all response curves is adjusted to zero , and spikes are deleted . all of these operations and the following parameter extraction by curve fitting can be performed using the biacore evaluation software ( ge healthcare ) or other programs like scrubber-2 ( myszka and collaborators ; biologic software ) [ 12 , 65 , 74 ] . to explain the kinetic principle behind a protein - dna interaction , we exclusively focus on the 1 : 1 model or langmuir isotherm . as already mentioned in section 2.2 , the kinetics can be described by the scheme : ( 3)dna+proteinkdkadnaprotein , where ka is the association rate constant and kd the dissociation rate constant . the resulting rate of the complex formation at the time t can be expressed using the following differential equation : ( 4)d(dnaprotein)dt = ka[dna][protein ] kd[dnaprotein ] , where [ dna ] , [ protein ] , and [ dnaprotein ] are the corresponding molar concentrations . there are three important ways to solve this equation : linearization , integration , and nonlinear regression ( numerical integration ) . the first and archaic way to analyze the data is linearization . the appliance of this method for surface plasmon resonance has been described among others by majka and speck , o'shannessy et al . , and morton et al . . substituting [ dna ] in ( 4 ) by [ dna]0 [ protein ] , where [ dna]0 is the concentration of the ligand at t = 0 , results in ( 5)d(dnaprotein)dt = ka([dna]0[protein])[protein ] kd[dnaprotein ] . the observed signal r approximates the formation of the protein - dna complex and the maximum signal rmax is proportional to the surface concentration of the pure oligonucleotide . therefore , in the case of an spr experiment , ( 5 ) can be written as ( 6)d(r)dt = ka(rmaxr)ckdr , where c is the analyte ( protein ) concentration . taking the natural logarithm of ( 6 ) , ( 7)lnd(r)dt = ln(karmaxc)(kac+kd)t and substituting ( 8)ks = kac+kd results in the final equation : ( 9)lnd(r)dt = ln(karmaxc)kst . ks , if the results obtained for the analyzed system follow a 1 : 1 kinetic model ( cf . the association rate constant can then be determined by plotting ks versus c. according to ( 8) , the slope of the corresponding linear function equals ka ( cf . figure 7(b ) ) . the dissociation rate constant kd has to be determined from the dissociation phase . equation ( 10 ) describes the rate of this process : ( 10)d(r)dt=kdr . the linearized form of ( 10 ) is ( 11)lnr0rt = kd(tt0 ) , where r0 is the response at t0 . in analogy to ( 9 ) , plotting of lnr0/rt versus ( t t0 ) gives a linear function with the slope kd , if the results obtained follow a 1 : 1 kinetic model ( cf . the data points are assumed to be scattered in a gaussian distribution around the regression line thereby exhibiting the same standard deviation . however , in most cases , transforming leads to an unequal distribution of the results . in conclusion , linear regression is less accurate [ 76 , 77 ] . the second method to evaluate the spr data involves direct analysis using the integrated form of the rate equation . although , in contrast to linearization , errors in the derived parameters approximate the errors in the measured results , several biological systems can not be described due to the fact that only simple bimolecular models can be evaluated using this method [ 75 , 76 ] . the third way to analyze the data usage of this method to analyze data obtained by surface plasmon resonance is described in the biaevaluation 3.0 software handbook and has been reviewed among others by morton et al . . using the marquardt - levenberg algorithm , kinetic models can be fitted to the data obtained . during this optimization process , the values of the corresponding kinetic variables ( e.g. , ka and kd ) of the fit are changed , until the lowest sum of the squared residuals s ( cf . these residuals are calculated from the vertical distances between the measured sensogram and the calculated curve of the fit following ( 12 ) : ( 12)s=ln(rfrm)2 , where rf is the fitted response value and rm the measured one at a certain point . to determine the goodness of the fit , the -value is used : ( 13)2=1(rfrm)2np . in ( 13 ) , n is the number of data points and p the number of fitted parameters . the lower the -value , the better the corresponding fit . acceptable values are 10 . plotting the residual versus the x - values ( in the case of spr x = time ) , sensograms should include an overlay of the fit to further demonstrate the congruency . in order to resolve even the last doubts , spr curves should be simulated using the model and the corresponding kinetic constants derived from the fit . only if all of these approaches match , an adequate fit is obtained . unlike linearization and integration , numerical integration offers the possibility to determine the rate constants with high accuracy by modeling a variety of complex kinetic mechanisms . it allows for the incorporation of effects like mass transport or rebinding that influence the data . in conclusion , it is the most generally applicable and robust method to extract kinetic parameters from spr results . there are two different possibilities to extract the equilibrium dissociation constant kd . in the case of the first method , kd can be calculated from the ratio of the association and dissociation constants derived from the kinetic analysis [ 60 , 65 ] : for the second approach , the response units in the equilibrium at different analyte concentrations are used . the resulting saturation curve is analyzed by nonlinear regression to extract the dissociation constant using ( 16 ) : ( 15)d(r)dt = ka(rmaxreq)ckdreq=0 ( 16)req=(rmaxreq)cc+kd / ka = ( rmaxreq)cc+kd , where req is the observed steady - state response and c the analyte concentration . calculating the equilibrium dissociation constant by nonlinear regression using the 1 : 1 model described by ( 16 ) or a bivalent binding model can be performed with software like graphpad prism ( graphpad software ) or origin ( originlab ) . although transforming the data into a linear form using the famous scatchard plot is also possible , nonlinear regression is definitely the method of choice due to the already - mentioned disadvantages of linearization . the corresponding list ranges from the investigation of single nucleotide mismatches using hybridization experiments and the research of triplexes consisting of dsdna and peptide nucleic acid ( pna ) to the kinetic analysis of small molecule - nucleic acid interactions involving binding of heterocyclic diamidines to at sequences . focusing on the applications connected with surface plasmon resonance in the field of protein - dna and rna interactions , several interesting implementations of the three spr methods ( spr , fo - spr , and spri ) described above are summarized below . one of the first publications outlining the analysis of protein - dna interactions by spr was published by jost et al . in 1991 . the authors immobilized a biotinylated oligonucleotide consisting of 40 bp on a streptavidin - coated chip and investigated binding of the two nuclear repressor proteins r1 and r2 . two years later in 1993 , bondeson et al . determined the kinetic rate constants and the equilibrium dissociation constant of the lactose repressor - operator complex formation using the linearization approach described above . since then , spr evolved to a powerful and meaningful method to study protein - dna and rna recognition . an extensive analysis of the dna - binding domain of the e.coli's dnaa protein was performed by blaesing et al . first of all , blaesing et al . optimized the spr method by using low amounts of dna ( 100 ru ) and a high flow rate ( 100 lmin ) to prevent mass transport effects . as ligand , biotinylated oligonucleotides consisting of 21 bp that contain the dnaa box sequence and flanking regions were immobilized on a streptavidin chip . as control , an oligonucleotide without a dnaa box was used . moreover , a blank flow cell was used for referencing . afterwards , purified dnaa and crude extract that contains the protein were used as analytes , respectively . then , binding of 36 different point mutants was investigated and the proteins divided into four different classes concerning their kd values : mutants with reduced wild - type like binding , mutants with low dissociation rates , mutants with high association and dissociation rates , and mutants without dna - binding activity . in summary , blaesing et al . were able to identify and to distinguish the dna - binding domain regions of dnaa that mediate sequence specificity from the ones that solely stabilize the dnaa box recognition . dna replication termination protein tus stops the process of chromosomal replication in the final stage in e.coli by forming a replication fork trap . the interaction between tus and its recognition sequence ( terb ) is the strongest known dna - protein interaction ( kd = 3.410 m ) . analyzed binding of tus to 9 different oligonucleotides , including terb , single - stranded dna molecules and nonspecific oligonucleotides that do not contain the terb sequence . moreover , binding contributions of four different point mutants , one from inside and three from outside the core binding domain , were elucidated . as expected from literature , a low ionic strength resulted in immeasurable fast association and immeasurable slow dissociation rates . the measurement of the four mutants that are characterized by binding constants differing by 4000 folds was feasible only at 250 mm kcl . successfully elucidated the kinetic and steady - state parameters and confirmed that tus binds with very high affinity to terb and nonspecifically to single - stranded oligonucleotides and dna molecules that do not contain the terb sequence . furthermore , the authors proposed on the basis of their data that structural changes in tus are involved in the binding process . in order to perform a detailed kinetic study of the proposed binding modes of dna polymerases , polymerase that lacks the 3-5-exonuclease activity binding of the enzyme towards different dna targets including single - stranded , blunt - end double - stranded , gapped and template - primer duplex dna - containing several different mismatches was analyzed by spr . the results first of all indicate that the polymerase recognizes single - stranded dna molecules with a higher affinity than blunt - end double - stranded oligonucleotides . using dna template - primer duplexes , the authors were able to show that polymerase binds in the template - primer region and in the single - stranded template overhang with a preference for the first one . the introduction of mismatches resulted in a decreasing affinity for the duplex region and an increase in the amount of protein bound to the overhanging single strand . the authors were able to show that polymerase recognizes several kinds of oligonucleotides but exhibits a considerable preference for template - primer duplexes . shumaker - parry et al . coated a gold surface with a sam consisting of oligo(ethylene glycol)-terminated thiol ( oeg ) and biotin - terminated thiol ( bat ) . using a commercial robotic microspotting system , the authors fabricated a 1012 array by spotting 120 oligonucleotides of 100 and 77 bp in length . in this proof - of - principle experiment , shumaker - parry et al . only used two different dna molecules : the binding site of the yeast transcription factor gal4 and an oligonucleotide that lacks the gal4 sequence . the authors used the latter as control spot for on - line referencing and analyzed the 120 spots simultaneously . in summary , shumaker - parry et al . reported a proof - of - principle for the usage of surface plasmon resonance imaging as high - throughput technique in the investigation of protein - dna interactions fang et al . analyzed the kinetics of the hydrolysis of rna - dna heteroduplexes by ribonuclease h ( rnase h ) using surface plasmon resonance imaging and surface plasmon resonance fluorescence spectroscopy ( spfs ) . in spfs , the enhanced field of the surface plasmon mode is used for the excitation of fluorophores attached to the immobilized ligand ( here : the immobilized single stranded rna ) . using a fluorescence detection unit , having already shown that spri can be used to detect the ribonuclease h reaction in 2004 , the authors were interested in a complete characterization of the enzymatic reaction . fabrication of the sensor array was performed using the muam / ssmcc method described above . to extract kinetic data from the sensograms , the authors created a model that includes the three rate constants enzyme adsorption ( ka ) , enzyme desorption ( kd ) , enzyme catalysis ( kcat ) , and a dimensionless diffusion parameter ( ) . the corresponding reaction scheme can be written as ( 17)e(x=)km e(x=0)+skdkaeskcat s+e(x=0 ) , where e(x= ) and e(x=0 ) are the bulk and surface enzyme species , respectively , km is the corresponding mass transport coefficient , s the rna - dna heteroduplex , es the enzyme - substrate complex , and s * the reaction product ( single stranded dna ) . a different illustration of the reaction scheme is presented in figure 8 . using this reaction model , fang et al . derived the following differential kinetic equation based on the relative surface coverages ( x ) : ( 18)s+es+s=1,ddt = ka(1ess)[e]b(kd+kcat)es1+(1ess),dsdt = kcates . by fitting the spri and spfs datasets using ( 18 ) the values of the constants ( ka , the authors successfully examined and described the surface enzyme reaction of rnase h using surface plasmon resonance techniques . the authors compared a coupling approach of an lc - ms instrument to an spr ( biacore 2000 ; ge healthcare ) and an spri system ( spri - plex ; genoptics ) . as evaluation system , binding of the bacterial nucleoid protein h - ns to high- and low - affinity sequences and the interaction between the integration host factor ( ihf ) and an oligonucleotide containing a single ihf binding site were analyzed . a direct coupling of the biacore 2000 ifc to the reverse phase hplc column of the lc - ms should facilitate the recovery and direct analysis of the analyte mixture . in the case of the spri , binding of the analyte mixture was first of all investigated using a standard protocol . the array was removed from the spri instrument , dried , and each spot independently treated with 1 l of the regeneration solution . then , the regeneration solution of every spot was recovered and spotted onto a proteinchip . after cocrystallization with a matrix , the proteinchip was analyzed using surface - enhanced laser desorption / ionization mass spectrometry ( seldi ) . were able to show that a satisfactory recovery and identification was not possible in the case of the biacore 2000 . however , using the spri - based method , the authors successfully recovered and analyzed both proteins ( h - ns and ihf ) using mass spectrometry after having quantified the interactions . rna - rna interactions between stem - loop structures are essential regulatory elements , for example , in prokaryotic organisms . in e.coli , two plasmid - encoded transcripts , rnai and rnaii , regulate the replication of the plasmid cole1 . interaction between the antisense rna , rnai , and the rna primer , rnaii , prevents the formation of the rna - dna hybrid , necessary for the replication initiation . a protein ( rop ) , also encoded by the plasmid , stabilizes the loop - loop interactions . to study this system , di primo immobilized biotinylated rnai on a streptavidin chip and saturated the chip with rnaii . afterwards , increasing concentrations of rop were injected . instead of performing several cycles that include the injection of one concentration , followed by a regeneration step , di primo used kinetic titration experiments by injecting three concentrations of rop sequentially . evaluation of the reaction between rop and the bimolecular rna complex was accomplished by keeping the rnaii concentration in the injected flow at a high constant level . although the first fiber - optic design was introduced by jorgenson and yee in 1993 , only a small number of biosensing applications ( especially concerning dna - protein interactions ) have been reported . moreover , the binding kinetics of the aptamer - higg interaction was determined by fo - spr and the values confirmed by affinity studies on capillary electrophoresis and a prism - based spr ( biacore 3000 ) . aberrant hypermethylation of cpg islands in promotor regions is a genome alteration frequently connected to human cancers . however , detection methods involving methylation - sensitive restriction digestion or methylation - specific pcr are laborious and time consuming . therefore , pan et al . developed a double - recognition method based on spr . the adenomatous polyposis coli ( acp ) gene promotor 1a that exhibits 31 cpg islands and has been confirmed in several cancers was used as detection model . in the first step of the method , single - stranded genomic dna was added to single - stranded biotinylated oligonucleotides that were immobilized on a streptavidin coated sensor chip and possess a certain sequence for a specific promotor region . in the second step , methyl - cpg binding domain ( mbd ) protein , that specifically binds symmetrically methylated oligonucleotides , was injected . to verify the specificity of the recognition process , poly(cga ) and methylated poly(mcga ) the authors successfully verified the methylation of the corresponding promoters by spr . transferring this method developed an spr - based method to determine the properties of antisense oligonucleotides using the endonuclease rnase h in an spr experiment . due to the ability of antisense molecules to hybridize sequence - specifically with single - stranded rna like mrna , injection of antisense strand into cells can result in the knockout of certain transcripts . in the first step of the dual assay , biotinylated chimeric oligonucleotides that consist of an rna sequence and a short dna strand ligated to its 3-end were immobilized on a streptavidin sensor chip ( cf . an antisense oligonucleotide , complementary to the ribonucleotide sequence of the immobilized molecule , was injected and a heteroduplex was formed ( cf . the enzyme recognizes the heteroduplex consisting of the rna sequence and the antisense strand and cleaves the rna part . the produced fragments were released into the solution and hybridize with complementary oligonucleotides immobilized in the following flow cell ( cf . the dna fragment of the chimeric dna molecule was necessary , to enhance the spr response in the first flow cell and to facilitate the specific hybridization with the immobilized ligands in the second flow cell . this method has the potential to screen the properties of antisense oligonucleotides containing chemical modifications . pollet et al . performed real - time monitoring of the amplification of an 80 base pair oligonucleotide by combining solid - phase pcr and fo - spr . in this first proof - of - concept report , the authors immobilized 5 thiol modified forward primer on the optical fiber and used standard pcr conditions ( taq polymerase , dntps , etc . ) . due to the negative impact on the overall performance caused by adsorption of the polymerase on the gold surface , mercaptoalkane compounds were immobilized , to prevent the nonspecific interactions of the enzyme . moreover , the sensitivity was increased by linking the reverse primer to gold nanoparticles ( cf . were able to determine the efficiency of the solid - phase amplification . compared to other reports of solid - phase pcr , , the authors described an innovative new readout mechanism for real - time pcr using spr . recently , we analyzed the interaction between the dna - binding domain of the bacterial transcription factor phob ( phob ) and its cognate dna sequence ( pho box ) by spr . we immobilized biotinylated 18 and 24 bp dsdna molecules that contain the entire or parts of the pho box of the regulon pst on a streptavidin surface . in addition to the wildtype phob protein , two point mutants were used as analyte , where amino acids involved in the dna recognition process were substituted by alanine . in spite of a systematic optimization ( e.g. , oligonucleotide length , surface concentration ) , an evaluation of the kinetic data using numerical integration did not result in a reliable fit . therefore , the equilibrium dissociation constants were elucidated using nonlinear regression to fit the response units in equilibrium at different analyte concentrations to a one - site binding model . in consideration of circular dichroism results of the dna - protein complexes , the spr data revealed new insights into the binding mechanism of phob . comparing the pho box sequences of different regulons that only differ in the minor groove additionally proved the dependency of the dna - protein interaction on the groove composition . beside the width of the corresponding minor groove , the bending properties of the dna molecule and certain interactions mediated by amino acid residues have to be considered .

several proteins , like transcription factors , bind to certain dna sequences , thereby regulating biochemical pathways that determine the fate of the corresponding cell . due to these key positions , it is indispensable to analyze protein - dna interactions and to identify their mode of action . surface plasmon resonance is a label - free method that facilitates the elucidation of real - time kinetics of biomolecular interactions . in this article , we focus on this biosensor - based method and provide a detailed guide how spr can be utilized to study binding of proteins to oligonucleotides . after a description of the physical phenomenon and the instrumental realization including fiber - optic - based spr and spr imaging , we will continue with a survey of immobilization methods . subsequently , we will focus on the optimization of the experiment , expose pitfalls , and introduce how data should be analyzed and published . finally , we summarize several interesting publications of the last decades dealing with protein - dna and rna interaction analysis by spr .

1. Introduction 2. General Principle of SPR 3. Setting Up the Experiment 4. Analyze and Publish Data 5. Applications

the variety of these mechanisms involves variations of the electrostatic potential due to groove narrowing or specific hydrogen bond donors and acceptors of the dna helix that are recognized by a complementary set of amino acids . label - based methods require the ligation of the analyte and/or ligand with reporters like enzymes , fluorescent dyes , or radioisotopes . in label - free approaches like atomic force microscopy - dynamic force spectroscopy experiments [ 4 , 5 ] , acoustic biosensors based on quartz crystal resonators , calorimetric biosensors , and surface plasmon resonance ( spr ) inherently properties ( e.g. this article will focus on the most widely used label - free detection method : surface plasmon resonance . in 2007 , 89% of all publications dealing with a beam of polarized light that propagates in a medium of high refractive index ( e.g. this phenomenon , called surface plasmon resonance , results in the observed dip of the light intensity . the wavevector of the plasmons depends on the refractive index of the conductor and the neighboring medium of low refractive index ( n2 ) . the wavevector of the photon depends on the wavelength of the polarized light and the angle of incidence ( ) . in conclusion , the refractive index n2 can be determined by measuring the intensity of the reflected light at different angles of incidence ( ) , if the wavelength of the polarized light and the refractive index of the conductor are both known [ 9 , 10 ] . in most practical applications of spr , the kretschmann - reather atr method that was already described in the last section is used . in order to evaluate the interactions of a protein with a nucleic acid that results in the formation of a protein - dna complex , one of the two interaction partners has to be immobilized on the surface of the conductor ( ligand in figure 1 ) . in biacore instruments , the resulting change in concentration of molecules at the gold surface due to the formation of the protein - dna complex is measured in resonance units ( rus ) and can be described according to ( 1 ) : ( 1)ru = nx=[riic]x=[(nc)ligandc]x . in this equation , n is the changing refractive index at the surface , x is a multiplier to convert n to ru , rii is the refractive index increment of the protein that is binding to the immobilized oligonucleotide , and c is the concentration of the protein . the typical shape of a sensogram that displays the change of the response units during the course of the experiment is shown in figure 2 . due to the formation of a protein - dna complex , the refractive index changes , resulting in a variation of the specific angle ( ) where the dip in intensity of the reflected light is at its minimum . after having finished the described cycle , another concentration of the analyte can be injected . in order to assemble a fiber - optic - based spr ( fo - spr ) , the silicon cladding has to be removed in a small area of the optical fiber . unlike prism - based spr instruments , sensing is accomplished by changing the wavelength instead of the angle of incidence . due to the fact that the width of the spr curve that affects the detection accuracy of the spr instrument depends on the number of reflections , fiber parameters like length , sensing region , and fiber core diameter are crucial for the performance . to further change the sensitivity , detection accuracy and operating range of fiber optic - based sprs , several modifications including a bimetallic coating , a tapered or u - shaped probe or the addition of dopants like geo2 have been used . although fiber - optic - based spr instruments have some advantages like the simple and miniaturized setup that lacks moving parts or the possibility to assemble an inexpensive disposable sensor for medical or sterile tasks , the sensitivity is limited in comparison to the kretschmann - reather setup [ 10 , 11 ] . a combination of protein arrays and techniques like spr would result in label - free alternatives to existing high - throughput methods that access kinetic data . in an spri experiment , it is either possible to substract an image that was taken before the injection start or to do on - line referencing by keeping one spot of the array blank . finally , a difference image of the array and a chart that displays the change in reflectivity for each probe during the course of time is obtained and can be evaluated further . due to the proportional dependency of the signal strength on the incoming light intensity , laser beams are the preferred source . however , in nearly all of the corresponding screening methods , also in the case of surface plasmon resonance , one of the interaction partners has to be immobilized . it is the most convenient and cost - effective way to immobilize short oligonucleotides as ligands on the surface to study protein - dna interactions . if the unspecific interactions between the sensor chip and the analyte are too big or if the interaction between a single protein and several oligonucleotides has to be analyzed , it might be necessary to reverse the alignment of the experimental setup by immobilizing the protein on the sensor chip . in general , it is essential for the quality , validity , and reproducibility of the results to test and select the optimum immobilization method and assembly . this hydrogel facilitates the application of several immobilization strategies through linker molecules that can be attached covalently . , biacore 's cm5 ) , the carboxyl groups can be activated by n - ethyl - n-(dimethylaminopropyl)-carbodiimide ( edc ) , and n - hydroxyl succinimide ( nhs ) . due to the fact that several proteins contain more than one lysine residue , coupling of amines will lead to a heterogeneous population of ligands with random orientation and eventually random accessibility of the interaction site . due to the lower abundance of cysteine residues in proteins , thiol - reactive surfaces combine the advantages of sensor chips equipped with sams with a more specific immobilization mechanism in comparison to the amine - based coupling reactions . if commercially obtained thiol - functionalized dna is used without further purification in any of the immobilization procedures described above , it has to be taken into consideration that the diversity of techniques for synthesis and purification can lead to a variety of impurities still present in the sample . moreover , they are easily regenerated at low ph , can be immobilized using most of the procedures presented here , and are commercially available for most antigens ( cf . short biotinylated oligonucleotides , the most frequently used ligands for the analysis of protein - dna interactions , can be readily purchased at every oligonucleotide supplier . preparing a fusion protein consisting of the desired ligand and sbp facilitates moderate binding of the ligand and a complete removal from the surface using 1-min injections of 50 mm naoh . , hexa - his , deca - his , double - his tags ) depends on the application of the spr experiment . site - directed immobilization of a fusion protein consisting of a ligand of interest and the dna binding domain of yeast gal4 or the bacterial lexa is an affinity immobilization method based on protein - dna interactions . in order to specifically recognize the desired transcriptional activator and to amplify the corresponding signals , a primary antibody against the protein and a secondary antibody concentration of the analyte is another very important aspect for reliable and high - quality results . due to the fact that the surface plasmon wave decays evanescently approximately 200 nm into the solution , even oligonucleotides of considerable length can be used as ligands . the interactions of the transcription factor leafy from arabidopsis thaliana to oligonucleotides that exhibit the sequences of the entire gene promoters apetala3 ( 2386 bp ) and apetala3 ( 3050 bp ) were successfully analyzed and the data quantitatively evaluated . one referencing method that significantly improves the quality of the results is called double referencing . due to the heterogeneity of the sensor chip surface , the transport of analyte through the microfluidic system and the nonstirred layer over the surface and the diffusion within the dextran matrix must be considered [ 3 , 62 ] . the corresponding amount of ligand immobilized on the surface can be calculated using ( 2 ) , where mligand / analyte is the corresponding molecular weight , ligand the valency of the ligand , and rligand the amount of immobilized ligand in ru : ( 2)rligand = rmaxmligandligandmanalyte . the corresponding value can be determined using a modified kinetic model of the biacore evaluation software . typical buffers for dna protein interaction analysis using surface plasmon resonance involve hbs - ep buffer ( 10 mm hepes , 150 mm nacl , 3 mm edta , 0.005% v / v polysorbate 20 , ph 7.4 ) , mes10 buffer ( 10 mm 2-(n - morpholino ) ethanesulfonic acid , 100 mm nacl , 1 mm edta , 0.005% v / v polysorbate 20 , ph 6.25 ) , or tris10 buffer ( 10 mm tris , 100 mm nacl , 1 mm edta , 0.005% v / v polysorbate 20 , ph 7.4 ) . changing the composition of the salt concentration influences the protein - dna interactions . the nonionic polyoxyethylene surfactant polysorbate 20 ( trade name : tween 20 ) is widely employed in immunoassays , afm , and spr to reduce nonspecific adsorption of proteins due to hydrophobic interactions . for the optimization of the regeneration protocol , it has to be kept in mind that enough data points of the dissociation should be recorded to ensure an accurate fit of the dissociation part of the sensogram , afterwards . a few potential solutions should be scouted to determine the most applicable regeneration buffer , by applying the corresponding conditions to approximately five cycles of analyte binding and regeneration . although the responses of the biosensor community varied , we definitely recommend every user to read the mighty binders and to reconsider ones ' own way of dealing with spr results critically [ 72 , 73 ] . different sample concentrations should be analyzed in a randomized fashion or high concentrations are analyzed at the beginning and additionally at the end of the experiment . taking into consideration that several factors like running buffer composition , regeneration conditions , immobilization procedures , and chemistry , potential impurities or even the analyte on its own might cause a degradation of the ligand or the sensor surface during the course of time , it is obvious , that the provisions mentioned above facilitate that the experimental setup is consistent . in order to prove this consistency and the reliability of the developed biosensor method , replicates of at least one series of measurements must be undertaken and an overlay of the results should be published . all of these operations and the following parameter extraction by curve fitting can be performed using the biacore evaluation software ( ge healthcare ) or other programs like scrubber-2 ( myszka and collaborators ; biologic software ) [ 12 , 65 , 74 ] . to explain the kinetic principle behind a protein - dna interaction , we exclusively focus on the 1 : 1 model or langmuir isotherm . the resulting rate of the complex formation at the time t can be expressed using the following differential equation : ( 4)d(dnaprotein)dt = ka[dna][protein ] kd[dnaprotein ] , where [ dna ] , [ protein ] , and [ dnaprotein ] are the corresponding molar concentrations . the appliance of this method for surface plasmon resonance has been described among others by majka and speck , o'shannessy et al . the observed signal r approximates the formation of the protein - dna complex and the maximum signal rmax is proportional to the surface concentration of the pure oligonucleotide . therefore , in the case of an spr experiment , ( 5 ) can be written as ( 6)d(r)dt = ka(rmaxr)ckdr , where c is the analyte ( protein ) concentration . although , in contrast to linearization , errors in the derived parameters approximate the errors in the measured results , several biological systems can not be described due to the fact that only simple bimolecular models can be evaluated using this method [ 75 , 76 ] . the third way to analyze the data usage of this method to analyze data obtained by surface plasmon resonance is described in the biaevaluation 3.0 software handbook and has been reviewed among others by morton et al . these residuals are calculated from the vertical distances between the measured sensogram and the calculated curve of the fit following ( 12 ) : ( 12)s=ln(rfrm)2 , where rf is the fitted response value and rm the measured one at a certain point . to determine the goodness of the fit , the -value is used : ( 13)2=1(rfrm)2np . in order to resolve even the last doubts , spr curves should be simulated using the model and the corresponding kinetic constants derived from the fit . in conclusion , it is the most generally applicable and robust method to extract kinetic parameters from spr results . the corresponding list ranges from the investigation of single nucleotide mismatches using hybridization experiments and the research of triplexes consisting of dsdna and peptide nucleic acid ( pna ) to the kinetic analysis of small molecule - nucleic acid interactions involving binding of heterocyclic diamidines to at sequences . focusing on the applications connected with surface plasmon resonance in the field of protein - dna and rna interactions , several interesting implementations of the three spr methods ( spr , fo - spr , and spri ) described above are summarized below . one of the first publications outlining the analysis of protein - dna interactions by spr was published by jost et al . the authors immobilized a biotinylated oligonucleotide consisting of 40 bp on a streptavidin - coated chip and investigated binding of the two nuclear repressor proteins r1 and r2 . determined the kinetic rate constants and the equilibrium dissociation constant of the lactose repressor - operator complex formation using the linearization approach described above . since then , spr evolved to a powerful and meaningful method to study protein - dna and rna recognition . then , binding of 36 different point mutants was investigated and the proteins divided into four different classes concerning their kd values : mutants with reduced wild - type like binding , mutants with low dissociation rates , mutants with high association and dissociation rates , and mutants without dna - binding activity . were able to identify and to distinguish the dna - binding domain regions of dnaa that mediate sequence specificity from the ones that solely stabilize the dnaa box recognition . in order to perform a detailed kinetic study of the proposed binding modes of dna polymerases , polymerase that lacks the 3-5-exonuclease activity binding of the enzyme towards different dna targets including single - stranded , blunt - end double - stranded , gapped and template - primer duplex dna - containing several different mismatches was analyzed by spr . in this proof - of - principle experiment , shumaker - parry et al . reported a proof - of - principle for the usage of surface plasmon resonance imaging as high - throughput technique in the investigation of protein - dna interactions fang et al . analyzed the kinetics of the hydrolysis of rna - dna heteroduplexes by ribonuclease h ( rnase h ) using surface plasmon resonance imaging and surface plasmon resonance fluorescence spectroscopy ( spfs ) . in spfs , the enhanced field of the surface plasmon mode is used for the excitation of fluorophores attached to the immobilized ligand ( here : the immobilized single stranded rna ) . using a fluorescence detection unit , having already shown that spri can be used to detect the ribonuclease h reaction in 2004 , the authors were interested in a complete characterization of the enzymatic reaction . the corresponding reaction scheme can be written as ( 17)e(x=)km e(x=0)+skdkaeskcat s+e(x=0 ) , where e(x= ) and e(x=0 ) are the bulk and surface enzyme species , respectively , km is the corresponding mass transport coefficient , s the rna - dna heteroduplex , es the enzyme - substrate complex , and s * the reaction product ( single stranded dna ) . by fitting the spri and spfs datasets using ( 18 ) the values of the constants ( ka , the authors successfully examined and described the surface enzyme reaction of rnase h using surface plasmon resonance techniques . as evaluation system , binding of the bacterial nucleoid protein h - ns to high- and low - affinity sequences and the interaction between the integration host factor ( ihf ) and an oligonucleotide containing a single ihf binding site were analyzed . in the case of the spri , binding of the analyte mixture was first of all investigated using a standard protocol . interaction between the antisense rna , rnai , and the rna primer , rnaii , prevents the formation of the rna - dna hybrid , necessary for the replication initiation . evaluation of the reaction between rop and the bimolecular rna complex was accomplished by keeping the rnaii concentration in the injected flow at a high constant level . moreover , the binding kinetics of the aptamer - higg interaction was determined by fo - spr and the values confirmed by affinity studies on capillary electrophoresis and a prism - based spr ( biacore 3000 ) . to verify the specificity of the recognition process , poly(cga ) and methylated poly(mcga ) the authors successfully verified the methylation of the corresponding promoters by spr . transferring this method developed an spr - based method to determine the properties of antisense oligonucleotides using the endonuclease rnase h in an spr experiment . performed real - time monitoring of the amplification of an 80 base pair oligonucleotide by combining solid - phase pcr and fo - spr . in this first proof - of - concept report , the authors immobilized 5 thiol modified forward primer on the optical fiber and used standard pcr conditions ( taq polymerase , dntps , etc . ) due to the negative impact on the overall performance caused by adsorption of the polymerase on the gold surface , mercaptoalkane compounds were immobilized , to prevent the nonspecific interactions of the enzyme . were able to determine the efficiency of the solid - phase amplification . compared to other reports of solid - phase pcr , , the authors described an innovative new readout mechanism for real - time pcr using spr . recently , we analyzed the interaction between the dna - binding domain of the bacterial transcription factor phob ( phob ) and its cognate dna sequence ( pho box ) by spr . comparing the pho box sequences of different regulons that only differ in the minor groove additionally proved the dependency of the dna - protein interaction on the groove composition .

childhood obesity has spread dramatically over the previous decades . to curtail this major health issue , , several studies have shown positive and encouraging outcomes of multidisciplinary approach for childhood obesity . a combination of dietary , physical activity and behavioural interventions compared to standard care or self - help can produce a significant and clinically meaningful reduction in obesity in children and adolescents [ 1 , 2 ] . however , in most studies , programs are limited to between 6 and 12 months of duration , and beneficial effects are partly lost from 6 to 12 months after completion , especially for severely obese children . long - term follow - up studies of paediatric obesity interventions show a mean 10% reduction in relative weight but also substantial relapse [ 5 , 6 ] . as obesity is a chronic disease , the question of the need of a chronic care program is raised . whether the continuation of the program will still be beneficial and how to implement this in a real - life situation remains to be answered identification of factors associated with better outcomes can help maximize the effectiveness of existing interventions , tailor treatment programs to the specific needs of the patients , and set realistic weight loss goals . treatment for children presents a unique challenge as nutrition education , physical activity , and behaviour modification must be presented to both the parents / caregiver and child . parental involvement and individual counselling have been recognised as an important feature of behavioural programs , particularly in preadolescent children [ 3 , 4 , 9 ] . it is thus relevant to examine the impact of family characteristics and psychosocial factors on children 's weight loss . in this paper , we first analyzed the 5-year results of an interdisciplinary long - term care program for childhood obesity . secondly , we determined the baseline factors ( medical , dietary , and psychosocial ) which were associated with weight loss . in 2000 , we set up an interdisciplinary approach for the treatment of childhood obesity . our approach is an individually adapted ( specific for each patient ) family - based , behavioural lifestyle and dietary intervention program . it consists in joint consultations where each child and his family are seen by both a psychologist and a paediatric endocrinologist at the same time . after a time together , the patient is examined ( weight , height , blood pressure , and tanner stage ) by the physician in a separate room which gives the opportunity to the child of having personal time with the paediatrician . during this time , the parents / family / caregivers are seen by the psychologist trained in family therapy . then , the child and the physician get back to the psychologist and the family for a resume of the situation in order to make some decisions . thereafter , the child and his family are taken by the psychologist to the dietician . the psychologist gives a summary of the situation , and then all the family is seen by the dietician . before each session , the interdisciplinary team reviews the situation of each patient . at the end of the visit a letter , including all the decisions taken with the child and his family , and some personal encouragement for the child is written and sent to the patient one month later . for further analysis , the implementation of those decisions was defined as adherence to the treatment . they participate in our program as long as they want or need . between two interdisciplinary visits , the patient and some patients are referred to a specific psychotherapist , and individual physiotherapy can also be prescribed in some situations to reintroduce physical activity especially if joining a sport club is difficult at the beginning of the treatment . the psychotherapy and the physiotherapy are defined in our approach as adjuvant therapies . our way to treat obese children the team develops realistic goals ( small step changes ) together with the child and his family rather than imposing ideas and assumptions about what they need to do to change their lifestyle . we start the treatment from their questions or their needs ( what can we do for you ? ) in order to stimulate them to be an active player in their own changes . we focus on the development of the confidence and competence of the parents or caregivers and of the children . our approach is also in agreement with the evidences published in the bmj by edmunds et al . in 2001 . we encourage the child to grow without gaining weight which decreases bmi slowly . the dietician does not prescribe any specific diet but focuses on healthy eating patterns ( decrease exposure to obesogenic foods , designate times for family meals , and allocate individual portions ) and on increasing the intake of healthy foods . adolescents who were educated about better food choices of moderate portion sizes had been described to be more successful in the long term than teenagers who were given a structured meal plan or restrictive diets . but , we mostly encourage regular daily activities such as riding a bicycle , walking the dog , dancing , gardening , using the stairs instead of elevators , and playing outside with friends who are more easily integrated into a child 's lifestyle than participation in organized sport teams . data suggested that less structured , more flexible lifestyle exercise may be superior to more structured and higher - intensity aerobic exercise for weight control . recommended activities must be enjoyable and consistent with the child 's and his / her family 's lifestyle and be rewarding irrespective of the health benefits . obesity may be reactive to an event of life ( divorce of their parents , difficulties at school ) . parents are encouraged to not focus on weight loss but address their and the child 's internal needs by expressing feelings and nurturing the child emotionally . the parents are targeted as the main agents of change , and they are responsible for inducing this change in the family home , not specifically at the target child . extended family members are included as a means of reaching all people who play a significant role in the child 's health . 428 medical files of children who entered the interdisciplinary consultations between 2002 and 2007 were retrospectively reviewed . inclusion criteria were to have participated in , at least , 2 interdisciplinary visits and to have at least one year of treatment . children with obesity due to an organic / syndromic cause or with type 2 diabetes were excluded . among the included patients : 73% were caucasian , 12% were hispanic , 10% were arabian , 2% were african , 2% were asiatic ( representative of the national population ) . the latest visit was defined as the most recent visit reported in the medical files when they were reviewed between 2007 and 2009 . thus , for some children the latest visit is the last visit before they were no longer monitored . for other children , the latest visit is not the last because they are still monitored by the team . weight was measured ( patient in socks with no shoes and wearing a light gown ) in kilograms to the nearest 0.1 kg using a medical weight scale ( seca nondigital medical scale ) , zeroed and calibrated before each weight . a stadiometer ( bmi ( kg / m ) were expressed relative to the cole population reference data . weight loss was defined by reduction of the bmi standard deviation score ( bmi - z - score ) since bmi is gender and age dependent in childhood . bmi - z - score standardizes an individual 's size , adjusting for age and sex , and allows comparison between values on an equivalent basis . the medical , dietary , and psychosocial factors characterizing the child and his family at baseline were assessed retrospectively by an external consultant ( who was not made aware of the patients weight evolution ) by reviewing the records ( standard home - made questionnaire ) filled in by the team at the first visit . as it is a retrospective and not a prospective study , a semiquantitative ( low / intermediate / high ) approach was used to evaluate each factor . for food consumption , no / low means not every day or never ; intermediate ( if applicable ) means every day but at a low ( 1 - 2 ) level ; yes / high means every day at a high ( > 2 ) level . physical activity means that the child attends a sports club or a youth organization at least twice a week ( yes / high ) , once a week ( intermediate ) or never ( no / low ) . delayed puberty was considered when a girl was assessed m1 > 13.5 years or a boy was assessed g1 > 14 years . obesity in the family means the child has at least one of the two parents who is obese ( bmi > 30 bad quality of sleep was assessed by snoring or short sleep duration ( < 9 h / night ) . family encouragement for the project and child 's motivation were assessed according to the involvement of the family and of the child in the project ( who had the idea to come here ? social integration was assessed as the participation of the child in out - of - school activities . family encouragement for leisure activities was assessed as activities realised by all the family out of the home . we use the quasilikelihood estimation with a linear or a logistic canonical link . for the measures of the same patient , we used an autoregressive correlation matrix and computed the covariance matrix by quasi - least - squares ( qlss ) . for the significance of the results , we used the sandwich variance matrix augmented by the correction proposed by morel et al . which may be evaluated by the normal distribution . for continuous independent variables , we worked with nonlinear regression by p splines ( b splines with penalization of degree 4 ) . out of 428 patients seen between 2002 and 2007 , 322 patients ( 75% ) were interested in our interdisciplinary treatment and attended a second visit . of those , 144 children ( 45% ) ( 59 boys ( 41.0% ) , 85 girls ( 59.0% ) ; mean age : 10.5 3.1 years ( range 419 years ; 105 ( 73% ) < 13 years , 39 ( 26% ) > 13 years ; mean bmi - z - score : 2.73 0.62 ) had at least a 1 year intervention program and were selected for our study . mean length of treatment was 2.2 years ( 16.7 years ) with an average of 3 1 visits per year . after 24 months , 72 children were still monitored ; 14 achieved a 48-month intervention . the length of treatment or assiduity ( number of visits ) did not depend on the initial weight loss ( bmi - z - score ) between the first , and the second visit ( p = 0.63 ) . sex , age and bmi - z - score at the first visit did also not influence the length of intervention ( p = 0.76 ; p = 0.09 and p = 0.43 resp . ) . table 1 described the percentage of patients where bmi - z - score decreased and was stable or increased at the second visit and at the latest visit . at the second visit ( approximately after 36 months ) , bmi - z - score was decreased in 53% of the patients , remained unchanged in 31% , and was increased in 16% . at the latest visit in fact , weight loss was mainly observed during the first 6 months of treatment ( figures 1(a ) and 1(b ) ) but was sustained long - term . the mean bmi - z - score 0.04 mean bmi - z - score ) of the initial mean bmi - z - score after a mean of intervention of 2.2 years and decreased by 12% 2% ( 0.28 0.06 mean bmi - z - score ) for patients with a 48-month treatment . initial bmi - z - score , age at the first visit , sex ( data of boys and girls were thus combined for further analysis ) and number of visit(s ) per year did not influence these results ( p = 0.73 ; p = 0.27 ; p = 0.95 and p = 0.89 resp . ) . furthermore , an additional weight loss was observed between 6 and 48 months of intervention whatever the bmi - z - score between the first and the second visit ( figure 2 ) . patients with a bmi - z - score reduction 0.3 units were 23% ( 2027 ; 95% ci ) of the population at 3 months versus 49% ( 4058 ; 95% ci ) at 48 months of treatment ( figure 3(a ) ) . however , 16% ( 1319 ; 95% ci ) of our patients gained weight after 3 months of treatment . the percentage was increased to 31% ( 2538 ; 95% ci ) of the patients reaching 48 months of treatment ( figure 3(b ) ) . the weight change between the first and the second visit was predictive of the additional weight change over the time ( figure 4 ) . no evidence of adverse effects on growth , eating disorder pathology or mental health , was found . we next investigated whether baseline medical , dietary , and psychosocial parameters reported at the first visit could influence the weight change over the time and which one could be associated with weight loss . patients who exercised in daily life before joining the interdisciplinary treatment were the most successful in term of weight loss ( table 2 ) . preexisting regular physical activity had a statistically significant ( p = 0.037 ) positive influence ( 0.42 0.11 of mean bmi - z - score ) on the weight evolution of the child , in comparison with those who did not exercise before starting the treatment ( 0.18 0.04 of mean bmi - z - score ) . having delayed puberty had a negative influence on the evolution of the mean bmi - z - score of the patients ( 0.02 0.10 of mean bmi - z - score ) in comparison with those who did not ( 0.23 0.03 of mean bmi - z - score , p = 0.046 ) . moreover , baseline daily water intake and daily soda intake had a statistical significant impact on the children 's weight outcome ( p = 0.046 and p = 0.00006 resp . ) we then determined whether the psychosocial context of the child at the first visit may influence the weight change observed later on ( table 4 ) . we showed that being a single child , having family encouragement for the project , the child 's motivation , the adherence to the treatment , and the compliance to adjuvant therapies had a statistically positive effect on the mean bmi - z - score at 9 months of intervention . the duration of the obesity and dual parent households did not impact the weight changes observed . this retrospective real - life study reported the outcomes of a long - term approach for treating childhood obesity and identified baseline predictors of weight changes . this intervention used interdisciplinary strategies ( with effective interaction between the team , not only juxtaposed competences ) but had the specificity to be individually adapted with a continuous care program . to our knowledge , this is the first time that the sustained benefit of a chronic intervention is reported and that the feasibility of a long - term intervention in real life is described in obese children . for example , family - based lifestyle behavioural treatment for obese children with similar clinical characteristics resulted in an average % decrease in overweight of ~7% after 6 months of treatment [ 3 , 4 ] which is comparable to the 8% decrease observed at 24 months in the current study . we described only a 4% bmi - z - score decrease at the second visit ( 3 - 6 months ) but a 12% bmi - z - score decrease at 48 months . in contrast , in the long term , the results of the abovementioned studies were not as promising as they were immediately after completion of the program ( ~ 3.5% decrease in weight at 12 months , 1% at 18 months ) . the beneficial effects of short intervention programs ( from 3 to 6 months ) were partly lost on the follow - up . even with a 12-month drug ( orlistat ) intervention combined to lifestyle , the initial weight loss was not maintained for more than 6 months . with this emphasis on acute short - term intervention , contemporary healthcare may not be well suited to meet the long - term needs of overweight children and their families fighting against this chronic disease . this indicates the need to develop chronic care models to optimize results , especially for severely obese children [ 4 , 7 ] . the addition of a 4-month maintenance treatment after short - term weight loss treatment resulted in better maintenance of weight loss compared with the no maintenance group ( 0.04 versus + 0.05 bmi - z - score ) but no additional weight loss was obtained over followup . in contrast , our program was still beneficial after 48 months of treatment . moreover , the percentage of patients with a 0.3 bmi - z - score reduction increased over time . at least 50% of children reaching a 36-month intervention presented a 0.3 reduction of bmi - z - score . the longer the treatment , the greater the proportion of patients who do not attend . this problem may be magnified with families , who may have more challenges in scheduling than individual adults , and where there are multiple people who may want to drop out of the treatment . in our study , 72 participants were still monitored at 24 months . mean drop - out rates in the literature are varying from 10% to 60% at 12 months of followup . for example , in an italian multicentric study of nutritional intervention in obese paediatric patients , drop - out rates ranged from 3034% to 9094% after 2 years . according to the literature , the main reasons for dropout are loss of interest , relocation , schedule conflict , transport difficulties , family issues for example , limited time for recurrent group sessions , and even for daily household demands [ 3 , 25 ] . . showed that participants in a 3-month brief multidisciplinary intervention still maintained an increased leisure - time physical activity compared to the control group subjects after 1 year of followup . even the weight benefit was modest after 1 year of followup ; this could help them to better general health in the long term . the fall in bmi observed in our study may be clinically relevant as demonstrated by many studies , even though not analysed here . short - term family - based treatment which combined nutrition education , behavioural modification and exercise was shown to improve body composition , lipids profile , blood pressure , and insulin resistance [ 4 , 25 , 26 ] . many of the obesity - associated complications can be reversed with a 5% decrease in age - adjusted bmi percentile . in adults with impaired glucose tolerance , the diabetes prevention program demonstrated that an intensive lifestyle program that reduced body weight by 7% delayed or prevented the development of type 2 diabetes . moreover , savoye et al . reported that obese adolescents with impaired glucose tolerance who were able to limit increases in bmi reverted to normal glucose tolerance 2 years later . thus , the bmi changes observed over time in our study are likely to be clinically significant as those changes were sustained over the longer term . we determined parameters characterizing the families and children at baseline conditions which were associated with a better weight control . indeed , for those less or not responsive patients , new research studies should try to devise new treatments to optimize long - term weight benefits . we demonstrated that those patients could be identified quickly according to the initial weight change observed between the first and the second visits . tanaka et al . also reported that a greater weight loss between the first and the second visits was a predictive factor in the success of the treatment . goldschmidt et al . also reported that early weight change seems to be related to treatment response through to the end of the treatment and also the 2-year followup . identification of factors that promote early weight change is critical because modification of these factors before initiation of the treatment may promote a better early response . in our study , similarly to reinehr et al . , reduction of overweight was independent of initial bmi - z - score , age at the first visit , and sex . it is well know that physical activity is related to long - term weight maintenance but , as suggested in another study , our data supports its role before the initiation of the treatment . children with large birth weight , gestational diabetes , no or short - term breastfeeding , parental obesity , asthma , and short sleep duration were described as having an increased risk of obesity [ 33 , 34 ] . baseline daily water and soda intake seemed to be a good predictor of early weight change . consumption of sugar sweetened drinks by adolescents is an independent variable associated with increasing bmi , but its role on early weight loss was never examined . healthy eating habits as eating breakfast and participating in programmed exercises were described to be correlated to healthful bmi , suggesting that these factors may be potentially protective against obesity in 1216-year old adolescents . our study extends those results by showing that prevention policy may also be helpful even for children who have to lose weight . our results demonstrated that motivated children given family encouragement were more likely to succeed in our treatment . interestingly , recent reports suggested greater weight loss in obese children when parents alone are targeted for intervention , which emphasizes the role of the parents in the child 's weight reduction . larger reductions in adult bmi were associated with more successful results , which indicates that working to enhance the adult role in child weight control programs may improve results . data from rhee et al . suggested that parents having an older child , believing that they themselves were overweight , perceiving that their child had a health problem were associated with greater parental readiness to make changes . emerging research also indicates that overweight children with psychosocial problems or the occurrence of maternal psychopathology are less responsive to weight - control intervention over the longer term the divorce rate has increased as well as the number of families with both parents working . our data suggested that the dual parent households did not affect weight changes observed at 9 months . this is in contrast with a recent study which showed a relationship between single - parents status and excess weight in children . further studies are needed to explore the dynamics of single - parent households and its influence on childhood diet and obesity . interestingly , our study showed that a family with an only child may expect a greater weight loss . other factors such as higher incomes and higher level of education for the mother were also reported to be associated with better results but were not analyzed in the current study . in conclusion , this study was a first step in determining whether weight loss was achievable with our interdisciplinary approach and highlighted potential success of a continuous care weight control program to lower bmi . an early weight change seems to be a marker for children 's long - term treatment response . preexisting regular physical activity , normal timing of puberty , baseline daily water and soda intake , motivation and some family characteristics predict the early response to the treatment . better prevention policy and parental support may thus improve the success of the childhood obesity treatment . our data may provide a better understanding of the factors involved in better weight control and may help to optimize / adapt our strategies for participants who do not benefit from treatment .

background . efforts are needed to improve the long - term efficiency of childhood obesity treatment . to adapt strategies , the identification of subgroups of patients with a greater weight loss may be useful . objective . to analyze the results of a chronic care program for childhood obesity and to determine baseline factors ( medical , dietary , and psychosocial ) associated with successful weight loss . subjects and method . we set up a family - targeted and individually adapted interdisciplinary long - term care program . we reviewed the medical files of 144 children ( 59 boys and 85 girls ; 10.5 3.1 y ; mean bmi - z - score : 2.73 0.62 ) who had 2 interdisciplinary visits and 1-year treatment . results . mean treatment length was 2.2 y ( 16.7 y ) with 3 1 visits / year . the duration of treatment did not depend on the initial weight loss , but this was predictive of the weight change over time . furthermore any additional weight loss was observed with time whatever the initial weight change . high levels of physical activity and daily water intake from baseline conditions were associated with a greater weight loss after 9 months of intervention . in contrast , a high baseline consumption of soft drinks resulted in lower weight loss . family specific factors such as being a single child or the child 's family support were identified as baseline factors which may contribute to better results . conclusion . our study suggests that the benefit of a chronic weight control program supports the need for its integration into the current concept of treatment . better prevention policy and parental support may improve the success of the childhood obesity treatment .

1. Introduction 2. Subjects and Methods 3. Results 4. Discussion

childhood obesity has spread dramatically over the previous decades . to curtail this major health issue , , several studies have shown positive and encouraging outcomes of multidisciplinary approach for childhood obesity . a combination of dietary , physical activity and behavioural interventions compared to standard care or self - help can produce a significant and clinically meaningful reduction in obesity in children and adolescents [ 1 , 2 ] . however , in most studies , programs are limited to between 6 and 12 months of duration , and beneficial effects are partly lost from 6 to 12 months after completion , especially for severely obese children . long - term follow - up studies of paediatric obesity interventions show a mean 10% reduction in relative weight but also substantial relapse [ 5 , 6 ] . as obesity is a chronic disease , the question of the need of a chronic care program is raised . whether the continuation of the program will still be beneficial and how to implement this in a real - life situation remains to be answered identification of factors associated with better outcomes can help maximize the effectiveness of existing interventions , tailor treatment programs to the specific needs of the patients , and set realistic weight loss goals . treatment for children presents a unique challenge as nutrition education , physical activity , and behaviour modification must be presented to both the parents / caregiver and child . it is thus relevant to examine the impact of family characteristics and psychosocial factors on children 's weight loss . in this paper , we first analyzed the 5-year results of an interdisciplinary long - term care program for childhood obesity . secondly , we determined the baseline factors ( medical , dietary , and psychosocial ) which were associated with weight loss . in 2000 , we set up an interdisciplinary approach for the treatment of childhood obesity . our approach is an individually adapted ( specific for each patient ) family - based , behavioural lifestyle and dietary intervention program . after a time together , the patient is examined ( weight , height , blood pressure , and tanner stage ) by the physician in a separate room which gives the opportunity to the child of having personal time with the paediatrician . during this time , the parents / family / caregivers are seen by the psychologist trained in family therapy . then , the child and the physician get back to the psychologist and the family for a resume of the situation in order to make some decisions . thereafter , the child and his family are taken by the psychologist to the dietician . the psychologist gives a summary of the situation , and then all the family is seen by the dietician . before each session , the interdisciplinary team reviews the situation of each patient . at the end of the visit a letter , including all the decisions taken with the child and his family , and some personal encouragement for the child is written and sent to the patient one month later . for further analysis , the implementation of those decisions was defined as adherence to the treatment . between two interdisciplinary visits , the patient and some patients are referred to a specific psychotherapist , and individual physiotherapy can also be prescribed in some situations to reintroduce physical activity especially if joining a sport club is difficult at the beginning of the treatment . our way to treat obese children the team develops realistic goals ( small step changes ) together with the child and his family rather than imposing ideas and assumptions about what they need to do to change their lifestyle . we focus on the development of the confidence and competence of the parents or caregivers and of the children . we encourage the child to grow without gaining weight which decreases bmi slowly . the dietician does not prescribe any specific diet but focuses on healthy eating patterns ( decrease exposure to obesogenic foods , designate times for family meals , and allocate individual portions ) and on increasing the intake of healthy foods . adolescents who were educated about better food choices of moderate portion sizes had been described to be more successful in the long term than teenagers who were given a structured meal plan or restrictive diets . but , we mostly encourage regular daily activities such as riding a bicycle , walking the dog , dancing , gardening , using the stairs instead of elevators , and playing outside with friends who are more easily integrated into a child 's lifestyle than participation in organized sport teams . data suggested that less structured , more flexible lifestyle exercise may be superior to more structured and higher - intensity aerobic exercise for weight control . recommended activities must be enjoyable and consistent with the child 's and his / her family 's lifestyle and be rewarding irrespective of the health benefits . obesity may be reactive to an event of life ( divorce of their parents , difficulties at school ) . parents are encouraged to not focus on weight loss but address their and the child 's internal needs by expressing feelings and nurturing the child emotionally . the parents are targeted as the main agents of change , and they are responsible for inducing this change in the family home , not specifically at the target child . extended family members are included as a means of reaching all people who play a significant role in the child 's health . 428 medical files of children who entered the interdisciplinary consultations between 2002 and 2007 were retrospectively reviewed . inclusion criteria were to have participated in , at least , 2 interdisciplinary visits and to have at least one year of treatment . among the included patients : 73% were caucasian , 12% were hispanic , 10% were arabian , 2% were african , 2% were asiatic ( representative of the national population ) . the latest visit was defined as the most recent visit reported in the medical files when they were reviewed between 2007 and 2009 . for other children , the latest visit is not the last because they are still monitored by the team . weight loss was defined by reduction of the bmi standard deviation score ( bmi - z - score ) since bmi is gender and age dependent in childhood . bmi - z - score standardizes an individual 's size , adjusting for age and sex , and allows comparison between values on an equivalent basis . the medical , dietary , and psychosocial factors characterizing the child and his family at baseline were assessed retrospectively by an external consultant ( who was not made aware of the patients weight evolution ) by reviewing the records ( standard home - made questionnaire ) filled in by the team at the first visit . as it is a retrospective and not a prospective study , a semiquantitative ( low / intermediate / high ) approach was used to evaluate each factor . for food consumption , no / low means not every day or never ; intermediate ( if applicable ) means every day but at a low ( 1 - 2 ) level ; yes / high means every day at a high ( > 2 ) level . physical activity means that the child attends a sports club or a youth organization at least twice a week ( yes / high ) , once a week ( intermediate ) or never ( no / low ) . obesity in the family means the child has at least one of the two parents who is obese ( bmi > 30 bad quality of sleep was assessed by snoring or short sleep duration ( < 9 h / night ) . family encouragement for the project and child 's motivation were assessed according to the involvement of the family and of the child in the project ( who had the idea to come here ? social integration was assessed as the participation of the child in out - of - school activities . family encouragement for leisure activities was assessed as activities realised by all the family out of the home . we use the quasilikelihood estimation with a linear or a logistic canonical link . for the significance of the results , we used the sandwich variance matrix augmented by the correction proposed by morel et al . which may be evaluated by the normal distribution . of those , 144 children ( 45% ) ( 59 boys ( 41.0% ) , 85 girls ( 59.0% ) ; mean age : 10.5 3.1 years ( range 419 years ; 105 ( 73% ) < 13 years , 39 ( 26% ) > 13 years ; mean bmi - z - score : 2.73 0.62 ) had at least a 1 year intervention program and were selected for our study . mean length of treatment was 2.2 years ( 16.7 years ) with an average of 3 1 visits per year . the length of treatment or assiduity ( number of visits ) did not depend on the initial weight loss ( bmi - z - score ) between the first , and the second visit ( p = 0.63 ) . sex , age and bmi - z - score at the first visit did also not influence the length of intervention ( p = 0.76 ; p = 0.09 and p = 0.43 resp . ) table 1 described the percentage of patients where bmi - z - score decreased and was stable or increased at the second visit and at the latest visit . at the second visit ( approximately after 36 months ) , bmi - z - score was decreased in 53% of the patients , remained unchanged in 31% , and was increased in 16% . at the latest visit in fact , weight loss was mainly observed during the first 6 months of treatment ( figures 1(a ) and 1(b ) ) but was sustained long - term . the mean bmi - z - score 0.04 mean bmi - z - score ) of the initial mean bmi - z - score after a mean of intervention of 2.2 years and decreased by 12% 2% ( 0.28 0.06 mean bmi - z - score ) for patients with a 48-month treatment . initial bmi - z - score , age at the first visit , sex ( data of boys and girls were thus combined for further analysis ) and number of visit(s ) per year did not influence these results ( p = 0.73 ; p = 0.27 ; p = 0.95 and p = 0.89 resp . ) furthermore , an additional weight loss was observed between 6 and 48 months of intervention whatever the bmi - z - score between the first and the second visit ( figure 2 ) . patients with a bmi - z - score reduction 0.3 units were 23% ( 2027 ; 95% ci ) of the population at 3 months versus 49% ( 4058 ; 95% ci ) at 48 months of treatment ( figure 3(a ) ) . however , 16% ( 1319 ; 95% ci ) of our patients gained weight after 3 months of treatment . the percentage was increased to 31% ( 2538 ; 95% ci ) of the patients reaching 48 months of treatment ( figure 3(b ) ) . the weight change between the first and the second visit was predictive of the additional weight change over the time ( figure 4 ) . we next investigated whether baseline medical , dietary , and psychosocial parameters reported at the first visit could influence the weight change over the time and which one could be associated with weight loss . patients who exercised in daily life before joining the interdisciplinary treatment were the most successful in term of weight loss ( table 2 ) . preexisting regular physical activity had a statistically significant ( p = 0.037 ) positive influence ( 0.42 0.11 of mean bmi - z - score ) on the weight evolution of the child , in comparison with those who did not exercise before starting the treatment ( 0.18 0.04 of mean bmi - z - score ) . having delayed puberty had a negative influence on the evolution of the mean bmi - z - score of the patients ( 0.02 0.10 of mean bmi - z - score ) in comparison with those who did not ( 0.23 0.03 of mean bmi - z - score , p = 0.046 ) . moreover , baseline daily water intake and daily soda intake had a statistical significant impact on the children 's weight outcome ( p = 0.046 and p = 0.00006 resp . ) we then determined whether the psychosocial context of the child at the first visit may influence the weight change observed later on ( table 4 ) . we showed that being a single child , having family encouragement for the project , the child 's motivation , the adherence to the treatment , and the compliance to adjuvant therapies had a statistically positive effect on the mean bmi - z - score at 9 months of intervention . the duration of the obesity and dual parent households did not impact the weight changes observed . this retrospective real - life study reported the outcomes of a long - term approach for treating childhood obesity and identified baseline predictors of weight changes . this intervention used interdisciplinary strategies ( with effective interaction between the team , not only juxtaposed competences ) but had the specificity to be individually adapted with a continuous care program . to our knowledge , this is the first time that the sustained benefit of a chronic intervention is reported and that the feasibility of a long - term intervention in real life is described in obese children . for example , family - based lifestyle behavioural treatment for obese children with similar clinical characteristics resulted in an average % decrease in overweight of ~7% after 6 months of treatment [ 3 , 4 ] which is comparable to the 8% decrease observed at 24 months in the current study . we described only a 4% bmi - z - score decrease at the second visit ( 3 - 6 months ) but a 12% bmi - z - score decrease at 48 months . in contrast , in the long term , the results of the abovementioned studies were not as promising as they were immediately after completion of the program ( ~ 3.5% decrease in weight at 12 months , 1% at 18 months ) . the beneficial effects of short intervention programs ( from 3 to 6 months ) were partly lost on the follow - up . even with a 12-month drug ( orlistat ) intervention combined to lifestyle , the initial weight loss was not maintained for more than 6 months . with this emphasis on acute short - term intervention , contemporary healthcare may not be well suited to meet the long - term needs of overweight children and their families fighting against this chronic disease . this indicates the need to develop chronic care models to optimize results , especially for severely obese children [ 4 , 7 ] . the addition of a 4-month maintenance treatment after short - term weight loss treatment resulted in better maintenance of weight loss compared with the no maintenance group ( 0.04 versus + 0.05 bmi - z - score ) but no additional weight loss was obtained over followup . in contrast , our program was still beneficial after 48 months of treatment . moreover , the percentage of patients with a 0.3 bmi - z - score reduction increased over time . at least 50% of children reaching a 36-month intervention presented a 0.3 reduction of bmi - z - score . the longer the treatment , the greater the proportion of patients who do not attend . this problem may be magnified with families , who may have more challenges in scheduling than individual adults , and where there are multiple people who may want to drop out of the treatment . for example , in an italian multicentric study of nutritional intervention in obese paediatric patients , drop - out rates ranged from 3034% to 9094% after 2 years . according to the literature , the main reasons for dropout are loss of interest , relocation , schedule conflict , transport difficulties , family issues for example , limited time for recurrent group sessions , and even for daily household demands [ 3 , 25 ] . even the weight benefit was modest after 1 year of followup ; this could help them to better general health in the long term . the fall in bmi observed in our study may be clinically relevant as demonstrated by many studies , even though not analysed here . short - term family - based treatment which combined nutrition education , behavioural modification and exercise was shown to improve body composition , lipids profile , blood pressure , and insulin resistance [ 4 , 25 , 26 ] . many of the obesity - associated complications can be reversed with a 5% decrease in age - adjusted bmi percentile . thus , the bmi changes observed over time in our study are likely to be clinically significant as those changes were sustained over the longer term . we determined parameters characterizing the families and children at baseline conditions which were associated with a better weight control . indeed , for those less or not responsive patients , new research studies should try to devise new treatments to optimize long - term weight benefits . we demonstrated that those patients could be identified quickly according to the initial weight change observed between the first and the second visits . also reported that a greater weight loss between the first and the second visits was a predictive factor in the success of the treatment . also reported that early weight change seems to be related to treatment response through to the end of the treatment and also the 2-year followup . identification of factors that promote early weight change is critical because modification of these factors before initiation of the treatment may promote a better early response . in our study , similarly to reinehr et al . , reduction of overweight was independent of initial bmi - z - score , age at the first visit , and sex . it is well know that physical activity is related to long - term weight maintenance but , as suggested in another study , our data supports its role before the initiation of the treatment . children with large birth weight , gestational diabetes , no or short - term breastfeeding , parental obesity , asthma , and short sleep duration were described as having an increased risk of obesity [ 33 , 34 ] . baseline daily water and soda intake seemed to be a good predictor of early weight change . consumption of sugar sweetened drinks by adolescents is an independent variable associated with increasing bmi , but its role on early weight loss was never examined . healthy eating habits as eating breakfast and participating in programmed exercises were described to be correlated to healthful bmi , suggesting that these factors may be potentially protective against obesity in 1216-year old adolescents . our study extends those results by showing that prevention policy may also be helpful even for children who have to lose weight . interestingly , recent reports suggested greater weight loss in obese children when parents alone are targeted for intervention , which emphasizes the role of the parents in the child 's weight reduction . larger reductions in adult bmi were associated with more successful results , which indicates that working to enhance the adult role in child weight control programs may improve results . suggested that parents having an older child , believing that they themselves were overweight , perceiving that their child had a health problem were associated with greater parental readiness to make changes . emerging research also indicates that overweight children with psychosocial problems or the occurrence of maternal psychopathology are less responsive to weight - control intervention over the longer term the divorce rate has increased as well as the number of families with both parents working . our data suggested that the dual parent households did not affect weight changes observed at 9 months . this is in contrast with a recent study which showed a relationship between single - parents status and excess weight in children . further studies are needed to explore the dynamics of single - parent households and its influence on childhood diet and obesity . interestingly , our study showed that a family with an only child may expect a greater weight loss . other factors such as higher incomes and higher level of education for the mother were also reported to be associated with better results but were not analyzed in the current study . in conclusion , this study was a first step in determining whether weight loss was achievable with our interdisciplinary approach and highlighted potential success of a continuous care weight control program to lower bmi . an early weight change seems to be a marker for children 's long - term treatment response . preexisting regular physical activity , normal timing of puberty , baseline daily water and soda intake , motivation and some family characteristics predict the early response to the treatment . better prevention policy and parental support may thus improve the success of the childhood obesity treatment . our data may provide a better understanding of the factors involved in better weight control and may help to optimize / adapt our strategies for participants who do not benefit from treatment .

da : i have a relatively broad area of scientific interest encompassing cancer genetics , mouse genetics and genome sequencing . with my cancer hat on , the paper that impressed me most was by sodir et al . , which showed that inhibition of myc can cause cancer regression even in advanced tumors . this work illustrates the critical role that myc plays in tumorigenesis , and clearly defines it as a therapeutic target . in terms of genome sequencing , i 'm very impressed by some of the new de novo assembly algorithms , such as li et al . , and i think our contribution of sequencing mouse genomes , keane et al . , is also important and will be of great use to the mouse genetics community . these sequences now make it possible to take a systems biology approach to mouse genetics and to link variants to phenotypes like never before . bb : i would say the foldit paper by david baker 's group because it shows a new model of research is possible . some large - scale problems such as protein folding remain challenging to solve with just computational approaches , and the problem is too difficult for even experts to solve manually . however , by designing tools that can break such problems into puzzles that people , even non - experts , can play with in their spare time can provide cutting - edge solutions . the first is the discovery and analysis of chromothripsis by peter campbell at the sanger institute . this idea that some of the complex chromosomal rearrangements observed in cancer cells can come from a single catastrophic event , where an entire chromosome gets shattered and stuck back together at random , is astounding . this has changed our understanding of cancer genome instability and repair : a truly novel finding and groundbreaking idea . the other cancer genetic study is the work of inder verma , rusty gage and colleagues at the salk institute demonstrating a new function for brca1 involved in heterochromatin chromatin formation and repressing satellite expression via the ubiquitylation of histone h2a . this is beautiful work , which might deeply change our understanding of brca1 's role in cancer . this adds to the numerous and recent evidence of the importance of chromatin - modifying processes in cancer . ch : one important recent paper is the work on the vaginal microbiome in reproductive - age women by jacques ravel and larry forney . this is a very well - structured and well - analyzed study that includes a variety of important findings . it establishes that diversity in the human microbiome is personalized and much more than ' noise ' , and that following individual subjects longitudinally can be extremely informative . it shows that the microbiota can be linked both to host phenotype and to host environment , but that neither of these alone is the whole story . in combination with other work , it shows that beyond community composition , the complete picture of microbial ecological structure , function and dynamics differs widely among humans and human body habitats . and it provides evidence that microbial community structure and its interaction with phenotype have potential links to genotype by way of racial background or ethnicity . finally , all of these results together suggest ways by which personalized medicine can be influenced by the microbiome . many of these are general results that have been shown in one or more previous studies , but this paper brings them all together nicely and applies them quite interpretably in the specific microbiological context of the vaginal community . one paper from the dekker and young labs demonstrated that mediator can form a complex with cohesin to connect enhancers and core promoters of active genes in mouse embryonic stem cells . predicting targets from transcription factor chip - seq has been a challenging problem , and perhaps mediator and cohesin binding could offer some insights into which bindings are functional with transcriptional outcomes . another paper , from howard chang 's group , developed a new technique called ' chirp - seq ' to identify the in vivo genome - wide location of long non - coding rnas and understand their chromatin regulation functions . the chang lab has shown exceptional ingenuity in both genomic technology development and biological mechanism discovery in recent years . a third paper , from peter laird 's group , showed that many cancer samples have small regions of hypermethylation within bigger domains of hypomethylation , and these domains coincide with lamin binding domains . it demonstrates the power of correlating unpublished genomics and epigenomics data with publicly available data to make interesting discoveries . cm : a recent paper that i think demonstrates an important point about mapping insights from functional genomic data in model organisms is mcgary et al . , in which the authors define the concept of ' phenologs ' , which are pairs of phenotypes across two species that share a set of orthologous genes . the key observation is that sets of functionally related genes are often conserved as functional modules across species , but that their corresponding phenotypes in the two species might have no obvious connection . thus , after constructing a phenolog map pairing phenotypes across species , the authors show that one can accurately predict new genes related to phenotypes in higher eukaryotes based on functional genomic data from more genetically tractable model organisms . for example , they identify a phenolog connecting yeast sensitivity to the drug lovastatin to abnormal angiogenesis in mice . this connection then enabled them to predict new genes with a role in angiogenesis based on yeast genes that showed the lovastatin phenotype , and the prediction was confirmed in the frog xenopus . this is an important paper because it provides a model for how we can leverage the wealth of data produced in model organisms that are more genetically tractable to gain insights about higher organisms and disease . the method itself was very simple but based on a powerful idea , and is one that will work in a number of different contexts . ao : a lot of my recent work has been on the analysis of rna - seq data . there are many opportunities provided by sequencing the transcriptome that have not been possible with previous technologies , such as detecting all expressed transcripts in a sample and annotating them , determining splicing variants and how isoforms change between samples , looking for allele - specific expression and looking at rna editing . however , i think there is still a lot of work to be done in order to develop analytical methods to capture all the different types of information that we might want to obtain from an experiment . one of the problems with developing analysis methods is actually showing that the method is doing the right thing and understanding possible biases and limitations of analysis strategies . in this context i believe a recent paper on spike - in standards for rna - seq experiments will be very influential , as these sorts of data sets enable us to identify and study biases in data generation and analysis and allow us to assess and compare methods . hopefully more of these ' truth ' data sets will be generated to enable us to further develop our understanding of the technology . jr : two recent papers have shown that a long non - coding rna termed coldair plays a key role in regulating plant flower formation . what 's remarkable is that this rna ' senses ' temperature and indicates to the plant when it is warm again and time to flower . it also highlights the importance of reading primary plant literature ; discoveries in plant science are often a decade ahead of the curve ! mw : there are so many wonderful papers in systems biology , which is by now quite a large discipline . it is hard to nail down only one ; my favorites that are destined to become classics include a paper from frank holstege 's group that identified different network topologies that can ensure robustness through paralog redundancy in yeast genetic and signaling networks . this paper is a beautiful example of true , integrative systems biology that takes advantage of different techniques , available datasets and modeling to gain systems - level insights into signaling network architecture . the second paper is from karen oegema 's group , in collaboration with fabio piano and kristin gunsalus . this paper utilizes the power of systems biology in the worm by combining phenotypic profiling with high - resolution imaging of defects in the gonad , and a new clustering method that provides unprecedented specificity . numerous functional annotations of genes were obtained by delineating more than 100 distinct and highly detailed gonadal phenotypes . da : i have worked with many wonderful colleagues . i 've fed off the contagious enthusiasm of jos jonkers and jonathan flint from amsterdam and oxford , respectively . both of them are outstanding scientists and fabulous collaborators who never seem to tire and always make me do better science . at the sanger institute , no experiment is too difficult or demanding for louise , and what she does n't know about the study of cancer genes in mice is not worth knowing ! i 've also learnt a lot from several people who i 've worked with who were incapable of managing people . while i 'm sure i do n't always get it right , i try hard to motivate people with the carrot rather than the stick and to make the lab a happy place to work . bb : i would say there were three people who have most influenced my career thus far , one mathematician and two biologists : my postdoctoral mentor daniel kleitman , who was professor of applied mathematics at mit , told me ' proteins , that 's what you should do ! ' when i was looking for interesting areas to apply my algorithms background - he encouraged me to initiate comparative genomics ; jonathan king , also at mit , who taught me much about protein folding and helped guide my group meetings through the first years ; last but not least , peter s kim , president of merck research laboratories , who worked tirelessly with me to define the new brand of research and publications in the burgeoning field of computational biology . oh : i am happy and fortunate to have worked with several brilliant and enthusiastic scientists , but i acknowledge the wonderful interactions i have had with dr kelly frazer for the past 4 years . beyond a fully accomplished genomic biologist , she has been an exemplary mentor , concerned both with traditional academic training , such as grant application and scientific communication , and also with more practical mentoring on how to manage successful and highly collaborative genomic projects . just like we all grow up to resemble our parents , i 've academically inherited much of olga 's lab management style . although we 're working in very different scientific areas now , my approaches to mentoring , organization and presentation remain heavily influenced by hers at princeton . in terms of scientific content , i give a great deal of credit and respect to dirk gevers at the broad institute , who 's been a phenomenal collaborator on many of the projects my group has tackled since starting my own lab . the best collaborations are with folks who both know the science and have fun doing it , and dirk and his team have been stellar on both counts . the first is my phd mentor , jun liu , who showed me the fun of methodology development in computational biology , which convinced me to adopt an academic career . jun has continued to help me over the years in research projects and career decisions , and taught me how to be a good mentor . the second person is my first collaborator , jason lieb , from whom i learned a lot about writing , leadership , and working as a team in a consortium . last but most importantly , i had a great collaborator , myles brown , now at dana - farber cancer institute . both of us are passionate about new genomic technologies and gene regulation questions , and it is really fun working together . we brainstorm on how to better adopt new genomic techniques , generate the data , develop the computational method , and apply them to important biological problems . i have learned so much from myles and his postdocs , and some of our postdocs continue to collaborate with each other after they become independent . he is also a role model for balancing work and life , and has given me much good career and life advice . i started my graduate studies at princeton with an entirely computational background and with little direction in terms of my research focus , other than being interested in machine learning , signal processing and data mining . olga introduced me to the exciting genomics revolution that was well underway and was willing to take a chance on me , even though i knew very little biology and had no previous experience in computational biology or bioinformatics . in her mentoring , olga emphasized establishing a solid foundation in computer science and statistics but also developed our skills to identify relevant and impactful biological questions , one of the biggest challenges of transitioning from a computer scientist to a ' computational biologist ' in my opinion . she established a lab environment at the lewis - sigler institute for integrative genomics that was centered on collaboration with experimentalists and developing practical bioinformatics solutions to problems faced in these collaborations . this perspective permanently shaped me as a scientist , and i certainly would not be where i am today without olga 's mentoring . ao : i have been very fortunate to have several great mentors in my career so far who have all been very influential over different aspects of my life and career . it 's very hard to single out people but i will just focus on two . firstly , my phd supervisor , professor rachel webster , has been very supportive of me and my career , giving me advice and guidance for the last 15 years . i did my phd in astrophysics and when i was considering leaving the field she fully supported that decision . since then she continues to both share her experiences and give me advice on many issues that are important to a research career , such as leading a research group , conducting collaborations , managing my time , employing people , and effectively combining a family and a successful career . even though we no longer work in the same discipline , i value highly her experience and advice and make an effort to catch up with her every few months . the second person who has had a major influence on my career is professor gordon smyth . he was the one who first gave me a postdoc position in bioinformatics even though i had no specific background in the area . he taught me the ropes of bioinformatics and statistics and guided me through the initial years in this field . i will always be grateful to him for taking that initial risk on me as i find genomics absolutely fascinating . my career has been guided by amazing mentors such as michael snyder , howard chang and eric lander [ 32 - 34 ] . his work was an infectious balance of scientific vigor and rigor , analytical yet creative interpretation with pioneering vision . his humble upbringing , ability to admit when he was wrong , and persistence for what was right has continued to inspire me . he continues to push the envelope in the field while sticking to his ambitious objective to attain complete protein - protein interaction networks in a variety of model systems . da : science needs to be your passion and you really need to love it and jump in with both feet . i 'm not talking about turning up and doing a few experiments between coffee breaks but making your science one of the most important things you do . i do n't know of any successful scientist who periodically does n't find themselves spending virtually every waking hour in the lab or in their office . surrounding yourself with people who are smarter than you also helps you raise your game . bb : published is better than perfect : it 's advisable to communicate intermediate results while you work to polish your system . also , computational biologists should try to become familiar with wet lab work in their chosen area of research . lastly , make the algorithms you develop available through tools that others can easily use . oh : beyond showing the necessary scientific achievements through regular publication , i would emphasize the importance of communication . communication is crucial to build a network and learn important lessons for a successful scientific career . talk to your mentor(s ) with an open mind and build a trusted and honest relationship with your colleagues . i have seen too many people frustrated due to communication issues with their mentor or colleagues . no one can blame you for it , as long as you are honest with yourself and others . also , be curious of other people 's work . when going to a couple meetings a year , make sure you talk to the senior investigators whose work you admire , and do not hesitate to tell them so . the next thing you know they will be writing you support letters and becoming collaborators . ch : stereotypes are often true , both the good ones and the bad ones . as a faculty member , there can be a lot of politics , you do spend a lot of time asking for money , and you are even busier than you were before . but on the flip side , you really can ' do anything ' , both in terms of choosing exciting scientific directions and in shaping your group and your environment . if there 's a question you want to work on , you can - there 's nothing to stop you except your own motivation to recruit a team and funding for the investigation . and there 's no one right way to do it - lab size , teaching / training balance , approach , methods , impact and sales pitch are all up to you . take advantage of your time as a student or postdoc to do as much of your own work as possible , though , since you 'll likely have fewer and fewer chances to perform the investigations with your own hands as time goes on . sl : in computational biology , you need to appreciate the biology as much as , if not more than , the statistical method or computer science . when starting out , get your hands as ' dirty ' as possible and as quickly as possible with data ; that is , dive into big datasets . without seeing enough data in detail and understanding the data characteristics find a good and niche research direction that : ( i ) is important in the long run ; ( ii ) you have a real interest in ; ( iii ) maximizes your existing expertise ( for example , previous training ) and advantages ( for example , timing , location , connections and other available resources ) . cm : my advice to young computational biologists is that the key to success in our field is to remain grounded in specific biology questions , particularly ones that are pursued in collaboration with experimentalists . the success of our field is ultimately measured by the impact we can have on our understanding of biology , and how we spend our time should reflect that . in my opinion , too many computational biology researchers are working in isolation on marginally relevant problems or making incremental improvements in areas that have already been well - populated by methods that are already adequate . meanwhile , there are pressing ' big data ' challenges being faced by our experimental colleagues , including everything from collection and normalization of massive datasets to scalable methods for integration of heterogeneous data types . often such collaborative projects involve some amount of what is often considered service work ( for example , processing / normalization of raw data , applying existing tools to new data , and so on ) , but this requires computational expertise and can have a major impact . furthermore , contributing our efforts to these service - oriented tasks almost always leads to exciting new issues and methods that generalize to other scenarios . the most exciting and impactful research is being accomplished by synergistic cross - disciplinary teams , so if you want to have an impact get involved in this sort of research . finally , remember that building collaborative relationships takes time - establishing the specific relevant expertise , effective communication and trust that are necessary for successful collaborations is hard work - but investing time in honing these skills will pay off and can serve as the basis for a successful and rewarding career . ao : i think one of the most important skills in research is the ability to communicate ideas . my advice would be to spend time practicing both writing skills and oral presentation skills . there is no point making great discoveries if no one knows about them or uses your discoveries . i think it is very important to understand the audience that you are communicating to and work out effective ways to explain concepts , especially in the current environment where there are many multidisciplinary teams with different background knowledge . what may seem obvious to you may not be obvious to other people that are potentially interested in your work . some people have a natural talent for communicating their ideas but for the majority of us it 's something we need to spend time working on . my way of doing this , at least initially , was to look at papers that i really like reading and work out why i think they are good and try to emulate the style in my own work . with regard to oral presentations , i always practice them out loud several times and try to get feedback from colleagues on which parts worked and which were n't clear or interesting . jr : i try not to give too much advice as i have seen many diverse paths met with great success in science . if there was one thing , i would say it 's the ability to understand both the experimental and the computational sciences . if trained as an experimentalist , i would learn the key principles in computational biology or at the very least the linguistics , and vice versa . mw : put yourself ' out there ' : ask questions at meetings , build a scientific network with colleagues , do not sacrifice quality for quantity of data . da : in cancer genetics the biggest challenge is integrating data from genome sequencing , transcriptomes and the epigenome , so that it makes sense . the problem is no longer acquiring the data but ' embracing the chaos ' . some of the boolean logic approaches are making inroads in this area . in mouse genetics the tal nucleases , which can be used to tailor the mouse genome with base - pair precision , potentially represent a big advance in this area , but we need to understand if they have off - target activity . in genome sequencing , read length really matters for assembly and while the last few years of short - read sequencing have been amazing we really need long - read technology that is truly scalable and accurate ( and cheap ) . bb : the mission of computational biology is to answer biological and biomedical questions by using computation in support of or in place of laboratory procedures , with one goal being to get more accurate answers at a greatly reduced cost . three major emerging challenges are : how to make sense of massively accumulating data , how to develop reasonable gold standards for testing our algorithms , and how best to integrate computational studies with real biological experiments ( on both sides ) . the past two decades have seen an exponential increase in genomic and biomedical data , which will soon outstrip advances in computing power to perform current methods of analysis . extracting new science from these massive datasets will require not only faster computers ; it will also require smarter algorithms . moore 's law has been a great friend of computational biologists : the amount of processing you can do per dollar of compute hardware is more or less doubling every year . back in the 1990s , . however , one way this balance is being disrupted is by the advent of next - generation sequencing . the size of genomic databases is going up by a factor of 10 every year , far outstripping the growth in our computational capacity . it 's tempting to think that cloud computing is going to solve this problem , but that 's not the case . it does n't change the problem that the data are increasing exponentially faster than computing power per dollar . in fact , you 've got to devise algorithms that are so fast that , in some cases , they ca n't even grow linearly in the size of the databases . another big challenge in computational biology is the determination of gold - standard datasets for training computational techniques . what we really want to identify are functional orthologs ( that is , genes that perform the same functions across various species ) . the most commonly available datasets capture this only indirectly by looking at , say , sequence similarity between the genes . there are many computational approaches that use this indirect data to predict such orthology relationships , but determining which one works best is difficult . one direction we have been exploring is using protein and genetic interaction data to improve orthology prediction by better capturing function correspondence . it would really help if we had even a limited set of proteins for which gold - standard orthology information was available . however , we are still some distance away from having any gold - standard orthology sets . there are many other problem domains where being able to generate good gold - standard datasets would significantly improve our ability to use computational methods . the final challenge is the need to improve the integration of biological and computational methods . in some domains , algorithmic thinking is already very tightly integrated into the process of experiment design , execution and interpretation . genome sequencing is a great example of where such integration has yielded great success . in other cases , however , biological methods use computational analysis only as an afterthought ; for example , many studies of cell signaling could benefit greatly from having knowledge of innovative computational techniques applied early in the design stage , so that the right data are available to enable the full power of these methods to be applied . far too often , enough biological details are abstracted away so that the solution loses its biological relevance . oh : access to adequate clinical research samples in cancer genetics is one of the most important challenges in cancer genetics . collection of samples by biopsy or surgical resection has been traditionally performed for clinical care only . it is currently extremely hard to use the same samples for research for various reasons , from preparation methods , to logistic or consent . several institutions like the university of california , san diego are developing master protocols to systematically consent a majority of oncology patients and collect samples from surgery or biopsy for investigational purposes . the resistance is high , in general legitimated by the patient 's protection , but people start to understand that it is the only way to eventually deliver the promises of personalized diagnostics and care . another challenge is to educate people about genomics and to tone down the natural hype of the genomics field . investigators involved in clinical and translational projects are in some way victims of the hype created by the fantastic and recent technological advances . i frequently talk to clinicians who are enthusiastic about sequencing their samples , but many projects often fall short due to the ignorance of the requirement of sample number or quality . for example , there is no good rationale to sequence the whole genome of thousands of samples except to make it a general resource for the community . biological questions might be better addressed with a more focused approach , such as sequencing exons or candidate regions in properly selected patients . the hype of the sequencing field is a wonderful catalyzer of novel ideas and provides much needed public exposure of our field , but we have to regularly educate prospective collaborators on basic notions of genetics or the reality of the sample preparation or data analysis . at our institution , my function in the university of california , san diego , clinical and translational research institute ( a clinical and translational science awards funded entity ) is to do just that : consult with people and help them with the design , preparation and analysis of their translational genomic experiments . traditional institutions expect faculty to lead independent projects typically funded through the r01 nih grants . however , genomics has traditionally functioned differently , following the principle of team science , where multiple principle investigators contribute to a large endeavor . this was the case for the human genome project and the hapmap project , and today the 1000 genome consortium and the cancer genome atlas , for example . this ' big science ' is usually financed through alternative sources of funding requiring collaborations and multiple principle investigators , and the results do not always lead to first or last author publications for the majority of the participants despite their essential roles . traditional institutions that promote faculty based on r01 awards and last author publications do not always recognize this aspect . some institutions , such as harvard or the ontario institute for cancer research , have established alternative academic review criteria that recognize participation in team science and allow investigators to successfully grow in this environment . at the time when funding is becoming scarce and more directed to specific projects , let 's hope that more institutions will follow these examples . ch : ( i ) understanding the systems - level ecological rules governing microbial community structure , ( ii ) relating the human microbiome to health and disease , and ( iii ) streamlining methods for turning next - generation data into actionable biology . addressing the combination of the first two challenges will help us realize some of the human microbiome 's potential as a means of diagnosis and therapeutic intervention . investigating the first challenge in particular should let us leverage systems biology 's successes in molecular biology during studies of microbial communities . the second will likewise feed back into the broader metagenomics community by identifying ' interesting ' microbiome properties , environments and phenotypes on which to focus . finally , the third challenge includes finding ways to collaborate on biological ' big science ' projects , to collectively analyze sequence data ( of all sorts , not just metagenomic ) , and to leverage shared computing resources . all of these continue to be necessary to solve the considerable data management and interpretation challenges brought about by next - generation sequencing technologies . these technologies will continue to accelerate biological discovery - but there are still many opportunities for computational methods to accelerate that acceleration . sl : now that it 's possible to profile transcription factor binding using chip - seq , the ability to predict the target genes and the direction of their expression changes upon factor activation or inactivation is still an important challenge . for factor binding , there are often thousands of genes nearby binding , but only a minority of the nearby genes really show differential expression and we do n't know why . also , for transcription factors with multiple functions such as ctcf ( for example , transcriptional repressor and insulator ) the challenge is whether we can differentiate their functions from chip - seq of other factors or histone marks . approaches such as hic and chia - pet can identify genome - wide higher - order chromatin interactions , which have the potential to answer this question , although there are still technical and cost challenges for these techniques to be widely adopted . performing chip - seq or dnase - seq with a small amount of starting currently one needs 100,00 to 500,000 cells to do a histone mark chip - seq , and 1 million to 2 million cells for transcription factor chip - seq . to make chip - seq or dnase - seq work well on tissues or tumors , it is important to start from smaller numbers of cells . the laboratories of peggy farnham and brad bernstein , and many other laboratories , have explored this issue . commercial companies like illumina are developing kits for library construction from < 1 ng of dna , and third - generation sequencing techniques promise to offer a better solution to working with small amounts of starting material . finally , there are many transcription factors , chromatin - modifying enzymes and histone marks functioning together to regulate gene expression . the specificity ( for example , which transcription factors specifically recruit which histone marks or histone modifying enzymes ) and the cooperativity ( for example , which factors are pioneering factors for the binding of other factors ) of these factors in different cells or conditions are still poorly understood . without understanding this , the effect of epigenetic drugs could be hard to interpret . as sequencing technologies increase throughput , multiplex chip - seq would allow us to investigate many more conditions in combination and we might have a better answer for this question . cm : in my specific area of interest , genetic interaction networks , there are a few challenges we face as a community . ( i ) scalable technology for mapping genetic interactions for other phenotypes , conditions and organisms , especially higher eukaryotes . the yeast community has been very successful in the past several years at developing technology for rapid construction of combinatorial mutants to map genetic interactions . specifically , the typical approach is to look for combinations of mutations that result in a surprising phenotype ( usually fitness defect ) given the phenotypes of the mutations introduced independently . these efforts have produced global interaction maps covering millions of combinatorial mutants , which have proven to be quite useful for understanding gene function and general organization of the cell . in yeast , efforts are underway to expand these maps to other phenotypes and other conditions ; this requires new scalable technologies given the space of possible experiments . such maps in higher eukaryotes will be important for understanding the genetic basis for complex phenotypes and disease and developing new therapeutic approaches , but the technology for mapping these interactions is still relatively limited in throughput . several exciting efforts are underway to address this challenge , most of them leveraging rna interference technology . the past year has produced new successes in drosophila and human cell lines but continued focus on improving and scaling the technology will be fruitful . ( ii ) translating insights about genetic interactions from perturbation studies to questions in population genomics . the focus of the genetic interaction community has largely been on precise combinatorial genetic perturbation in single individuals ( for example , standard lab strains ) . this approach is attractive because the effects of perturbations can be studied in a controlled genetic background . however , we would ultimately like to leverage this knowledge about how genetic variations combine to influence phenotype to understand the link between genotypic and phenotypic variation across individuals in a population . the latter challenge is of course the main goal of genome - wide association studies in humans ; to date these have struggled to explain large portions of the heritable phenotypic variation . applying insights derived from large - scale perturbation studies to the population genomics questions will be an interesting direction , especially as the mapping technologies become more feasible in higher eukaryotes . there are new opportunities and the necessary data to make progress on this front in yeast with the recent sequencing and phenotyping of several saccharomyces cerevisiae strains . the combination of this information on genomic and phenotypic variation , combined with extensive functional studies on the reference genome , will provide a good testing ground for new methods in this area . , a more general challenge is the problem of leveraging functional genomic data across species to speed the process of functional characterization . even the most basic question in systems biology , ' what are all of the genetic components related to biological process x ? ' , has not been answered comprehensively in most species , particularly in higher eukaryotes . enormous resources have been spent generating functional data in model organisms , but these data are relatively underutilized for mapping functions in other species . the paper from mcgary et al . provides a nice demonstration of how insights from relatively data - rich model systems can be used to direct experimental investigation of genes related to specific phenotypes in more complex organisms , and i suspect similar approaches can be developed in other settings . accomplishing this will require new computational infrastructure and tools to support integration and comparative analysis of functional genomic data . ao : getting a handle on the propensity and type of rna editing that is occurring is a fascinating area which , as yet , has not been fully resolved . it has been documented that the sequence of rna can be modified post - transcriptionally , resulting in an rna sequence that is different from the dna from which it was derived . high - throughput sequencing technologies give us the opportunity to study rna editing on a genome - wide scale and there have been several publications recently on this topic . however , there is quite a debate about how frequently this actually occurs . in my view , results are probably influenced by biases in mapping procedures ( see joe pickrell 's blog and the recent paper by schrider et al . ) and it will be fascinating to see how this debate gets resolved in the near future and what the results mean for the diversity of the transcriptome . one of the major scientific challenges right now is the integration of different types of data to explain a biological phenomenon . for example , the encode project is producing genome - wide expression , trascription factor and epigenetic data on many different cell types . making sense of even just a small fraction of these data sets is extremely challenging and will require major breakthroughs in analysis and interpretation . in particular , i believe the integration of epigenetic and expression data will be a major challenge over the next few years and there are many specific questions that are unresolved . there are two questions that i think are particularly interesting in the area of data integration . ( i ) how can we describe the epigenetic landscape and how is it related to development and disease ? there are over 100 epigentic histone modifications known to date and more are being discovered all the time . therefore , there are millions of possible epigenetic combinations that could be predictive of expression and function , and most probably only a small fraction of these are important , however . recently there has been some excellent work published on combining epigenetic marks to annotate the genome . ( ii ) how is alternative spicing controlled and what is the role of epigenetics ? next - generation sequencing has shown that many genes in the genome have multiple isoforms ; however , the mechanisms that control the switching between alternative transcripts are not well understood . recently there have been extremely fascinating observations that show an important role for epigenetics in controlling splicing events ( for example , ) . there is still a long way to go in order to try and integrate epigenetic and expression data on a genome - wide scale . jr : ( i ) what properties of large non - coding rna genes would identify subfamilies and classes ? imagine the text book had already been written for non - coding rnas and someone recently discovered protein genes . one of the first things to do is identify functional domains ( for example , helix - loop - helix , dna - binding domains , and so on ) that could be extrapolated to families related by functional properties . with rna it 's a bit trickier but initial progress is being made for large non - coding rnas using co - expression with proteins , a process termed ' guilt by association ' . we recently got a glimpse of some of the first emerging global properties after mapping and characterizing 8,000 long non - coding rnas . they are strikingly more tissue - specific than protein - coding genes , an interesting feature that we could potentially use in medical diagnostics . it 's clear that there are numerous functional large non - coding rnas but almost the entire genome is transcribed . progress is being made by more global loss - of - function and gain - of - function experiments . ( iii ) what do these non - coding rnas do and how do they do it ? we have identified an emerging theme of non - coding rna interacting with proteins and modulating their function . if we could learn how these rnas work , we could envision engineering them to guide stem cells into distinct cell types . mw : ( i ) single cell systems biology : many labs are making good progress toward this , from cell biological imaging screens , to measuring parameters in single cells . this will result in high resolution of biological information and will provide important insights into cell - to - cell variability in cellular networks . ( ii ) dynamic networks : currently , many available networks collapse all measured interactions into a single graph . however , it is clear that only parts of the network are active in different cells or under particular conditions . visualization is an important component of this , as is measuring reaction kinetics and the concentration of different biomolecules . ( iii ) developing integrative networks that combine metabolism , protein - protein interactions , genetics and regulatory networks . . however , it is clear that many biological processes are controlled by a flow of information through different types of molecules , and thus networks , and often result in differences in cellular and organismal metabolism . to better capture the events important to a biological process , it will be important to combine all relevant , active networks into a single graph da : if money were no object , i 'd sequence all the athletes in the olympic village and all the dogs at crufts ! bb : i would do much larger - scale genome - wide association studies to have sensitivities at the levels of small multifactorial effects ( that is , leverage statistical power to be able to detect a signal in millions of human genomes that is n't currently distinguishable from noise ) . assuming one can do the analysis efficiently with my lab 's compressive genomics paradigm , whole genome sequences offer the ability to directly infer causal variants , as opposed to single - nucleotide polymorphisms , which currently just map marker regions ( using linkage disequilibrium ) to phenotypes . i would also like to do a genome - wide epigenome scan for discordant twins , to find out why they are different despite near identical genomes . oh : i would study cancer genetics at a much higher resolution : at the single - cell level to study the process of clonal evolution and selection ; at the environment or niche level to study the effect of the stroma and surrounding cellular environment on cancer progression ; and finally , at the diploid level to determine the differential role of each allele in cancer progression . ch : there are two directions in which i 'd like to go with such a grant , one computational and the other translational . the former is a bit prosaic , in that the field has now completed enough human microbiome projects to have a good idea of what data and metadata are useful . we need to develop a platform to standardize and automate the process of boiling microbial sequence down to functional information , then comparing that to subject phenotype , much as has been done successfully for environmental metagenomes up to this point . translationally , this would enable a sustainable effort to track the human microbiome longitudinally , in a large prospective cohort . epidemiology in such cohorts has been uniquely successful in characterizing dietary and environmental influences on health ; the microbiome is a big component of our everyday environment that 's so far not been assessed in such a manner . building and maintaining such a cohort over time would take more than just my group and a few million dollars , but it 's a project i 'd be excited to see happen soon ! sl : i would examine the epigenetic status at regulatory sequences ( especially enhancers ) and see how this changes between conditions ( for example , development , stimulation and drug treatment ) and between individuals ( for example , tumor tissues ) . cm : i 'd invest in tools for perturbing and characterizing mutants derived from a reference ' normal ' human genome , so that we could enable true systems biology in humans . to do this , we 'd need to improve technology for single and combinatorial knockdown of transcripts genome - wide , quantitative read - outs for various cell states ( for example , transcription levels , protein levels , protein / chromatin modifications , protein - protein interactions ) and single - cell quantitative phenotyping . in addition to improving the technology on all of these fronts , i would invest significant resources in applying them systematically and globally on a set of carefully chosen reference cell lines . i think the only way we will understand how the genotype determines phenotype - the basis of human disease - is through systematic functional genomics . ao : as i 'm interested in epigenetics and gene regulation , i would love to spend some money building up interesting epigenetic and expression data sets using chip - seq , rna - seq and dna - seq to look at histone modifications , transcription factors , rna expression , micrornas and rna - protein interactions , all with a large number of controls . however , i would probably spend most money on employing and training people to develop methods to analyze and visualize the data in new and imaginative ways and to collaborate on exciting projects . there are so many different possibilities for exploring the vast amounts of genomic data that are now being produced . however , i believe the biggest bottleneck is the bioinformatics and the shortage of researchers in the field social insects fascinate me , and i have a hunch that maternal and/or paternal rna storage might be involved in establishing which larva becomes a worker or a queen . more practically , i would use the money to perform combinatorial genetic engineering of stem cells , aiming to guide and tweak their metamorphoses into other types of cells . mw : if money ( and time ! ) were no object , i would measure all metabolites in a system of interest ( caenorhabditis elegans in my case ) , and repeat this upon perturbation of gene regulatory networks by transcription factor rna interference and under a variety of environmental and nutritional conditions . the data would then be used to create comprehensive and integrative network models that link metabolism to gene expression . david j adams ( da ) is a faculty member at the wellcome trust sanger institute . he performs forward genetic screens to uncover cancer genes and cancer pathways and is also leading a program to use new - technology sequencing to decode the genomes of several mouse strains that form the basis of mouse experimental genetics . bonnie berger ( bb ) is professor of applied math and computer science at massachusetts institute of technology . her recent work focuses on designing algorithms to gain biological insights from advances in automated data collection and the subsequent large data sets drawn from them . she works on a diverse set of problems , including network inference , protein folding , comparative genomics and medical genomics . olivier harismendy is an assistant professor of pediatrics at the university of california , san diego and member of the moores ucsd cancer center ; he works on several projects , including investigating the function of regulatory dna variants in cardiovascular diseases and cancer , the identification of dna markers of cancer progression and the development and implementation of genomic assays and analysis in the clinic to guide personalized cancer care . curtis huttenhower ( ch ) is assistant professor of computational biology and bioinformatics at harvard university . his research is concerned with the discovery of useful biological knowledge in large collections of genomic data . this requires the development of computational methodology that is efficient enough to deal with billions of data points while remaining biologically rich enough to capture the complexities of molecular biology . x shirley liu ( sl ) is an associate professor at the department of biostatistics and computational biology at the dana - farber cancer institute and harvard school of public health . a computational biologist , with expertise on the integrative modeling of transcription and epigenetic regulation , she and her colleagues have developed a number of widely used algorithms for transcription factor motif discovery . chad l myers ( cm ) is a principal investigator at the university of minnesota . the research in his laboratory focuses on machine - learning approaches for integrating diverse genomic data to make inferences about biological networks , the main purpose of which is to further understanding of gene function and how genes or proteins interact to carry out cellular processes . alicia oshlack ( ao ) is the head of the bioinformatics research group at the murdoch childrens research institute . she is an expert in developing analysis methods for high - throughput genetic technologies and works on many collaborative projects throughout the institute . john l rinn ( jr ) is an assistant professor of stem cell and regenerative biology at harvard university and medical school , and senior associate member of the broad institute . his research aims to understand the role of long intergenic non - coding rnas in establishing the distinct epigenetic states of adult and embryonic cells and their misregulation in diseases such as cancer . her laboratory uses a variety of experimental and computational systems biology approaches to map and characterize gene regulatory networks and to understand how regulatory circuitry controls animal development , function and homeostasis . ultimately , her aim is to understand how dysfunctional networks affect or cause diseases such as diabetes , obesity and cancer .

as we come to the end of 2011 , genome biology has asked some members of our editorial board for their views on the state of play in genomics . what was their favorite paper of 2011 ? what are the challenges in their particular research area ? who has had the biggest influence on their careers ? what advice would they give to young researchers embarking on a career in research ?

What in your opinion was the most important paper published in your field in the past year? Who has had the most influence in your career so far? What advice would you give to young scientists starting off in a research career today, or what advice would you give to your younger self? What in your opinion are the top three challenges in your field right now (and what progress is being made to address them)? If money were no object, what study would you love to perform? About the contributors

in terms of genome sequencing , i 'm very impressed by some of the new de novo assembly algorithms , such as li et al . , and i think our contribution of sequencing mouse genomes , keane et al . , is also important and will be of great use to the mouse genetics community . bb : i would say the foldit paper by david baker 's group because it shows a new model of research is possible . however , by designing tools that can break such problems into puzzles that people , even non - experts , can play with in their spare time can provide cutting - edge solutions . the first is the discovery and analysis of chromothripsis by peter campbell at the sanger institute . the other cancer genetic study is the work of inder verma , rusty gage and colleagues at the salk institute demonstrating a new function for brca1 involved in heterochromatin chromatin formation and repressing satellite expression via the ubiquitylation of histone h2a . this adds to the numerous and recent evidence of the importance of chromatin - modifying processes in cancer . ch : one important recent paper is the work on the vaginal microbiome in reproductive - age women by jacques ravel and larry forney . this is a very well - structured and well - analyzed study that includes a variety of important findings . it establishes that diversity in the human microbiome is personalized and much more than ' noise ' , and that following individual subjects longitudinally can be extremely informative . it shows that the microbiota can be linked both to host phenotype and to host environment , but that neither of these alone is the whole story . finally , all of these results together suggest ways by which personalized medicine can be influenced by the microbiome . predicting targets from transcription factor chip - seq has been a challenging problem , and perhaps mediator and cohesin binding could offer some insights into which bindings are functional with transcriptional outcomes . it demonstrates the power of correlating unpublished genomics and epigenomics data with publicly available data to make interesting discoveries . cm : a recent paper that i think demonstrates an important point about mapping insights from functional genomic data in model organisms is mcgary et al . the key observation is that sets of functionally related genes are often conserved as functional modules across species , but that their corresponding phenotypes in the two species might have no obvious connection . thus , after constructing a phenolog map pairing phenotypes across species , the authors show that one can accurately predict new genes related to phenotypes in higher eukaryotes based on functional genomic data from more genetically tractable model organisms . for example , they identify a phenolog connecting yeast sensitivity to the drug lovastatin to abnormal angiogenesis in mice . this is an important paper because it provides a model for how we can leverage the wealth of data produced in model organisms that are more genetically tractable to gain insights about higher organisms and disease . the method itself was very simple but based on a powerful idea , and is one that will work in a number of different contexts . ao : a lot of my recent work has been on the analysis of rna - seq data . jr : two recent papers have shown that a long non - coding rna termed coldair plays a key role in regulating plant flower formation . what 's remarkable is that this rna ' senses ' temperature and indicates to the plant when it is warm again and time to flower . it also highlights the importance of reading primary plant literature ; discoveries in plant science are often a decade ahead of the curve ! numerous functional annotations of genes were obtained by delineating more than 100 distinct and highly detailed gonadal phenotypes . da : i have worked with many wonderful colleagues . i 've fed off the contagious enthusiasm of jos jonkers and jonathan flint from amsterdam and oxford , respectively . both of them are outstanding scientists and fabulous collaborators who never seem to tire and always make me do better science . at the sanger institute , no experiment is too difficult or demanding for louise , and what she does n't know about the study of cancer genes in mice is not worth knowing ! in terms of scientific content , i give a great deal of credit and respect to dirk gevers at the broad institute , who 's been a phenomenal collaborator on many of the projects my group has tackled since starting my own lab . the first is my phd mentor , jun liu , who showed me the fun of methodology development in computational biology , which convinced me to adopt an academic career . jun has continued to help me over the years in research projects and career decisions , and taught me how to be a good mentor . last but most importantly , i had a great collaborator , myles brown , now at dana - farber cancer institute . both of us are passionate about new genomic technologies and gene regulation questions , and it is really fun working together . i have learned so much from myles and his postdocs , and some of our postdocs continue to collaborate with each other after they become independent . he is also a role model for balancing work and life , and has given me much good career and life advice . olga introduced me to the exciting genomics revolution that was well underway and was willing to take a chance on me , even though i knew very little biology and had no previous experience in computational biology or bioinformatics . in her mentoring , olga emphasized establishing a solid foundation in computer science and statistics but also developed our skills to identify relevant and impactful biological questions , one of the biggest challenges of transitioning from a computer scientist to a ' computational biologist ' in my opinion . it 's very hard to single out people but i will just focus on two . i did my phd in astrophysics and when i was considering leaving the field she fully supported that decision . since then she continues to both share her experiences and give me advice on many issues that are important to a research career , such as leading a research group , conducting collaborations , managing my time , employing people , and effectively combining a family and a successful career . the second person who has had a major influence on my career is professor gordon smyth . he was the one who first gave me a postdoc position in bioinformatics even though i had no specific background in the area . his work was an infectious balance of scientific vigor and rigor , analytical yet creative interpretation with pioneering vision . his humble upbringing , ability to admit when he was wrong , and persistence for what was right has continued to inspire me . da : science needs to be your passion and you really need to love it and jump in with both feet . i 'm not talking about turning up and doing a few experiments between coffee breaks but making your science one of the most important things you do . i do n't know of any successful scientist who periodically does n't find themselves spending virtually every waking hour in the lab or in their office . surrounding yourself with people who are smarter than you also helps you raise your game . also , computational biologists should try to become familiar with wet lab work in their chosen area of research . lastly , make the algorithms you develop available through tools that others can easily use . i have seen too many people frustrated due to communication issues with their mentor or colleagues . also , be curious of other people 's work . when going to a couple meetings a year , make sure you talk to the senior investigators whose work you admire , and do not hesitate to tell them so . ch : stereotypes are often true , both the good ones and the bad ones . but on the flip side , you really can ' do anything ' , both in terms of choosing exciting scientific directions and in shaping your group and your environment . sl : in computational biology , you need to appreciate the biology as much as , if not more than , the statistical method or computer science . cm : my advice to young computational biologists is that the key to success in our field is to remain grounded in specific biology questions , particularly ones that are pursued in collaboration with experimentalists . the success of our field is ultimately measured by the impact we can have on our understanding of biology , and how we spend our time should reflect that . in my opinion , too many computational biology researchers are working in isolation on marginally relevant problems or making incremental improvements in areas that have already been well - populated by methods that are already adequate . ao : i think one of the most important skills in research is the ability to communicate ideas . my advice would be to spend time practicing both writing skills and oral presentation skills . there is no point making great discoveries if no one knows about them or uses your discoveries . what may seem obvious to you may not be obvious to other people that are potentially interested in your work . my way of doing this , at least initially , was to look at papers that i really like reading and work out why i think they are good and try to emulate the style in my own work . with regard to oral presentations , i always practice them out loud several times and try to get feedback from colleagues on which parts worked and which were n't clear or interesting . if trained as an experimentalist , i would learn the key principles in computational biology or at the very least the linguistics , and vice versa . da : in cancer genetics the biggest challenge is integrating data from genome sequencing , transcriptomes and the epigenome , so that it makes sense . some of the boolean logic approaches are making inroads in this area . in mouse genetics the tal nucleases , which can be used to tailor the mouse genome with base - pair precision , potentially represent a big advance in this area , but we need to understand if they have off - target activity . in genome sequencing , read length really matters for assembly and while the last few years of short - read sequencing have been amazing we really need long - read technology that is truly scalable and accurate ( and cheap ) . the past two decades have seen an exponential increase in genomic and biomedical data , which will soon outstrip advances in computing power to perform current methods of analysis . back in the 1990s , . the size of genomic databases is going up by a factor of 10 every year , far outstripping the growth in our computational capacity . it 's tempting to think that cloud computing is going to solve this problem , but that 's not the case . it would really help if we had even a limited set of proteins for which gold - standard orthology information was available . genome sequencing is a great example of where such integration has yielded great success . far too often , enough biological details are abstracted away so that the solution loses its biological relevance . oh : access to adequate clinical research samples in cancer genetics is one of the most important challenges in cancer genetics . investigators involved in clinical and translational projects are in some way victims of the hype created by the fantastic and recent technological advances . i frequently talk to clinicians who are enthusiastic about sequencing their samples , but many projects often fall short due to the ignorance of the requirement of sample number or quality . for example , there is no good rationale to sequence the whole genome of thousands of samples except to make it a general resource for the community . the hype of the sequencing field is a wonderful catalyzer of novel ideas and provides much needed public exposure of our field , but we have to regularly educate prospective collaborators on basic notions of genetics or the reality of the sample preparation or data analysis . at our institution , my function in the university of california , san diego , clinical and translational research institute ( a clinical and translational science awards funded entity ) is to do just that : consult with people and help them with the design , preparation and analysis of their translational genomic experiments . traditional institutions expect faculty to lead independent projects typically funded through the r01 nih grants . this was the case for the human genome project and the hapmap project , and today the 1000 genome consortium and the cancer genome atlas , for example . this ' big science ' is usually financed through alternative sources of funding requiring collaborations and multiple principle investigators , and the results do not always lead to first or last author publications for the majority of the participants despite their essential roles . ch : ( i ) understanding the systems - level ecological rules governing microbial community structure , ( ii ) relating the human microbiome to health and disease , and ( iii ) streamlining methods for turning next - generation data into actionable biology . addressing the combination of the first two challenges will help us realize some of the human microbiome 's potential as a means of diagnosis and therapeutic intervention . investigating the first challenge in particular should let us leverage systems biology 's successes in molecular biology during studies of microbial communities . the second will likewise feed back into the broader metagenomics community by identifying ' interesting ' microbiome properties , environments and phenotypes on which to focus . approaches such as hic and chia - pet can identify genome - wide higher - order chromatin interactions , which have the potential to answer this question , although there are still technical and cost challenges for these techniques to be widely adopted . the laboratories of peggy farnham and brad bernstein , and many other laboratories , have explored this issue . as sequencing technologies increase throughput , multiplex chip - seq would allow us to investigate many more conditions in combination and we might have a better answer for this question . cm : in my specific area of interest , genetic interaction networks , there are a few challenges we face as a community . ( i ) scalable technology for mapping genetic interactions for other phenotypes , conditions and organisms , especially higher eukaryotes . the yeast community has been very successful in the past several years at developing technology for rapid construction of combinatorial mutants to map genetic interactions . specifically , the typical approach is to look for combinations of mutations that result in a surprising phenotype ( usually fitness defect ) given the phenotypes of the mutations introduced independently . such maps in higher eukaryotes will be important for understanding the genetic basis for complex phenotypes and disease and developing new therapeutic approaches , but the technology for mapping these interactions is still relatively limited in throughput . several exciting efforts are underway to address this challenge , most of them leveraging rna interference technology . however , we would ultimately like to leverage this knowledge about how genetic variations combine to influence phenotype to understand the link between genotypic and phenotypic variation across individuals in a population . applying insights derived from large - scale perturbation studies to the population genomics questions will be an interesting direction , especially as the mapping technologies become more feasible in higher eukaryotes . there are new opportunities and the necessary data to make progress on this front in yeast with the recent sequencing and phenotyping of several saccharomyces cerevisiae strains . the combination of this information on genomic and phenotypic variation , combined with extensive functional studies on the reference genome , will provide a good testing ground for new methods in this area . , a more general challenge is the problem of leveraging functional genomic data across species to speed the process of functional characterization . even the most basic question in systems biology , ' what are all of the genetic components related to biological process x ? ' provides a nice demonstration of how insights from relatively data - rich model systems can be used to direct experimental investigation of genes related to specific phenotypes in more complex organisms , and i suspect similar approaches can be developed in other settings . ao : getting a handle on the propensity and type of rna editing that is occurring is a fascinating area which , as yet , has not been fully resolved . high - throughput sequencing technologies give us the opportunity to study rna editing on a genome - wide scale and there have been several publications recently on this topic . in my view , results are probably influenced by biases in mapping procedures ( see joe pickrell 's blog and the recent paper by schrider et al . ) and it will be fascinating to see how this debate gets resolved in the near future and what the results mean for the diversity of the transcriptome . one of the major scientific challenges right now is the integration of different types of data to explain a biological phenomenon . making sense of even just a small fraction of these data sets is extremely challenging and will require major breakthroughs in analysis and interpretation . there are over 100 epigentic histone modifications known to date and more are being discovered all the time . there is still a long way to go in order to try and integrate epigenetic and expression data on a genome - wide scale . we recently got a glimpse of some of the first emerging global properties after mapping and characterizing 8,000 long non - coding rnas . they are strikingly more tissue - specific than protein - coding genes , an interesting feature that we could potentially use in medical diagnostics . it 's clear that there are numerous functional large non - coding rnas but almost the entire genome is transcribed . progress is being made by more global loss - of - function and gain - of - function experiments . we have identified an emerging theme of non - coding rna interacting with proteins and modulating their function . mw : ( i ) single cell systems biology : many labs are making good progress toward this , from cell biological imaging screens , to measuring parameters in single cells . ( ii ) dynamic networks : currently , many available networks collapse all measured interactions into a single graph . however , it is clear that only parts of the network are active in different cells or under particular conditions . to better capture the events important to a biological process , it will be important to combine all relevant , active networks into a single graph da : if money were no object , i 'd sequence all the athletes in the olympic village and all the dogs at crufts ! ch : there are two directions in which i 'd like to go with such a grant , one computational and the other translational . the former is a bit prosaic , in that the field has now completed enough human microbiome projects to have a good idea of what data and metadata are useful . epidemiology in such cohorts has been uniquely successful in characterizing dietary and environmental influences on health ; the microbiome is a big component of our everyday environment that 's so far not been assessed in such a manner . in addition to improving the technology on all of these fronts , i would invest significant resources in applying them systematically and globally on a set of carefully chosen reference cell lines . i think the only way we will understand how the genotype determines phenotype - the basis of human disease - is through systematic functional genomics . however , i would probably spend most money on employing and training people to develop methods to analyze and visualize the data in new and imaginative ways and to collaborate on exciting projects . however , i believe the biggest bottleneck is the bioinformatics and the shortage of researchers in the field social insects fascinate me , and i have a hunch that maternal and/or paternal rna storage might be involved in establishing which larva becomes a worker or a queen . were no object , i would measure all metabolites in a system of interest ( caenorhabditis elegans in my case ) , and repeat this upon perturbation of gene regulatory networks by transcription factor rna interference and under a variety of environmental and nutritional conditions . the data would then be used to create comprehensive and integrative network models that link metabolism to gene expression . bonnie berger ( bb ) is professor of applied math and computer science at massachusetts institute of technology . she works on a diverse set of problems , including network inference , protein folding , comparative genomics and medical genomics . his research is concerned with the discovery of useful biological knowledge in large collections of genomic data . x shirley liu ( sl ) is an associate professor at the department of biostatistics and computational biology at the dana - farber cancer institute and harvard school of public health . a computational biologist , with expertise on the integrative modeling of transcription and epigenetic regulation , she and her colleagues have developed a number of widely used algorithms for transcription factor motif discovery . ultimately , her aim is to understand how dysfunctional networks affect or cause diseases such as diabetes , obesity and cancer .

rapid palatal expansion ( rpe ) has been widely used in the field of orthodontics since the mid-1960s for increasing the transverse dimensions of the maxilla in growing patients.1 rapid palatal expansion enables the separation of the midpalatal suture , which is followed by skeletal orthopedic expansion.2 surgically assisted rpe ( sarpe ) is a treatment modality that helps overcome increased resistance from the bony palate and zygomatic buttress in adults.34 however , sarpe has several limitations , including high cost , a complex treatment process , and surgical morbidity,5 and most patients are reluctant to undergo this surgical procedure . therefore , several efforts have been made to minimize the surgical risks and limitations of rpe . previous histological studies have shown that the midpalatal suture begins to obliterate during the juvenile stage , with a marked degree of closure observed in the third decade of life.67 therefore , conventional rpe can produce unwanted effects in adults , such as expansion failure , alveolar bone dehiscence , buccal crown tipping , root resorption , reduction in buccal bone thickness , and marginal bone loss.8 to minimize these side effects , orthopedic expansion of the basal bone is essential in non - growing patients.910 nonsurgical maxillary expansion can be achieved through conventional , bone - anchored , or combination - type rpe . bone - anchored devices have been reported to successfully expand the maxilla after lateral osteotomy . 411 to ensure expansion of the basal bone without surgical intervention and maintain the separated bone in consolidation , lee et al.12 introduced miniscrew - assisted rpe ( marpe ) and reported successful expansion of the maxilla through opening of the midpalatal suture . . however , the movements of each tooth and its alveolus are barely identifiable on conventional two - dimensional ( 2d ) radiographs such as lateral or posteroanterior ( pa ) cephalograms . cone - beam computed tomography ( cbct ) allows imaging at relatively low radiation dosages and presents a clear view of bony structures , with minimal image distortion.13 skeletal and dentoalveolar changes after conventional tooth - borne and tooth - bone - borne rpe have been investigated using cbct in growing patients.141516 in contrast , there is limited information regarding nonsurgical expansion using bone - borne techniques such as marpe in young adults . therefore , cbct can be used to accurately assess not only the changes in each tooth and its alveolus , but also quantitative three - dimensional ( 3d ) changes in the maxillofacial complex after marpe.17 the aim of the present study was to evaluate the following null hypothesis : short - term skeletal and dentoalveolar measurements obtained before and after marpe in young adults do not differ significantly . to test the hypothesis , cbct data acquired before and after marpe were compared . the cbct records of 19 patients with maxillary constriction who had undergone maxillary expansion by marpe between january 2012 and october 2013 and had a complete set of cbct images acquired before ( t1 ) and after ( t2 ) expansion were retrieved from the archives of the department of orthodontics , yonsei university dental hospital . maxillary constriction was diagnosed at maxillomandibular transverse differential index values > 19.6 mm.18 of the 19 patients , 5 were excluded on the basis of the following exclusion criteria : failure of opening of the midpalatal suture ( n = 3 ) , systemic diseases , craniofacial anomalies ( n = 1 ) , and history of orthodontic treatment ( n = 1 ) . finally , 14 patients ( male , 9 ; female , 5 ) with a mean age of 20.1 2.4 years ( range , 1626 years ) were retrospectively enrolled in the study . the mean duration of expansion was 27 days ( range , 1835 days ) , and the mean amount of expansion was 6.7 mm ( range , 4.58.8 mm ) . the second set of cbct images were acquired within 5 weeks ( mean duration , 10.7 days ; range , 135 days ) of completion of expansion . the mean duration between t1 and t2 was 38 days ( range , 2466 days ) . this study was approved by the institutional review board of yonsei university dental hospital ( no . 2 - 2015 - 0017 ) . because of the retrospective nature of the study , the institutional review board waived the requirement for written informed consent from patients . the marpe device was fabricated by modifying the conventional hyrax - type rpe device.12 four rigid connectors , composed of 0.8-mm stainless steel wire with helical hooks , were soldered onto the base of the conventional hyrax screw body ( hyrax click ; dentaurum , ispringen , germany ) . two anterior hooks were positioned in the rugae area , and two posterior hooks were positioned in the para - midsagittal area . following cementation of the appliance to the maxillary first premolars and molars , four orthodontic miniscrews ( orlus ; ortholution , seoul , korea ) , with a collar diameter of 1.8 mm and length of 7 mm , were placed at the center of each helical hook ( figure 1).12 maxillary expansion was initiated on the day after marpe device placement . the appliance was activated at a rate of one turn per day ( 0.2 mm per turn ) until the required expansion was achieved . the cbct device ( alphard vega ; asahi roentgen ind , kyoto , japan ) was set at 5.0 ma and 80 kv , and images were acquired for 17 seconds , with a voxel size of 0.3 mm . during image acquisition , the patients were seated upright , with the frankfort horizontal plane parallel to the floor and the patient 's head stabilized by an ear rod . patients were asked to open their mouth slightly during image acquisition to prevent overlapping of the maxillary and mandibular teeth . the images were imported as digital imaging and communications in medicine ( dicom ) files using invivo5 software ( anatomage , san jose , ca , usa ) and reoriented with the palatal plane parallel to the floor in the sagittal and coronal planes . interpremolar ( ipmw ) and intermolar ( imw ) widths , defined as the distances between the right and left buccal / mesiobuccal cusp tips of the first premolars and first molars , respectively , were measured on 3d tooth images ( figure 2 ) . for measurement of transverse dimensions , bilateral landmarks ( z , n , j , ma , c6 , and ag ) were identified in these images , and the distances between right and left corresponding landmarks were measured ( figure 3 ) . three - dimensional skull images acquired at t1 and t2 were superimposed using a point - to - point and volumetric registration method with specifically normalized mutual data based on the anterior cranial base ( figure 4).19 upon volumetric registration , the t1 and t2 images shared the same coordinate system , which compensated for discrepancies and minimized the risk of measurement errors . on each image , six pairs of bony landmarks were identified on the basis of a previous report,19 following which , each landmark was coordinated . displacements in the maxilla were analyzed along the x , y , and z axes by calculating the deviation of each landmark between t1 and t2 . the distances between right and left corresponding landmarks were measured , and the differences between the measurements in t1 and t2 were calculated . two coronal scans were obtained perpendicular to the midsagittal plane , passing through the buccal / mesiobuccal cusp tips and furcations of the maxillary first premolars and molars . for the maxillary first premolars with a single root , coronal scans were obtained perpendicular to the midsagittal plane , passing through the buccal and palatal cusp tips . for measurement of nasal cavity and basal bone widths , the anterior - most slice showing the entire palatal roots of the maxillary right first premolars and molars was selected ( figure 5 ) . on each image , the following parameters were measured : nasal cavity width , defined as the transverse width between the lateral - most points of each nasal cavity ; basal bone width , defined as the transverse width between the right and left intersection points of the maxillary lateral border and a line passing through the nasal floor ; interdental angle , defined as an angle between the right and left tooth axes determined by connecting the central fossa and palatal root apex ; buccal bone thickness , defined as the distance from the buccal root surface to the outer border of the alveolar bone , along a horizontal line passing through the furcation ; and buccal alveolar height , defined as the distance from the buccal / mesiobuccal cusp tip to the buccal alveolar crest . buccal bone thickness and alveolar height were measured on the right and left sides , and the mean values of the two measurements were used for statistical analyses . comparison of skeletal and dentoalveolar measurements before and after marpe was performed using paired t - tests or wilcoxon signed - rank tests , according to the normality of data distribution . all statistical analyses were performed using the spss version 15.0 software ( spss inc . , the resultant intraclass correlation coefficient ( icc ) indicated high reliability ( icc > 0.90 ) . the cbct records of 19 patients with maxillary constriction who had undergone maxillary expansion by marpe between january 2012 and october 2013 and had a complete set of cbct images acquired before ( t1 ) and after ( t2 ) expansion were retrieved from the archives of the department of orthodontics , yonsei university dental hospital . maxillary constriction was diagnosed at maxillomandibular transverse differential index values > 19.6 mm.18 of the 19 patients , 5 were excluded on the basis of the following exclusion criteria : failure of opening of the midpalatal suture ( n = 3 ) , systemic diseases , craniofacial anomalies ( n = 1 ) , and history of orthodontic treatment ( n = 1 ) . finally , 14 patients ( male , 9 ; female , 5 ) with a mean age of 20.1 2.4 years ( range , 1626 years ) were retrospectively enrolled in the study . the mean duration of expansion was 27 days ( range , 1835 days ) , and the mean amount of expansion was 6.7 mm ( range , 4.58.8 mm ) . the second set of cbct images were acquired within 5 weeks ( mean duration , 10.7 days ; range , 135 days ) of completion of expansion . the mean duration between t1 and t2 was 38 days ( range , 2466 days ) . this study was approved by the institutional review board of yonsei university dental hospital ( no . 2 - 2015 - 0017 ) . because of the retrospective nature of the study , the institutional review board waived the requirement for written informed consent from patients . the marpe device was fabricated by modifying the conventional hyrax - type rpe device.12 four rigid connectors , composed of 0.8-mm stainless steel wire with helical hooks , were soldered onto the base of the conventional hyrax screw body ( hyrax click ; dentaurum , ispringen , germany ) . two anterior hooks were positioned in the rugae area , and two posterior hooks were positioned in the para - midsagittal area . following cementation of the appliance to the maxillary first premolars and molars , four orthodontic miniscrews ( orlus ; ortholution , seoul , korea ) , with a collar diameter of 1.8 mm and length of 7 mm , were placed at the center of each helical hook ( figure 1).12 maxillary expansion was initiated on the day after marpe device placement . the appliance was activated at a rate of one turn per day ( 0.2 mm per turn ) until the required expansion was achieved . the cbct device ( alphard vega ; asahi roentgen ind , kyoto , japan ) was set at 5.0 ma and 80 kv , and images were acquired for 17 seconds , with a voxel size of 0.3 mm . during image acquisition , the patients were seated upright , with the frankfort horizontal plane parallel to the floor and the patient 's head stabilized by an ear rod . patients were asked to open their mouth slightly during image acquisition to prevent overlapping of the maxillary and mandibular teeth . the images were imported as digital imaging and communications in medicine ( dicom ) files using invivo5 software ( anatomage , san jose , ca , usa ) and reoriented with the palatal plane parallel to the floor in the sagittal and coronal planes . interpremolar ( ipmw ) and intermolar ( imw ) widths , defined as the distances between the right and left buccal / mesiobuccal cusp tips of the first premolars and first molars , respectively , were measured on 3d tooth images ( figure 2 ) . for measurement of transverse dimensions , bilateral landmarks ( z , n , j , ma , c6 , and ag ) were identified in these images , and the distances between right and left corresponding landmarks were measured ( figure 3 ) . three - dimensional skull images acquired at t1 and t2 were superimposed using a point - to - point and volumetric registration method with specifically normalized mutual data based on the anterior cranial base ( figure 4).19 upon volumetric registration , the t1 and t2 images shared the same coordinate system , which compensated for discrepancies and minimized the risk of measurement errors . on each image , six pairs of bony landmarks were identified on the basis of a previous report,19 following which , each landmark was coordinated . displacements in the maxilla were analyzed along the x , y , and z axes by calculating the deviation of each landmark between t1 and t2 . the distances between right and left corresponding landmarks were measured , and the differences between the measurements in t1 and t2 were calculated . two coronal scans were obtained perpendicular to the midsagittal plane , passing through the buccal / mesiobuccal cusp tips and furcations of the maxillary first premolars and molars . for the maxillary first premolars with a single root , coronal scans were obtained perpendicular to the midsagittal plane , passing through the buccal and palatal cusp tips . for measurement of nasal cavity and basal bone widths , the anterior - most slice showing the entire palatal roots of the maxillary right first premolars and molars was selected ( figure 5 ) . on each image , the following parameters were measured : nasal cavity width , defined as the transverse width between the lateral - most points of each nasal cavity ; basal bone width , defined as the transverse width between the right and left intersection points of the maxillary lateral border and a line passing through the nasal floor ; interdental angle , defined as an angle between the right and left tooth axes determined by connecting the central fossa and palatal root apex ; buccal bone thickness , defined as the distance from the buccal root surface to the outer border of the alveolar bone , along a horizontal line passing through the furcation ; and buccal alveolar height , defined as the distance from the buccal / mesiobuccal cusp tip to the buccal alveolar crest . buccal bone thickness and alveolar height were measured on the right and left sides , and the mean values of the two measurements were used for statistical analyses . comparison of skeletal and dentoalveolar measurements before and after marpe was performed using paired t - tests or wilcoxon signed - rank tests , according to the normality of data distribution . all statistical analyses were performed using the spss version 15.0 software ( spss inc . , the resultant intraclass correlation coefficient ( icc ) indicated high reliability ( icc > 0.90 ) . the ipmw and imw had increased by 5.5 and 5.4 mm , respectively , after marpe ( p < 0.001 ; table 2 ) . the midpalatal suture was separated , and the maxilla exhibited statistically significant lateral movement ( p < 0.05 ) . the zygomatic arch and nasal cavity were widened by 0.8 and 1.4 mm , respectively , and the lateral contour of the maxillary alveolus exhibited an expansion of 2.03.2 mm ( p < 0.001 ) . the amount of expansion decreased with the superior positioning of anatomical structures , indicating a pyramidal pattern of maxillary expansion . according to the findings of superimposition of the 3d skull models , all of the evaluated landmarks exhibited significant lateral movement ( p < 0.01 ; table 3 ) . the alare and ectocanine had shifted forward ( p < 0.01 ) , while the prosthion and ectomolare had shifted upward ( p < 0.05 ) . the distances between all of the right and left corresponding landmarks had increased after expansion ( p < 0.01 ; table 4 ) . evaluation of coronal images of the first premolars and first molars demonstrated an increase in the nasal cavity width and basal bone width , buccal tipping of the maxillary first molars , and a decrease in the buccal bone thickness and alveolar bone height ( p < 0.01 ; table 5 ) . the increase in width of the basal bone was greater compared to that of the nasal cavity , which further confirmed the pyramidal pattern of maxillary expansion . in the present study , we evaluated skeletal and dentoalveolar changes after marpe in young adults with transverse maxillary discrepancy using cbct . marpe effectively achieved dentoalveolar as well as skeletal expansion by separation of the midpalatal suture . the increase in imw width ( 5.4 mm ) accounted for 37.0% of the skeletal expansion at the j - point ( 2.0 mm ) , 22.2% of the alveolar expansion at the cementoenamel junction ( 1.2 mm ) , and 40.7% of the dental expansion at the cusp tip ( 2.2 mm ) . despite the decrease in thickness and height of the buccal alveolus and the buccal tipping of the maxillary first molar , the changes observed after marpe in the present study were similar to those observed after conventional rpe and can be considered clinically insignificant.82021 two - dimensional pa cephalograms and periapical or occlusal radiographs are considered adequate for ensuring the opening of the midpalatal suture . apart from the increased radiation dose involved in cbct , metal artifacts , voxel size , and superimposition errors may affect the spatial resolution of cbct images , resulting in measurement errors . nevertheless , the accuracy of cbct has been well documented,131722 and several reports on the assessment of conventional rpe outcomes using computed tomography or cbct in growing patients have been published.141516 in the present study , we evaluated cbct data , the accuracy of which has been verified,1317 to analyze the 3d movements of anatomical landmarks as well as the changes in the buccal alveolar bone , which is not possible with 2d modalities . the bone - borne nature of the marpe device can result in skeletal and alveolar expansion in young adults despite the increased resistance of the midpalatal and circumaxillary sutures from the age of 1416 years.623 among the 19 patients treated by marpe in the present study , only 3 exhibited failure of opening of the midpalatal suture and were excluded , resulting in a success rate of 84.2% . skeletal expansion observed in the present study included the expansion of the zygomatic arch as well as nasal cavity . while the zygomatic arch expanded by less than 1 mm , the expansion of the nasal cavity was more evident and would have resulted in increased air flow and improved nasal breathing.224 thus , in the coronal plane , maxillary expansion followed a pyramidal pattern , similar to the expansion pattern reported with conventional rpe or sarpe.82021 in the axial plane , however , the expansion did not demonstrate a pyramidal pattern , as previously reported.215 the amount of expansion was similar between the anterior and posterior regions at the crown ( ipmw and imw ) and alveolar crest ( prosthion , ectocanine , and ectomolare ) levels . however , nasal cavity width in the premolar region had increased more obviously compared to that in the molar region , which may be attributed to the variations in nasal concha . in similar age groups treated with sarpe , while parallel expansion of the maxilla was observed upon the release of pterygoid plates,11 the amount of anterior expansion was found to be greater than the amount of posterior expansion when only the pterygoid plates were not released.425 in the sagittal and vertical directions , marpe did not result in clinically significant changes . on the 3d skull images , some of the landmarks exhibited forward or upward movements of less than 1 mm . these movements might have resulted from changes in not only circumaxillary sutures , but also the spheno - occipital synchondrosis and orbital structures.26 the activation force of an rpe device initially results in the compression of the periodontal ligament , bending of the alveolar bone , and tipping of the anchored teeth.2 therefore , a 124-increase in molar inclination is inevitable , probably because of alveolar bending and/or tipping of the posterior teeth.27 previous studies regarding rpe and sarpe reported buccal tipping of 4.956.9 of the maxillary first molar.1528 these values are similar to those observed in the present study . the degree of buccal tipping of the first molar was greater compared to that of the first premolar . however , the first premolar exhibited similar or slightly greater increases in imw , ipmw , nasal cavity , and basal bone widths than the first molar . garib et al.21 also reported greater changes in the degree of molar inclination than that of premolar inclination . the higher density of the buccal cortical bone in the maxillary canine and premolar regions29 might have resulted in the greater buccal inclination of the first molar in comparison with that of the first premolar . tipping movements might cause changes in the alveolar bone.1030 in the present study , we observed decreases in buccal plate thickness ( 0.61.1 mm ) and buccal alveolar crest height ( 1.72.2 mm ) after marpe . however , the buccal alveolar height might have been underestimated because of the tipping movements . in previous studies , rpe and sarpe were found to induce a similar decrease in buccal bone plate thickness.2021 in one of the previous studies,20 the teeth appeared to have moved through the alveolus , resulting in decreased buccal bone thickness and increased lingual bone thickness . tipping movements , which indicate greater lateral movement at the cervical level than at the apical level , along with the decrease in the alveolar bone thickness , might lead to the decrease in buccal alveolar crest height , which can eventually result in gingival recession . in children , the decrease in buccal alveolar crest height at the maxillary first molar after conventional rpe was reported to be approximately 1.23 mm,31 which is comparable to the corresponding value observed in the present study . despite the lack of evidence regarding alveolar bone loss and subsequent gingival recession due to rpe,32 the amount of marginal bone loss after rpe might be an indicator of future gingival recession , which calls for attention from the attending clinician . the amount of appliance expansion ( 6.7 mm ) was greater than the increase in the ipmw / imw ( 5.5/5.4 mm ) . this could indicate errors in counting of the number of appliance turns , because the amount of expansion was calculated on the basis of patient reports . alternatively , it could indicate deformation of the appliance . because of the controversies regarding nonsurgical expansion in adults,23 we were unable to address the effects of conventional rpe in patients with maxillary constriction . additionally , because of ethical considerations , we could not include a control group of untreated patients in our study . our study sample comprised only 14 young adults , which places emphasis on the necessity of further prospective studies involving larger numbers of patients and long - term evaluation of stability and periodontal adaptation after marpe . the null hypothesis was rejected . within the limitations of this study , our results suggest the following . marpe can be an effective treatment modality for the correction of maxillary transverse deficiency in young adults through separation of the midpalatal suture.maxillary expansion achieved with marpe exhibits a pyramidal pattern . in the present study , the degrees of skeletal , alveolar , and dental expansion were 37.0% , 22.2% , and 40.7% , respectively.buccal tipping of maxillary teeth upon marpe leads to the decrease in buccal alveolar bone thickness and crest height . marpe can be an effective treatment modality for the correction of maxillary transverse deficiency in young adults through separation of the midpalatal suture . the degrees of skeletal , alveolar , and dental expansion were 37.0% , 22.2% , and 40.7% , respectively . buccal tipping of maxillary teeth upon marpe leads to the decrease in buccal alveolar bone thickness and crest height .

objectivethe aim of this study was to evaluate the skeletal and dentoalveolar changes after miniscrew - assisted rapid palatal expansion ( marpe ) in young adults by cone - beam computed tomography ( cbct).methodsthis retrospective study included 14 patients ( mean age , 20.1 years ; range , 1626 years ) with maxillary transverse deficiency treated with marpe . skeletal and dentoalveolar changes were evaluated using cbct images acquired before and after expansion . statistical analyses were performed using paired t - test or wilcoxon signed - rank test according to normality of the data.resultsthe midpalatal suture was separated , and the maxilla exhibited statistically significant lateral movement ( p < 0.05 ) after marpe . some of the landmarks had shifted forwards or upwards by a clinically irrelevant distance of less than 1 mm . the amount of expansion decreased in the superior direction , with values of 5.5 , 3.2 , 2.0 , and 0.8 mm at the crown , cementoenamel junction , maxillary basal bone , and zygomatic arch levels , respectively ( p < 0.05 ) . the buccal bone thickness and height of the alveolar crest had decreased by 0.61.1 mm and 1.72.2 mm , respectively , with the premolars and molars exhibiting buccal tipping of 1.12.9.conclusionsour results indicate that marpe is an effective method for the correction of maxillary transverse deficiency without surgery in young adults .

INTRODUCTION MATERIALS AND METHODS Subjects Measurements Statistical analysis RESULTS DISCUSSION CONCLUSION

rapid palatal expansion ( rpe ) has been widely used in the field of orthodontics since the mid-1960s for increasing the transverse dimensions of the maxilla in growing patients.1 rapid palatal expansion enables the separation of the midpalatal suture , which is followed by skeletal orthopedic expansion.2 surgically assisted rpe ( sarpe ) is a treatment modality that helps overcome increased resistance from the bony palate and zygomatic buttress in adults.34 however , sarpe has several limitations , including high cost , a complex treatment process , and surgical morbidity,5 and most patients are reluctant to undergo this surgical procedure . previous histological studies have shown that the midpalatal suture begins to obliterate during the juvenile stage , with a marked degree of closure observed in the third decade of life.67 therefore , conventional rpe can produce unwanted effects in adults , such as expansion failure , alveolar bone dehiscence , buccal crown tipping , root resorption , reduction in buccal bone thickness , and marginal bone loss.8 to minimize these side effects , orthopedic expansion of the basal bone is essential in non - growing patients.910 nonsurgical maxillary expansion can be achieved through conventional , bone - anchored , or combination - type rpe . 411 to ensure expansion of the basal bone without surgical intervention and maintain the separated bone in consolidation , lee et al.12 introduced miniscrew - assisted rpe ( marpe ) and reported successful expansion of the maxilla through opening of the midpalatal suture . cone - beam computed tomography ( cbct ) allows imaging at relatively low radiation dosages and presents a clear view of bony structures , with minimal image distortion.13 skeletal and dentoalveolar changes after conventional tooth - borne and tooth - bone - borne rpe have been investigated using cbct in growing patients.141516 in contrast , there is limited information regarding nonsurgical expansion using bone - borne techniques such as marpe in young adults . therefore , cbct can be used to accurately assess not only the changes in each tooth and its alveolus , but also quantitative three - dimensional ( 3d ) changes in the maxillofacial complex after marpe.17 the aim of the present study was to evaluate the following null hypothesis : short - term skeletal and dentoalveolar measurements obtained before and after marpe in young adults do not differ significantly . to test the hypothesis , cbct data acquired before and after marpe were compared . the cbct records of 19 patients with maxillary constriction who had undergone maxillary expansion by marpe between january 2012 and october 2013 and had a complete set of cbct images acquired before ( t1 ) and after ( t2 ) expansion were retrieved from the archives of the department of orthodontics , yonsei university dental hospital . maxillary constriction was diagnosed at maxillomandibular transverse differential index values > 19.6 mm.18 of the 19 patients , 5 were excluded on the basis of the following exclusion criteria : failure of opening of the midpalatal suture ( n = 3 ) , systemic diseases , craniofacial anomalies ( n = 1 ) , and history of orthodontic treatment ( n = 1 ) . finally , 14 patients ( male , 9 ; female , 5 ) with a mean age of 20.1 2.4 years ( range , 1626 years ) were retrospectively enrolled in the study . the mean duration of expansion was 27 days ( range , 1835 days ) , and the mean amount of expansion was 6.7 mm ( range , 4.58.8 mm ) . the second set of cbct images were acquired within 5 weeks ( mean duration , 10.7 days ; range , 135 days ) of completion of expansion . two anterior hooks were positioned in the rugae area , and two posterior hooks were positioned in the para - midsagittal area . following cementation of the appliance to the maxillary first premolars and molars , four orthodontic miniscrews ( orlus ; ortholution , seoul , korea ) , with a collar diameter of 1.8 mm and length of 7 mm , were placed at the center of each helical hook ( figure 1).12 maxillary expansion was initiated on the day after marpe device placement . during image acquisition , the patients were seated upright , with the frankfort horizontal plane parallel to the floor and the patient 's head stabilized by an ear rod . interpremolar ( ipmw ) and intermolar ( imw ) widths , defined as the distances between the right and left buccal / mesiobuccal cusp tips of the first premolars and first molars , respectively , were measured on 3d tooth images ( figure 2 ) . for measurement of transverse dimensions , bilateral landmarks ( z , n , j , ma , c6 , and ag ) were identified in these images , and the distances between right and left corresponding landmarks were measured ( figure 3 ) . displacements in the maxilla were analyzed along the x , y , and z axes by calculating the deviation of each landmark between t1 and t2 . the distances between right and left corresponding landmarks were measured , and the differences between the measurements in t1 and t2 were calculated . two coronal scans were obtained perpendicular to the midsagittal plane , passing through the buccal / mesiobuccal cusp tips and furcations of the maxillary first premolars and molars . for measurement of nasal cavity and basal bone widths , the anterior - most slice showing the entire palatal roots of the maxillary right first premolars and molars was selected ( figure 5 ) . on each image , the following parameters were measured : nasal cavity width , defined as the transverse width between the lateral - most points of each nasal cavity ; basal bone width , defined as the transverse width between the right and left intersection points of the maxillary lateral border and a line passing through the nasal floor ; interdental angle , defined as an angle between the right and left tooth axes determined by connecting the central fossa and palatal root apex ; buccal bone thickness , defined as the distance from the buccal root surface to the outer border of the alveolar bone , along a horizontal line passing through the furcation ; and buccal alveolar height , defined as the distance from the buccal / mesiobuccal cusp tip to the buccal alveolar crest . buccal bone thickness and alveolar height were measured on the right and left sides , and the mean values of the two measurements were used for statistical analyses . comparison of skeletal and dentoalveolar measurements before and after marpe was performed using paired t - tests or wilcoxon signed - rank tests , according to the normality of data distribution . all statistical analyses were performed using the spss version 15.0 software ( spss inc . the cbct records of 19 patients with maxillary constriction who had undergone maxillary expansion by marpe between january 2012 and october 2013 and had a complete set of cbct images acquired before ( t1 ) and after ( t2 ) expansion were retrieved from the archives of the department of orthodontics , yonsei university dental hospital . maxillary constriction was diagnosed at maxillomandibular transverse differential index values > 19.6 mm.18 of the 19 patients , 5 were excluded on the basis of the following exclusion criteria : failure of opening of the midpalatal suture ( n = 3 ) , systemic diseases , craniofacial anomalies ( n = 1 ) , and history of orthodontic treatment ( n = 1 ) . finally , 14 patients ( male , 9 ; female , 5 ) with a mean age of 20.1 2.4 years ( range , 1626 years ) were retrospectively enrolled in the study . the mean duration of expansion was 27 days ( range , 1835 days ) , and the mean amount of expansion was 6.7 mm ( range , 4.58.8 mm ) . the second set of cbct images were acquired within 5 weeks ( mean duration , 10.7 days ; range , 135 days ) of completion of expansion . this study was approved by the institutional review board of yonsei university dental hospital ( no . following cementation of the appliance to the maxillary first premolars and molars , four orthodontic miniscrews ( orlus ; ortholution , seoul , korea ) , with a collar diameter of 1.8 mm and length of 7 mm , were placed at the center of each helical hook ( figure 1).12 maxillary expansion was initiated on the day after marpe device placement . during image acquisition , the patients were seated upright , with the frankfort horizontal plane parallel to the floor and the patient 's head stabilized by an ear rod . the images were imported as digital imaging and communications in medicine ( dicom ) files using invivo5 software ( anatomage , san jose , ca , usa ) and reoriented with the palatal plane parallel to the floor in the sagittal and coronal planes . interpremolar ( ipmw ) and intermolar ( imw ) widths , defined as the distances between the right and left buccal / mesiobuccal cusp tips of the first premolars and first molars , respectively , were measured on 3d tooth images ( figure 2 ) . displacements in the maxilla were analyzed along the x , y , and z axes by calculating the deviation of each landmark between t1 and t2 . the distances between right and left corresponding landmarks were measured , and the differences between the measurements in t1 and t2 were calculated . two coronal scans were obtained perpendicular to the midsagittal plane , passing through the buccal / mesiobuccal cusp tips and furcations of the maxillary first premolars and molars . for measurement of nasal cavity and basal bone widths , the anterior - most slice showing the entire palatal roots of the maxillary right first premolars and molars was selected ( figure 5 ) . on each image , the following parameters were measured : nasal cavity width , defined as the transverse width between the lateral - most points of each nasal cavity ; basal bone width , defined as the transverse width between the right and left intersection points of the maxillary lateral border and a line passing through the nasal floor ; interdental angle , defined as an angle between the right and left tooth axes determined by connecting the central fossa and palatal root apex ; buccal bone thickness , defined as the distance from the buccal root surface to the outer border of the alveolar bone , along a horizontal line passing through the furcation ; and buccal alveolar height , defined as the distance from the buccal / mesiobuccal cusp tip to the buccal alveolar crest . buccal bone thickness and alveolar height were measured on the right and left sides , and the mean values of the two measurements were used for statistical analyses . comparison of skeletal and dentoalveolar measurements before and after marpe was performed using paired t - tests or wilcoxon signed - rank tests , according to the normality of data distribution . all statistical analyses were performed using the spss version 15.0 software ( spss inc . the ipmw and imw had increased by 5.5 and 5.4 mm , respectively , after marpe ( p < 0.001 ; table 2 ) . the midpalatal suture was separated , and the maxilla exhibited statistically significant lateral movement ( p < 0.05 ) . the zygomatic arch and nasal cavity were widened by 0.8 and 1.4 mm , respectively , and the lateral contour of the maxillary alveolus exhibited an expansion of 2.03.2 mm ( p < 0.001 ) . the amount of expansion decreased with the superior positioning of anatomical structures , indicating a pyramidal pattern of maxillary expansion . according to the findings of superimposition of the 3d skull models , all of the evaluated landmarks exhibited significant lateral movement ( p < 0.01 ; table 3 ) . the alare and ectocanine had shifted forward ( p < 0.01 ) , while the prosthion and ectomolare had shifted upward ( p < 0.05 ) . the distances between all of the right and left corresponding landmarks had increased after expansion ( p < 0.01 ; table 4 ) . evaluation of coronal images of the first premolars and first molars demonstrated an increase in the nasal cavity width and basal bone width , buccal tipping of the maxillary first molars , and a decrease in the buccal bone thickness and alveolar bone height ( p < 0.01 ; table 5 ) . the increase in width of the basal bone was greater compared to that of the nasal cavity , which further confirmed the pyramidal pattern of maxillary expansion . in the present study , we evaluated skeletal and dentoalveolar changes after marpe in young adults with transverse maxillary discrepancy using cbct . the increase in imw width ( 5.4 mm ) accounted for 37.0% of the skeletal expansion at the j - point ( 2.0 mm ) , 22.2% of the alveolar expansion at the cementoenamel junction ( 1.2 mm ) , and 40.7% of the dental expansion at the cusp tip ( 2.2 mm ) . despite the decrease in thickness and height of the buccal alveolus and the buccal tipping of the maxillary first molar , the changes observed after marpe in the present study were similar to those observed after conventional rpe and can be considered clinically insignificant.82021 two - dimensional pa cephalograms and periapical or occlusal radiographs are considered adequate for ensuring the opening of the midpalatal suture . nevertheless , the accuracy of cbct has been well documented,131722 and several reports on the assessment of conventional rpe outcomes using computed tomography or cbct in growing patients have been published.141516 in the present study , we evaluated cbct data , the accuracy of which has been verified,1317 to analyze the 3d movements of anatomical landmarks as well as the changes in the buccal alveolar bone , which is not possible with 2d modalities . the bone - borne nature of the marpe device can result in skeletal and alveolar expansion in young adults despite the increased resistance of the midpalatal and circumaxillary sutures from the age of 1416 years.623 among the 19 patients treated by marpe in the present study , only 3 exhibited failure of opening of the midpalatal suture and were excluded , resulting in a success rate of 84.2% . skeletal expansion observed in the present study included the expansion of the zygomatic arch as well as nasal cavity . while the zygomatic arch expanded by less than 1 mm , the expansion of the nasal cavity was more evident and would have resulted in increased air flow and improved nasal breathing.224 thus , in the coronal plane , maxillary expansion followed a pyramidal pattern , similar to the expansion pattern reported with conventional rpe or sarpe.82021 in the axial plane , however , the expansion did not demonstrate a pyramidal pattern , as previously reported.215 the amount of expansion was similar between the anterior and posterior regions at the crown ( ipmw and imw ) and alveolar crest ( prosthion , ectocanine , and ectomolare ) levels . in similar age groups treated with sarpe , while parallel expansion of the maxilla was observed upon the release of pterygoid plates,11 the amount of anterior expansion was found to be greater than the amount of posterior expansion when only the pterygoid plates were not released.425 in the sagittal and vertical directions , marpe did not result in clinically significant changes . on the 3d skull images , some of the landmarks exhibited forward or upward movements of less than 1 mm . these movements might have resulted from changes in not only circumaxillary sutures , but also the spheno - occipital synchondrosis and orbital structures.26 the activation force of an rpe device initially results in the compression of the periodontal ligament , bending of the alveolar bone , and tipping of the anchored teeth.2 therefore , a 124-increase in molar inclination is inevitable , probably because of alveolar bending and/or tipping of the posterior teeth.27 previous studies regarding rpe and sarpe reported buccal tipping of 4.956.9 of the maxillary first molar.1528 these values are similar to those observed in the present study . the degree of buccal tipping of the first molar was greater compared to that of the first premolar . the higher density of the buccal cortical bone in the maxillary canine and premolar regions29 might have resulted in the greater buccal inclination of the first molar in comparison with that of the first premolar . tipping movements might cause changes in the alveolar bone.1030 in the present study , we observed decreases in buccal plate thickness ( 0.61.1 mm ) and buccal alveolar crest height ( 1.72.2 mm ) after marpe . however , the buccal alveolar height might have been underestimated because of the tipping movements . in previous studies , rpe and sarpe were found to induce a similar decrease in buccal bone plate thickness.2021 in one of the previous studies,20 the teeth appeared to have moved through the alveolus , resulting in decreased buccal bone thickness and increased lingual bone thickness . tipping movements , which indicate greater lateral movement at the cervical level than at the apical level , along with the decrease in the alveolar bone thickness , might lead to the decrease in buccal alveolar crest height , which can eventually result in gingival recession . in children , the decrease in buccal alveolar crest height at the maxillary first molar after conventional rpe was reported to be approximately 1.23 mm,31 which is comparable to the corresponding value observed in the present study . the amount of appliance expansion ( 6.7 mm ) was greater than the increase in the ipmw / imw ( 5.5/5.4 mm ) . this could indicate errors in counting of the number of appliance turns , because the amount of expansion was calculated on the basis of patient reports . marpe can be an effective treatment modality for the correction of maxillary transverse deficiency in young adults through separation of the midpalatal suture.maxillary expansion achieved with marpe exhibits a pyramidal pattern . in the present study , the degrees of skeletal , alveolar , and dental expansion were 37.0% , 22.2% , and 40.7% , respectively.buccal tipping of maxillary teeth upon marpe leads to the decrease in buccal alveolar bone thickness and crest height . marpe can be an effective treatment modality for the correction of maxillary transverse deficiency in young adults through separation of the midpalatal suture . the degrees of skeletal , alveolar , and dental expansion were 37.0% , 22.2% , and 40.7% , respectively . buccal tipping of maxillary teeth upon marpe leads to the decrease in buccal alveolar bone thickness and crest height .

c57bl/6 is the best - known inbred mouse strain and has been used as the genetic background for spontaneous and induced mutations . to produce knockout mice , embryonic stem ( es ) cells derived from 129 mouse substrains were used initially to manipulate the mouse genome ; however , these substrains were not suitable for most biomedical studies , especially in immunology , neurobiology , and physiology [ 4 , 13 , 28 ] . backcrossing to c57bl/6 mice is carried out frequently to generate congenic strains to facilitate phenotypic analyses , but this procedure requires additional cost and time . in addition , the targeted locus from the original es cell genome remains in the congenic mice and may confound the results of studies using these animals . therefore , es cells with a pure c57bl/6 genetic background are more useful for the generation of knockout mice . importantly , es cells derived from c57bl/6n mice maintained their pluripotency after homologous recombination [ 18 , 25 ] , and the methods used to generate germline - transmitting chimeric mice have been improved . the international knockout mouse consortium ( ikmc ) conducted large - scale mutagenesis to mutate all of the protein - coding genes in mice using gene trapping and targeting in es cells [ 9 , 22 ] . since then , several mouse es cells derived from the c57bl/6ntac strain have been used as standard es cells for the production of mutant alleles [ 5 , 18 ] . moreover , the international mouse phenotyping consortium has used the ikmc - targeted c57bl/6n es cell clones to undertake the broad - based phenotyping of 20,000 mouse genes . since the 1950s , the c57bl/6 strain has diverged into several substrains , including two major groups , c57bl/6j and c57bl/6n . currently , more than 20 inbred substrains derived from c57bl/6j and c57bl/6n mice have been established and distributed worldwide [ 1 , 16 ] . the c57bl/6j strain has many specific single nucleotide polymorphisms ( snps ) that distinguish it from other inbred strains , and snps that can differentiate c57bl/6j substrains have also been identified [ 14 , 32 ] . in addition , several phenotypic differences have been reported among c57bl/6j substrains [ 8 , 20 , 23 , 24 ] . c57bl/6j - specific snp information is useful for the genetic monitoring of mouse strains with a c57bl/6j - derived background and interpretation of phenotypic data . however , genetic variation among c57bl/6n - derived substrains , including c57bl/6ntac , which was used to generate the ikmc es cells has not yet been verified . previously , c57bl/6j - specific snps detected by comparing the reference c57bl/6j sequence with other inbred mouse strains have been reported [ 7 , 17 , 26 , 27 , 29 , 31 ] ; however , c57bl/6n was not included in these snp data . recently , the wellcome trust sanger institute ( wtsi ) published whole genome resequencing data of 17 key mouse inbred strains including c57bl/6nj , which enabled us to identify c57bl/6nj - specific snps through comparisons with other inbred strains [ 10 , 30 ] . in this study , we searched for snps specific to the c57bl/6nj strain using the resequence database of the wtsi . moreover , in light of the branching history of c57bl/6n - derived substrains , we found variation in the number of accumulated c57bl/6nj - specific snps among the c57bl/6n - derived substrains , which can be used to differentiate the substrains . snp genotyping was conducted in 11 c57bl/6n and 7 c57bl/6j - derived inbred substrains available from different breeders and holders around the world ( table 1table 1.c57bl/6 substrains investigated in this studysubstrainsourcec57bl/6n substrainsc57bl/6njthe jackson laboratory ( bar harbor , ma , usa)c57bl/6ncrsimsimonsen laboratories , inc . ( hamamatsu , japan)c57bl/6bythe jackson laboratory ( bar harbor , ma , usa)c57bl/6byjthe jackson laboratory ( bar harbor , ma , usa)c57bl/6j substrainsc57bl/6jthe jackson laboratory ( bar harbor , ma , usa ) via charls river laboratories japan , inc . ( hamamatsu , japan)c57bl/6jeijthe jackson laboratory ( bar harbor , ma , usa)c57bl/6jolahsdharlan laboratories , inc . ( new york , ny , usa)nomenclatured strain names of each c57bl/6 substrain were in accordance with jax notes . ) . as for the c57bl/6nj , c57bl/6by , c57bl/6byj , and c57bl/6jeij strains , genomic dna from one animal of each strain was obtained from the jackson laboratory mouse dna resources ( stock # 005304 , # 000663 , # 001139 , and # 000924 , respectively ; bar harbor , me ) . as for the other strains , live mice or frozen tissue from two animals of the c57bl/6n - derived substrain and one animal of the c57bl/6j - derived substrain were used , respectively . genomic dna was extracted from the tail tips or kidneys using an autogen na-2000 automatic nucleic acid isolation system ( kurabo industries ltd . , osaka , japan ) and/or a dneasy blood & tissue kit ( qiagen gmbh , hilden , germany ) . all animal experiments were conducted in accordance with the regulations for animal experiments of riken ( october 1 , 2003 rule no . our experimental protocols , including those involving animals ( exp10 - 002 ) , were approved by the animal experiments committee of the riken tsukuba institute . informative snps were selected from the public snp database at the mouse genome project , wtsi ( http://www.sanger.ac.uk/sanger/mouse_snpviewer/rel-1303 ) , by comparing the sequence data of c57bl/6nj with that of the c57bl/6j reference strain . high confidence call on the database were extracted , and then candidate c57bl/6nj - specific snps were selected through a comparison with sequence data from the 12 other inbred mouse strains in the database ( 129p2/olahsd , 129s1/svimj , 129s5/svevbrd , a / j , akr / j , balb / cj , c3h / hej , cba / j , dba/2j , lp / j , nod / shiltj , and nzo / hlltj ) . to confirm whether the in silico - selected c57bl/6nj - specific candidate snps were present in mouse dna samples , the snp loci of the c57bl/6nj and c57bl/6j strains were genotyped by pcr and direct sequencing . target regions containing the candidate snps were amplified by pcr with flanking primers designed by using batchprimer3 v1.0 ( probes.pw.usda.gov/batchprimer3/index.html ) . pcr was performed using a qiagen multiplex pcr kit ( qiagen gmbh ) according to the manufacturer s protocol . the pcr products were electrophoresed and separated on an e - gel clonewell 0.8% sybr safe gel using an e - gel ibase power system ( both from life technologies , carlsbad , ca ) . sequencing reactions were performed in a dna engine and dyad ptc-220 peltier thermal cycler ( bio - rad , laboratories , inc . , hercules , ca ) using an abi bigdye terminator v3.1 cycle sequencing kit with amplitaq dna polymerase ( life technologies ) , following the protocols supplied by the manufacturers . the fluorescently labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol . the samples were resuspended in distilled water and then subjected to electrophoresis in an abi 3730 l sequencer ( life technologies ) . after the snps were confirmed in both strains , the other c57bl/6 substrains were genotyped by the same method . snp genotyping was conducted in 11 c57bl/6n and 7 c57bl/6j - derived inbred substrains available from different breeders and holders around the world ( table 1table 1.c57bl/6 substrains investigated in this studysubstrainsourcec57bl/6n substrainsc57bl/6njthe jackson laboratory ( bar harbor , ma , usa)c57bl/6ncrsimsimonsen laboratories , inc . ( hamamatsu , japan)c57bl/6bythe jackson laboratory ( bar harbor , ma , usa)c57bl/6byjthe jackson laboratory ( bar harbor , ma , usa)c57bl/6j substrainsc57bl/6jthe jackson laboratory ( bar harbor , ma , usa ) via charls river laboratories japan , inc . ( hamamatsu , japan)c57bl/6jeijthe jackson laboratory ( bar harbor , ma , usa)c57bl/6jolahsdharlan laboratories , inc . ( new york , ny , usa)nomenclatured strain names of each c57bl/6 substrain were in accordance with jax notes . ) . as for the c57bl/6nj , c57bl/6by , c57bl/6byj , and c57bl/6jeij strains , genomic dna from one animal of each strain was obtained from the jackson laboratory mouse dna resources ( stock # 005304 , # 000663 , # 001139 , and # 000924 , respectively ; bar harbor , me ) . as for the other strains , live mice or frozen tissue from two animals of the c57bl/6n - derived substrain and one animal of the c57bl/6j - derived substrain were used , respectively . genomic dna was extracted from the tail tips or kidneys using an autogen na-2000 automatic nucleic acid isolation system ( kurabo industries ltd . , osaka , japan ) and/or a dneasy blood & tissue kit ( qiagen gmbh , hilden , germany ) . all animal experiments were conducted in accordance with the regulations for animal experiments of riken ( october 1 , 2003 rule no . our experimental protocols , including those involving animals ( exp10 - 002 ) , were approved by the animal experiments committee of the riken tsukuba institute . informative snps were selected from the public snp database at the mouse genome project , wtsi ( http://www.sanger.ac.uk/sanger/mouse_snpviewer/rel-1303 ) , by comparing the sequence data of c57bl/6nj with that of the c57bl/6j reference strain . high confidence call on the database were extracted , and then candidate c57bl/6nj - specific snps were selected through a comparison with sequence data from the 12 other inbred mouse strains in the database ( 129p2/olahsd , 129s1/svimj , 129s5/svevbrd , a / j , akr / j , balb / cj , c3h / hej , cba / j , dba/2j , lp / j , nod / shiltj , and nzo / hlltj ) . to confirm whether the in silico - selected c57bl/6nj - specific candidate snps were present in mouse dna samples , the snp loci of the c57bl/6nj and c57bl/6j strains were genotyped by pcr and direct sequencing . target regions containing the candidate snps were amplified by pcr with flanking primers designed by using batchprimer3 v1.0 ( probes.pw.usda.gov/batchprimer3/index.html ) . pcr was performed using a qiagen multiplex pcr kit ( qiagen gmbh ) according to the manufacturer s protocol . the pcr products were electrophoresed and separated on an e - gel clonewell 0.8% sybr safe gel using an e - gel ibase power system ( both from life technologies , carlsbad , ca ) . sequencing reactions were performed in a dna engine and dyad ptc-220 peltier thermal cycler ( bio - rad , laboratories , inc . , hercules , ca ) using an abi bigdye terminator v3.1 cycle sequencing kit with amplitaq dna polymerase ( life technologies ) , following the protocols supplied by the manufacturers . the fluorescently labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol . the samples were resuspended in distilled water and then subjected to electrophoresis in an abi 3730 l sequencer ( life technologies ) . after the snps were confirmed in both strains , the other c57bl/6 substrains were genotyped by the same method . from the snp database , 1,361 informative snps on all of the chromosomes except y were screened , and the snps were able to distinguish c57bl/6nj from the other 12 inbred strains in silico ( table 2table 2.confirmation of c57bl/6nj - specific snps by direct sequencing of pcr productschromosomenumber of snpsselectednumber of snpssequenceddirect sequencingsucceededconfirmeddata discrepancy19191676072989867616389452825347418880564161311267148211011757169908821398196812880106114108211501188012801277013851398114541298115591188016691177017621044018671088019209541x60161073total136148631527738snp : single nucleotide polymorphism . ) . no informative snps on chromosome y were found in the database . among the informative snps , 486 candidate snps from chromosome 1x were selected to include approximately 1 snp locus per 10 mb , and the genotypes of the selected snp loci were examined by direct sequencing of c57bl/6j and c57bl/6nj dna samples . as a result , 315 snp loci were sequenced successfully and 277 snps were confirmed to be specific to c57bl/6nj . the genotypes of the remaining 38 snp loci were not consistent with the data in the snp database . according to the ensembl mouse genome server ( www.ensembl.org/mus_musculus/info/index ) , 10 of the 277 snps were nonsynonymous variant snps , such as missense or stop - gain variants , which were predicted to affect the amino acid sequence of the protein ( table 3table 3.status of 10 nonsynonymous variant snpsdbsnp idchromosomeposition ( bp)(grcm38)linked genec57bl/6jallelec57bl/6njallelevariant typeof c57bl/6nj allelers262569844589,775,351adamts3c / ct / tmissense ( val199ile)rs2297125655112,762,721myo18bc / ct / tmissense ( arg1935his)rs2435755097102,973,309olfr577c / ct / tmissense ( val228ile)rs2462742901088,091,833pmcht / tc / cmissense ( ile132thr)rs2406174011146,222,615cyfip2g / ga / amissense ( ser968phe)rs2388931571190,480,671stxbp4c / ct / tmissense ( ala535thr)rs24299160913119,477,8084833420g17rikc / ca / amissense ( thr484lys)rs2481576001470,586,204fam160b2g / gt / tmissense ( ser575arg)rs2460334091511,336,383adamts12g / gt / tmissense ( cys1518phe)rs2305964091364,921,972spata31c / ct / tstop gained ( arg645ter)information of the variant type was obtained from the ensembl mouse genome server.snp : single nucleotide polymorphism . ) . next , 100 snps for genetic monitoring of c57bl/6n - derived substrains were selected from the 277 snps to include 1 snp locus per 1040 mb to cover all of the chromosomes except y ( table 4table 4.position of the 100 selected snp loci and their flanking primer sequencelocusno.dbsnpidchromosomeposition ( bp)(grcm38)c57bl/6jallelec57bl/6njalleleflanking primer sequence ( 5 to 3)forward primerreverse primer1rs246236360111,996,705c / ct / taccccctgaaccttcaattctttcccatggaattctgctc2rs246490354114,344,561t / ta / aggggaagagtgggatgactatggtcagcatattccagtgc3rs212521754116,968,405c / ca / agcaacgaaggaaattgaagctgttgaggcatgtccctttt4rs227394849119,544,960a / at / tgggcagaacttccttttccttctcacctgagtccctggat5rs232920323121,639,642c / cg / ggaaatagcacaggtccatcaaacccagcagacaagagacaaa6rs213024334130,167,141c / ct / tggaatctcaacctaagcagcatggaaaattgaagcaaccag7rs244794780140,107,883t / ta / atctgttgctctccagcattgctacaccctggcctgacact8rs260670033150,146,448c / ct / tagcagaaatgccaaaatgcttcagacccaaaaggacatgc9rs249907793161,950,812c / ca / accagtgggttaagtgggattatcaaatggggtggcattta10rs265151779183,182,637g / ga / aattctgctaactgcggaggattgttcaccctcttccccta11rs229124202189,861,338g / gt / tggaaggcagatcaccaactctctatggtggccctaggatg12rs237656339199,547,673g / ga / atggctcctgacatctttcctgctcctggatcggcatatta13rs2235407541110,024,886g / gc / cgaccaaatgccttgaaaatgactctccccatcccttttctt14rs2596836381119,116,297c / ct / taggtcttgggctctttagggacttgctggctgactccttc15rs2299112891132,980,179t / ta / attttatttcctccgcattggactcgggaacacacaagctc16rs2156227031142,008,378c / ca / atttttgtgttggccaaggatcctttctcttcagaggggtttt17rs2390173981154,474,620c / ct / tcagatcccggctcaattttaagctcattagcctggcatgt18rs2142540721161,859,644c / ct / tgtttgctcctccccctactctgtatgaaaatgtgcaggttctg19rs2559148941172,611,934g / ga / aatgccggtgtaccttcagagccccagtaaccattctcctg20rs2223038181179,503,532g / ga / actgcccatactcctgtccatagggcctggtactgagaaca21rs2622826751188,434,376a / at / taccctttgatggttcccattaattttgctaggcccatgaat22rs251979693211,214,185c / ct / tccccacatttgcttatccaggccaattgtgaggaatgctt23rs230600693221,681,174g / gt / tggtccagcattattggcattgtgatcccatctgccatctt24rs242780245230,188,489a / ac / cccactgtcaccagcacattcccaccactcctctccgaata25rs228546410241,205,764t / ta / aatgcccacaatgcaaacatatagcccctctgactgtccac26rs254996546251,969,852c / ct / tccgtgaccaagtatgcacag gctgagggttcagttgtggt27rs256541267270,251,451c / ct / tctgaagaaaggcctgtttggcgaattcaatgctgccaata28rs248280077280,873,138c / ct / ttgtgccgattcctctagcttctgcaccaattagcagcaa29rs214356625296,674,180g / gt / tgtgtatgcccccaacctttaccagtgattgcatttcacctt30rs2243445632102,710,505a / at / tcaggacaggagagggtcaagatcccaggccataggatttt31rs2519335042112,966,408t / tc / cgctcggtctgaaaggtcaacggaagcaagagcttggaaga32rs2585082212122,708,738t / ta / aactttgtgccttttgcaaccgagggggatccaaggataag33rs2550141102132,432,999c / ct / tcagcacagatggtttcatggagatgcacaagtggctctga34rs2424139242140,793,056t / tg / gaaattttcttccccacagcagtgagccagtacaggggaga35rs2174437742152,781,403a / ag / gctcttcttcctgcccttcctagccattgagtgaggtgctt36rs2532121972164,813,748c / ct / tctgaactgcaaccctcatcaagtgtagccctccctgtcct37rs2133762332170,240,435g / ga / agctaagtggtcttgggatgcgccaccacacacagctaattt38rs2647192472180,149,012a / ag / gttctgctaggcttcctggtgctccctcacaacaggctcat39rs2215213922181,868,891c / ct / ttctttttgcctcttgttggaacgtgcttgtgagctctctga40rs25652080938,498,163g / ga / atggcagaagtttgtttcaggccatctggggctgaatactt41rs214801792332,773,111g / gc / ctgctggggtagttttccactggagggagtcaggtgcaata42rs222821429366,305,330c / ct / tcacccaacacccacagagtatgtcttttcaaagggccaga43rs243656799372,616,062g / ga / acccattggacacgaaaacttcactgctgctcattggttca44rs2628279303109,597,274a / at / tggcagtttggcctgtaggtactttactggcttgcctcacc45rs2541452193147,657,255c / ct / tcagcaggatatgcgtcctctgcttcccctcccataatttc46rs219227155419,328,298t / tc / cacacaagaactggcacatggttgggacctgtcagcctatc47rs235104023456,463,984c / ct / tagcagttggtgtgtttgctgcccccattgctttgtgtcta48rs2618792874104,973,294t / tg / ggacgagggaaaatgagtggacaaatggcatgttcgtttga49rs256724446535,701,259g / ga / aattcattcctgacccatcca cttcctcaattcccctccat50rs260260338580,026,465g / ga / atggggaaagaatgtgcctacttggtccaacatcaaactacctt51rs2172979945117,118,668g / ga / accaaggagcagccctactaacaactcctggtcaacgctct52rs2219906685150,224,989g / gt / tcttgtagaacccaggccatc gtccccacccattacatcag53rs257294810639,971,164g / ga / agcattcagctctccttcctg gagacctgggcacaatgact54rs224069095674,169,211c / ca / actcatcatgacacaaggagcacatgtgtggcccctagttct55rs375404556113,159,679g / ga / aattcctggccagccttagat tgttggtgagagtccttcca56rs2175440766144,513,005g / gc / ccacacatccatctgcctctg gcagccggagtattagcaag57rs212452109716,595,985a / at / tgagttcaatccctgggacaagtgtcactgtggctggctta58rs224103578753,390,545c / ct / tggagggaatgtgtcagtaacgcctgacctcagtgtgcagaa59rs2435755097102,973,309c / ct / ttcatcacaggagggaagaggggctatctgtcgtcctttgc60rs2293401857140,821,590a / at / tcttcaggcccttcacgagtagattcctattggctggcttg61rs263791105822,903,742g / ga / acgaatgtgcatttgtcatccgcccttccaactacctcaca62rs255341040858,790,625g / ga / aggcactgtttatcttgggaactgccaaacagcactcagaag63rs239219835879,117,401g / ga / ataaatggcccgaattcacattgtgcaccttcctttgttca64rs256624163894,046,068g / ga / atcagagcccacagaaaaaggccatgggtttcacacattca65rs2117501478118,442,679g / ga / atcgggggcttaatttctctttgcctagacctggatttggt66rs52003732910,125,248t / tc / cttctcccctctgtgagcaagttgccaccacctcaaaaact67rs214490504960,662,109g / ga / atcatcccggaacataaatggagtctcgccaatacgactgc68rs2435001469116,160,235c / ct / tcagaaggatcctggacttgc ctcttatctccccgccaga69rs511230661011,070,460g / gt / tcaaggcccctgtaaatccttcgagggcctagcatatttca70rs2194899731041,944,745g / ga / acctcggctaacttcaagcacgcctgtgcgcttactttgat71rs2135838721049,357,252g / gt / tgttgcacaggctgagaatgacccaaatgaattgcaaaggt72rs2462742901088,091,833t / tc / ccacagaacacaggctccaaagccaacatggtcggtagact73rs2238570791112,253,003a / at / tctggtttggaggtgagcatt aaaagctccggaaggtgaat74rs2406174011146,222,615g / ga / atgacccccaatcacacattagccagcttatccatctgcac75rs2316564571229,886,947t / tg / gtcttggttaaggtggcaaggattcacaaatgtcggcatca76rs2174227771270,772,479c / ct / tccgggaaaaacatacacaccaccctgctctccttgacaaa77rs2213454421297,702,669a / at / ttcctctcacccgtatctgctgcgtcttcagagaccttctca78rs2263104241341,494,375g / gc / ccatctccatggtgctcgatatccacagttcagccaaagg79rs2305964091364,921,972c / ct / tggtgttgaccatgagccttcctggggtgagcttaggtctg80rs25150721713101,112,155a / at / tccctgtaccgtccaatcatcttctccccacctctgatgtc81rs24299160913119,477,808c / ca / attttggctgtgcaattcttgcacacagaggtcgcctatca82rs2651932701439,164,780c / ct / tggccatctcatcagtgcataaggctgacatggttttgagc83rs2354286821475,727,727g / ga / aacatctccagcttccagacc gaggcggtgactatgaagga84rs22260727514117,850,332g / ga / attgtggtttcaggaatgtcgggcaaacttcttgcctcaga85rs2432458031522,748,238t / tg / gtgtttcctgagagggtttgctttgggaaagacaagggaga86rs2434005121555,816,925c / ct / ttcagaggctgaagtgacagcgggcagtctgtctgtggaag87rs2313211251597,760,563c / cg / gctctcacgaggacatgagcaggctccccagtaaaacatga88rs2302438641620,458,800c / ct / ttgggggcttatcttgttcacacttaaccacaagcccagga89rs2409488961661,450,798g / ga / atggagcatgacaaggaatcatttgcaaaatccatgattgg90rs2400679571740,854,409c / ct / ttgctcatggtaaatgctggatcagcactcaggtgatttcc91rs2591440331769,131,609c / ct / tcatgcacacggcagtagaagcagaggtggaaccaggaaga92rs2259637801822,530,101g / gt / tagcggtatgcttgctttgatacaagggccaaatattgctg93rs2146383311841,344,993t / tc / cctgccagataagccaccaattcaccaatgacagagcaaaaa94rs2557892421859,519,801c / ct / tttcccctagcttggaaaccttctttcctggagttgcccta95rs2636879611890,448,757a / ag / gttcccattgtggtcattgaatgagctaaatttggagcaagc96rs2324143571923,329,888c / ct / tcagccctcccctttatcttcgtatgcccctgttgggtcta97rs2306561701940,364,531g / ga / aagcgctcgctttgacataattgggacaggaggaggttaca98rs246037535x84,805,631c / ct / tccctagggcaacatggtaaacattccgtgcaaatgagatg99rs266019057x112,095,948a / ag / gggtggcagagatggaaacatctgtcttgcttggtcgctaa100rs212226666x157,445,480t / ta / atgcacttgcacatcctacagggggtttgggttttcattttsnp : single nucleotide polymorphism . ) , and these snps were genotyped in the other 10 c57bl/6n and 6 c57bl/6j - derived substrains . all c57bl/6n and c57bl/6j - derived substrains were homozygous for these 100 snp loci . in addition , when two samples from the same strain were genotyped in the c57bl/6n - derived substrains , they were found to be completely identical . the genotyping results for the c57bl/6j - derived substrains were consistent with the c57bl/6j reference sequence in the database . snp genotyping demonstrated variation in the number of c57bl/6nj - specific snps among the c57bl/6n - derived substrains ( table 5table 5.snps among c57bl/6 substrains ) . 10 , 13 , 14 , 22 , 30 , 42 , 44 , 45 , 54 , 58 , 63 , 68 , 84 , and 93 were shared commonly , while the remaining 86 snps were only shared partly among the 11 c57bl/6n - derived substrains . the relationship between the genealogy of the c57bl/6n - derived substrains used in this study and the number of the 100 c57bl/6nj - specific snps in the substrains is summarized in fig . 1.genealogy of c57bl/6n - derived substrains ( left ) and a bar graph for the number of c57bl/6nj - specific snps ( single nucleotide polymorphisms ) ( right ) . abbreviations in the genealogy : n : national institutes of health ; by : dr . donald bailey ; j : the jackson laboratory ; cr : national cancer institute ; jms : institute of medical science , the university of tokyo ; slc : japan slc ; crl : charles river laboratories ; crlj : charles river laboratories japan ; seac : kyudo ; hsd : harlan laboratories ; jic : central institute for experimental animals ; jcl : clea japan ; sim : simonsen laboratories ; tac : taconic farm the c57bl/6nhsd and c57bl/6ncrl strains were derived independently from the nih on unknown dates in 1974 . on the right ( bar graph ) , the number of c57bl/6nj - specific snps of each c57bl/6n - derived substrain is indicated as a solid bar . the number of snps is also indicated at the right side of each bar .. milestones in the establishment of the strains were obtained from the product catalogs of the breeders and previous reports [ 14 , 16 ] . the c57bl/6 strain was separated from the c57bl parental strain at the end of the 1940s and introduced to the jackson laboratory . a few years later , c57bl/6 mice were sent to the national institutes of health ( nih ; bethesda , md ) from the jackson laboratory , and the c57bl/6n mice were separated into the other c57bl/6n substrains , including the c57bl/6by strain , at different times . c57bl/6ncrsim mice from simonsen laboratories ( gilroy , ca ) were derived from c57bl/6n mice at the nih in 1995 . c57bl/6ntac mice from taconic farms ( new york , ny ) were derived from the nih animal genetic resource at f151 in 1991 . c57bl/6njcl mice were introduced to the central institute for experimental animals ( kawasaki , japan ) from the nih at f121 in 1978 , and then transferred to clea japan ( tokyo , japan ) at f146 in 1988 . charles river laboratories ( wilmington , ma ) obtained c57bl/6n mice from the nih in 1974 . the c57bl/6ncrl mice were further transferred to charles river laboratories japan ( yokohama , japan ) at f101 in 1976 , and since then , the mice have been distributed as c57bl/6ncrlcrlj . c57bl/6nseac mice were introduced to kyudo ( tosu , japan ) from the charles river laboratory japan in 1981 . c57bl/6ncrslc mice were introduced to the institute of medical science , the university of tokyo ( tokyo , japan ) in 1972 by mr . samuel m. poiey , and then the mice were transferred to japan slc ( hamamatsu , japan ) in 1975 . genealogy of c57bl/6n - derived substrains ( left ) and a bar graph for the number of c57bl/6nj - specific snps ( single nucleotide polymorphisms ) ( right ) . abbreviations in the genealogy : n : national institutes of health ; by : dr . donald bailey ; j : the jackson laboratory ; cr : national cancer institute ; jms : institute of medical science , the university of tokyo ; slc : japan slc ; crl : charles river laboratories ; crlj : charles river laboratories japan ; seac : kyudo ; hsd : harlan laboratories ; jic : central institute for experimental animals ; jcl : clea japan ; sim : simonsen laboratories ; tac : taconic farm . doted line : the year of introduction was unknown . the c57bl/6nhsd and c57bl/6ncrl strains were derived independently from the nih on unknown dates in 1974 . on the right ( bar graph ) , the number of c57bl/6nj - specific snps of each c57bl/6n - derived substrain is indicated as a solid bar . the number of snps is also indicated at the right side of each bar . the number of c57bl/6nj - specific snps in each c57bl/6n - derived substrain was well correlated with its branching date from the original c57bl/6n strain ( fig . 1 ) . each c57bl/6n - derived substrain , except c57bl/6ntac and c57bl/6ncrsim , could only share c57bl/6nj - specific snps that had arisen before its branching from the original c57bl/6n strain . the oldest c57bl/6by and c57bl/6byj strains may well preserve the genotype of c57bl/6n in approximately 1961 and only share 14 snps with other later substrains . as for the c57bl/6ntac and c57bl/6ncrsim strains , they only shared c57bl/6nj - specific snps that had arisen before the c57bl/6nj mice branched from the original c57bl/6n strain in 1984 . eleven snps that were detected only in the c57bl/6nj strain were assumed to have arisen in c57bl/6nj after it branched from the c57bl/6n strain in 1984 . in other words , we could determine retrospectively when the c57bl/6nj - specific snps had arisen by comparing the branching history and accumulated c57bl/6nj - specific snps in different c57bl/6n - derived substrains . similar observations have been reported previously in c57bl/6j - derived substrains [ 14 , 32 ] . the c57bl/6ncrlcrlj and c57bl/6nseac substrains that branched directly from c57bl/6ncrl and c57bl/6ncrlcrlj , respectively , but not directly from the original c57bl/6n , had only 70 c57bl/6nj - specific snps , which is less than that of c57bl/6ncrl ( fig . this corresponds to the number of discontinuous distribution patterns of snps seen in table 5 . one possible explanation for the discontinuous distribution of snps is that the 9 snp loci , nos . 20 , 32 , 38 , 46 , 48 , 52 , 66 , 70 , and 72 ( table 5 ) , were heterozygous just before the branching of the c57bl/6nhsd and c57bl/6ncrl strains in 1974 . then , the snps were independently fixed to either c57bl/6nj type or c57bl/6j type in the subsequent inbreeding of each substrain . finally , 6 snp loci ( nos . 20 , 38 , 46 , 48 , 52 and 66 ) of c57bl/6nhsd and 8 snp loci ( nos . 32 , 38 , 46 , 48 , 52 , 66 , 70 and 72 ) of c57bl/6ncrl were thought to be fixed to c57bl/6nj type , while the 9 snp loci of c57bl/6ncrlcrlj and c57bl/6nseac were fixed to c57bl/6j type . although there was no significant difference in the pattern of the 100 selected snps between c57bl/6by and c57bl/6byj , and c57bl/6ntac and c57bl/6ncrsim , informative snps that can be used to discriminate these substrains can be found by genotyping additional candidate snps in the future . in the selected 277 snps , 10 nonsynonymous snps were identified that were predicted to affect the amino acid sequence of the protein ( table 3 ) . according to the ensembl mouse genome server , 4 of these 10 snp variants , which occurred in the myo18b , olfr577 , cyfip2 , and adamts12 genes , were predicted to affect adversely each protein s function . notably , the ser968phe variant of cyfip2 in c57bl/6n mice was reported recently by kumar et al . ; this mutation destabilizes cyfip2 protein and leads to acute and sensitized cocaine - response phenotypes . although the c57bl/6n - derived substrains are closely related strains , such genetic differences would undoubtedly affect the phenotypes in various studies and interact with targeted mutations . other phenotypic differences in behavior have been also reported among several c57bl/6n - derived substrains [ 3 , 12 ] . as for the c57bl/6j and c57bl/6n strains , a relationship between genome variation and phenotypic changes has also been demonstrated . in the future , advanced phenotypic analyses among the c57bl/6n - derived substrains will reveal novel functions of the genome variations detected among these substrains . all of the snps detected in this report , including several nonsynonymous snps , will also be instrumental in the accurate identification of the c57bl/6n - derived substrain status of mutant mice and avoid the incorrect interpretation of data due to background effects . furthermore , the selected snp markers will be useful for quickly producing congenic strains to move a targeted mutation from one c57bl/6n - derived substrain to another substrain . hence , our findings will serve as useful markers for the accurate and sophisticated genetic monitoring of c57bl/6n - derived congenic strains .

c57bl/6n inbred mice are used as the genetic background for producing knockout mice in large - scale projects worldwide ; however , the genetic divergence among c57bl/6n - derived substrains has not been verified . here , we identified novel single nucleotide polymorphisms ( snps ) specific to the c57bl/6nj strain and selected useful snps for the genetic monitoring of c57bl/6n - derived substrains . informative snps were selected from the public snp database at the wellcome trust sanger institute by comparing sequence data from c57bl/6nj and c57bl/6j mice . a total of 1,361 candidate snps from the snp database could distinguish the c57bl/6nj strain from 12 other inbred strains . we confirmed 277 c57bl/6nj - specific snps including 10 nonsynonymous snps by direct sequencing , and selected 100 useful snps that cover all of the chromosomes except y. genotyping of 11 c57bl/6n - derived substrains at these 100 snp loci demonstrated genetic differences among the substrains . this information will be useful for accurate genetic monitoring of mouse strains with a c57bl/6n - derived background .

Introduction Materials and Methods Animals In silico selection of informative SNPs for C57BL/6NJ Experimental confirmation of SNPs and genotyping Results Discussion

from the snp database , 1,361 informative snps on all of the chromosomes except y were screened , and the snps were able to distinguish c57bl/6nj from the other 12 inbred strains in silico ( table 2table 2.confirmation of c57bl/6nj - specific snps by direct sequencing of pcr productschromosomenumber of snpsselectednumber of snpssequenceddirect sequencingsucceededconfirmeddata discrepancy19191676072989867616389452825347418880564161311267148211011757169908821398196812880106114108211501188012801277013851398114541298115591188016691177017621044018671088019209541x60161073total136148631527738snp : single nucleotide polymorphism . ) next , 100 snps for genetic monitoring of c57bl/6n - derived substrains were selected from the 277 snps to include 1 snp locus per 1040 mb to cover all of the chromosomes except y ( table 4table 4.position of the 100 selected snp loci and their flanking primer sequencelocusno.dbsnpidchromosomeposition ( bp)(grcm38)c57bl/6jallelec57bl/6njalleleflanking primer sequence ( 5 to 3)forward primerreverse primer1rs246236360111,996,705c / ct / taccccctgaaccttcaattctttcccatggaattctgctc2rs246490354114,344,561t / ta / aggggaagagtgggatgactatggtcagcatattccagtgc3rs212521754116,968,405c / ca / agcaacgaaggaaattgaagctgttgaggcatgtccctttt4rs227394849119,544,960a / at / tgggcagaacttccttttccttctcacctgagtccctggat5rs232920323121,639,642c / cg / ggaaatagcacaggtccatcaaacccagcagacaagagacaaa6rs213024334130,167,141c / ct / tggaatctcaacctaagcagcatggaaaattgaagcaaccag7rs244794780140,107,883t / ta / atctgttgctctccagcattgctacaccctggcctgacact8rs260670033150,146,448c / ct / tagcagaaatgccaaaatgcttcagacccaaaaggacatgc9rs249907793161,950,812c / ca / accagtgggttaagtgggattatcaaatggggtggcattta10rs265151779183,182,637g / ga / aattctgctaactgcggaggattgttcaccctcttccccta11rs229124202189,861,338g / gt / tggaaggcagatcaccaactctctatggtggccctaggatg12rs237656339199,547,673g / ga / atggctcctgacatctttcctgctcctggatcggcatatta13rs2235407541110,024,886g / gc / cgaccaaatgccttgaaaatgactctccccatcccttttctt14rs2596836381119,116,297c / ct / taggtcttgggctctttagggacttgctggctgactccttc15rs2299112891132,980,179t / ta / attttatttcctccgcattggactcgggaacacacaagctc16rs2156227031142,008,378c / ca / atttttgtgttggccaaggatcctttctcttcagaggggtttt17rs2390173981154,474,620c / ct / tcagatcccggctcaattttaagctcattagcctggcatgt18rs2142540721161,859,644c / ct / tgtttgctcctccccctactctgtatgaaaatgtgcaggttctg19rs2559148941172,611,934g / ga / aatgccggtgtaccttcagagccccagtaaccattctcctg20rs2223038181179,503,532g / ga / actgcccatactcctgtccatagggcctggtactgagaaca21rs2622826751188,434,376a / at / taccctttgatggttcccattaattttgctaggcccatgaat22rs251979693211,214,185c / ct / tccccacatttgcttatccaggccaattgtgaggaatgctt23rs230600693221,681,174g / gt / tggtccagcattattggcattgtgatcccatctgccatctt24rs242780245230,188,489a / ac / cccactgtcaccagcacattcccaccactcctctccgaata25rs228546410241,205,764t / ta / aatgcccacaatgcaaacatatagcccctctgactgtccac26rs254996546251,969,852c / ct / tccgtgaccaagtatgcacag gctgagggttcagttgtggt27rs256541267270,251,451c / ct / tctgaagaaaggcctgtttggcgaattcaatgctgccaata28rs248280077280,873,138c / ct / ttgtgccgattcctctagcttctgcaccaattagcagcaa29rs214356625296,674,180g / gt / tgtgtatgcccccaacctttaccagtgattgcatttcacctt30rs2243445632102,710,505a / at / tcaggacaggagagggtcaagatcccaggccataggatttt31rs2519335042112,966,408t / tc / cgctcggtctgaaaggtcaacggaagcaagagcttggaaga32rs2585082212122,708,738t / ta / aactttgtgccttttgcaaccgagggggatccaaggataag33rs2550141102132,432,999c / ct / tcagcacagatggtttcatggagatgcacaagtggctctga34rs2424139242140,793,056t / tg / gaaattttcttccccacagcagtgagccagtacaggggaga35rs2174437742152,781,403a / ag / gctcttcttcctgcccttcctagccattgagtgaggtgctt36rs2532121972164,813,748c / ct / tctgaactgcaaccctcatcaagtgtagccctccctgtcct37rs2133762332170,240,435g / ga / agctaagtggtcttgggatgcgccaccacacacagctaattt38rs2647192472180,149,012a / ag / gttctgctaggcttcctggtgctccctcacaacaggctcat39rs2215213922181,868,891c / ct / ttctttttgcctcttgttggaacgtgcttgtgagctctctga40rs25652080938,498,163g / ga / atggcagaagtttgtttcaggccatctggggctgaatactt41rs214801792332,773,111g / gc / ctgctggggtagttttccactggagggagtcaggtgcaata42rs222821429366,305,330c / ct / tcacccaacacccacagagtatgtcttttcaaagggccaga43rs243656799372,616,062g / ga / acccattggacacgaaaacttcactgctgctcattggttca44rs2628279303109,597,274a / at / tggcagtttggcctgtaggtactttactggcttgcctcacc45rs2541452193147,657,255c / ct / tcagcaggatatgcgtcctctgcttcccctcccataatttc46rs219227155419,328,298t / tc / cacacaagaactggcacatggttgggacctgtcagcctatc47rs235104023456,463,984c / ct / tagcagttggtgtgtttgctgcccccattgctttgtgtcta48rs2618792874104,973,294t / tg / ggacgagggaaaatgagtggacaaatggcatgttcgtttga49rs256724446535,701,259g / ga / aattcattcctgacccatcca cttcctcaattcccctccat50rs260260338580,026,465g / ga / atggggaaagaatgtgcctacttggtccaacatcaaactacctt51rs2172979945117,118,668g / ga / accaaggagcagccctactaacaactcctggtcaacgctct52rs2219906685150,224,989g / gt / tcttgtagaacccaggccatc gtccccacccattacatcag53rs257294810639,971,164g / ga / agcattcagctctccttcctg gagacctgggcacaatgact54rs224069095674,169,211c / ca / actcatcatgacacaaggagcacatgtgtggcccctagttct55rs375404556113,159,679g / ga / aattcctggccagccttagat tgttggtgagagtccttcca56rs2175440766144,513,005g / gc / ccacacatccatctgcctctg gcagccggagtattagcaag57rs212452109716,595,985a / at / tgagttcaatccctgggacaagtgtcactgtggctggctta58rs224103578753,390,545c / ct / tggagggaatgtgtcagtaacgcctgacctcagtgtgcagaa59rs2435755097102,973,309c / ct / ttcatcacaggagggaagaggggctatctgtcgtcctttgc60rs2293401857140,821,590a / at / tcttcaggcccttcacgagtagattcctattggctggcttg61rs263791105822,903,742g / ga / acgaatgtgcatttgtcatccgcccttccaactacctcaca62rs255341040858,790,625g / ga / aggcactgtttatcttgggaactgccaaacagcactcagaag63rs239219835879,117,401g / ga / ataaatggcccgaattcacattgtgcaccttcctttgttca64rs256624163894,046,068g / ga / atcagagcccacagaaaaaggccatgggtttcacacattca65rs2117501478118,442,679g / ga / atcgggggcttaatttctctttgcctagacctggatttggt66rs52003732910,125,248t / tc / cttctcccctctgtgagcaagttgccaccacctcaaaaact67rs214490504960,662,109g / ga / atcatcccggaacataaatggagtctcgccaatacgactgc68rs2435001469116,160,235c / ct / tcagaaggatcctggacttgc ctcttatctccccgccaga69rs511230661011,070,460g / gt / tcaaggcccctgtaaatccttcgagggcctagcatatttca70rs2194899731041,944,745g / ga / acctcggctaacttcaagcacgcctgtgcgcttactttgat71rs2135838721049,357,252g / gt / tgttgcacaggctgagaatgacccaaatgaattgcaaaggt72rs2462742901088,091,833t / tc / ccacagaacacaggctccaaagccaacatggtcggtagact73rs2238570791112,253,003a / at / tctggtttggaggtgagcatt aaaagctccggaaggtgaat74rs2406174011146,222,615g / ga / atgacccccaatcacacattagccagcttatccatctgcac75rs2316564571229,886,947t / tg / gtcttggttaaggtggcaaggattcacaaatgtcggcatca76rs2174227771270,772,479c / ct / tccgggaaaaacatacacaccaccctgctctccttgacaaa77rs2213454421297,702,669a / at / ttcctctcacccgtatctgctgcgtcttcagagaccttctca78rs2263104241341,494,375g / gc / ccatctccatggtgctcgatatccacagttcagccaaagg79rs2305964091364,921,972c / ct / tggtgttgaccatgagccttcctggggtgagcttaggtctg80rs25150721713101,112,155a / at / tccctgtaccgtccaatcatcttctccccacctctgatgtc81rs24299160913119,477,808c / ca / attttggctgtgcaattcttgcacacagaggtcgcctatca82rs2651932701439,164,780c / ct / tggccatctcatcagtgcataaggctgacatggttttgagc83rs2354286821475,727,727g / ga / aacatctccagcttccagacc gaggcggtgactatgaagga84rs22260727514117,850,332g / ga / attgtggtttcaggaatgtcgggcaaacttcttgcctcaga85rs2432458031522,748,238t / tg / gtgtttcctgagagggtttgctttgggaaagacaagggaga86rs2434005121555,816,925c / ct / ttcagaggctgaagtgacagcgggcagtctgtctgtggaag87rs2313211251597,760,563c / cg / gctctcacgaggacatgagcaggctccccagtaaaacatga88rs2302438641620,458,800c / ct / ttgggggcttatcttgttcacacttaaccacaagcccagga89rs2409488961661,450,798g / ga / atggagcatgacaaggaatcatttgcaaaatccatgattgg90rs2400679571740,854,409c / ct / ttgctcatggtaaatgctggatcagcactcaggtgatttcc91rs2591440331769,131,609c / ct / tcatgcacacggcagtagaagcagaggtggaaccaggaaga92rs2259637801822,530,101g / gt / tagcggtatgcttgctttgatacaagggccaaatattgctg93rs2146383311841,344,993t / tc / cctgccagataagccaccaattcaccaatgacagagcaaaaa94rs2557892421859,519,801c / ct / tttcccctagcttggaaaccttctttcctggagttgcccta95rs2636879611890,448,757a / ag / gttcccattgtggtcattgaatgagctaaatttggagcaagc96rs2324143571923,329,888c / ct / tcagccctcccctttatcttcgtatgcccctgttgggtcta97rs2306561701940,364,531g / ga / aagcgctcgctttgacataattgggacaggaggaggttaca98rs246037535x84,805,631c / ct / tccctagggcaacatggtaaacattccgtgcaaatgagatg99rs266019057x112,095,948a / ag / gggtggcagagatggaaacatctgtcttgcttggtcgctaa100rs212226666x157,445,480t / ta / atgcacttgcacatcctacagggggtttgggttttcattttsnp : single nucleotide polymorphism . )

thp-1 cells were obtained from the german collection of microorganisms and cell cultures ( dsmz ) and cultured overnight at a cell density of 0.3 10/ml at 37c and 5% co2 in roswell park memorial institute 1640 medium with glutamax ( invitrogen ) , 10% fetal bovine serum , and 50 m -mercaptoethanol . five nanomolar human recombinant sdf-1 ( r&d systems ) was preincubated with nox - a12 in various concentrations in hank 's balanced salt solution ( invitrogen ) containing 1 mg / ml bovine serum albumin ( bsa ) and 20 mm 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid ( hepes ) ( hbh ) in the lower compartments of a 96-transwell plate with 5-m pores ( costar corning , # 3388 ) at 37c for 20 min . added to the upper compartments were 1 10 thp-1 cells in hbh buffer , which were allowed to migrate at 37c for 3 h. migrated cells were quantified by resazurin ( sigma ) fluorescence . relative fluorescence intensity was normalized and plotted against nox - a12 concentrations for determination of half - maximal inhibitory concentration ( ic50 ) using graphpad prism . pathhunter express cxcr7 activated g protein coupled receptor ( gpcr ) internalization cells ( discoverx ) were incubated with 10 nm human recombinant sdf-1 ( r&d systems ) and various concentrations of nox - a12 at 37c for 3 h. cxcr7 internalization was quantified according to the instructions of the supplier , and relative luminescence intensity was normalized and plotted against nox - a12 concentrations for ic50 determination using graphpad prism software . human umbilical vein endothelial cells ( huvecs ) were obtained from lonza and cultured in endothelial cell growth medium2 ( lonza ) at 37c and 5% co2 . one day before the experiment , huvecs were seeded in a 10-cm culture plate at 40% confluency and incubated overnight . cells were washed once with phosphate buffered saline ( pbs ) and incubated with diic12(3 ) fluorescent dye ( bd biosciences ) at a concentration of 5 g / ml in endothelial cell basal medium2 ( lonza ) containing 0.5% bsa and 0.5% fetal bovine serum for 15 min at 37c . cells were washed twice with pbs , mildly trypsinized , washed , and resuspended in dulbecco 's modified eagle 's medium ( dmem ) without phenol red containing 20 mm hepes and 0.1% bsa at a concentration of 3.2 10 cells / ml . cells were incubated at 37c and 5% co2 at least 60 min before adding to the fluoroblok transwell inserts with fluorescent blocking pet membrane and 3-m pores ( bd biosciences , falcon , # 351151 ) . the lower side of the inserts was coated with 5 g / ml human fibronectin ( r&d systems ) at room temperature for 1 h , washed once with pbs , and then incubated with 1 g / ml human sdf-1 ( r&d systems ) and nox - a12 in dmem without phenol red and 20 mm hepes at room temperature for 2 h. the filters were washed once and inserted in a bd falcon 24-well companion plate containing 750 l dmem without phenol red , 20 mm hepes , and 0.1% bsa . two hundred fifty microliters of stained and equilibrated huvecs as we have described were added to the upper compartment . cell migration was quantified using a bottom fluorescence reading plate reader ( synergy2 , biotek ) and plotted against incubation time . injection of 50 mg / kg of the carcinogen enu ( sigma ) into sprague - dawley rats ( taconic farms ) at gestational day 18 . during each experiment , at least one control pregnant rat received saline vehicle under identical conditions . pups were born and weaned at normal age , after which they were monitored at least weekly for signs of illness , which usually developed after 120 days of age , at which time the pups started to show signs of brain lesions ( loss of weight , hind limb paralysis , and lethargy ) . in the first study , designed only to evaluate survival , the rats were assigned randomly to the various groups on day 115 of age , just before any rats started to die . in the second study , designed to evaluate tumor response by mri as well as survival , tumor size was estimated by mri starting on day 130 of age , and the rats were assigned to groups so as to have approximately the same total tumor volume in each group . all animal procedures were approved by stanford university 's administrative panel on laboratory animal care . whole brain irradiation was performed with a phillips x - ray unit operated at 200 kvp with a dose rate of 1.21 gy / min ( 20 ma with added filtration of 0.5 mm copper , distance from x - ray source to the target of 31 cm , and a half value layer of 1.3 mm copper ) . the rats were anesthetized and placed in individual lead boxes with a cutout that allowed the brain to be irradiated tangentially with full shielding of the buccal cavity as well as the rest of the body . to ensure the maximum uniformity of the dose delivered , the animals were turned 180 degrees halfway through each irradiation ( giving the equivalent of parallel opposed fields ) . sdf-1 spiegelmer nox - a12 ( primary sequence : 5-gcguggugugaucuagauguauuggcugauccuagucagguacgc-3 , modified with 40 kda peg at the 5-terminus and synthesized at noxxon pharma , berlin , germany ) ; adequate exposure of the drug was confirmed in a separate pharmacokinetics study ( supplemental fig . the compound was dissolved at a concentration of 10 mg / ml in sterile 5% glucose solution with occasional shaking for up to 30 min at room temperature . for the 5 mg / kg group , the frozen solution was thawed on the day of injection and diluted to 5 mg / ml , and 1 ml / kg was subcutaneously administered every 2 days for 8 weeks ; for the 20 mg / kg group , the 10 mg / ml solution was used directly and 2 ml / kg was administered for 4 or 8 weeks ; for the 10 mg / kg group , the 10 mg / kg solution was used directly and 1 ml / kg was administered for 10 weeks . in the study in which nox - a12 was combined with temozolomide ( tmz ) , the tmz ( 10 mg / kg i.p ; sigma - aldrich ) was given 5 days / week for 3 weeks . mri measurements ( t2-weighted images ) were performed on a 1 t machine ( bruker ) commencing on day 130 after birth and then repeated every 2 weeks until death . rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group . rats were securely fixed to an mri head holder ( stanford center of biomedical imaging ) , and t2-weighted coronal images of 1.5 mm thickness were obtained . images were read by a reviewer who was blinded to treatment group using an osirix computer program . each imaging slice that contained tumor was outlined freehand and the volume calculated ; the sum of all the slice volumes was considered the tumor volume for that subject . brains were removed and positioned for cutting using the same stereotactic readings from the bregma and lambda , so that they were at the precise angle in which they underwent mri and brains removed . fifty - micrometer sections were cut from 2.2 mm anterior to 7.5 mm posterior to the bregma ; all sections were stained with standard hematoxylin and eosin . using the serial imaging for verification statistical analyses were performed by the 2-tailed student 's t - test or 1-way anova to determine statistical significance . meier curves and the log - rank test were used to compare survival times among the groups . thp-1 cells were obtained from the german collection of microorganisms and cell cultures ( dsmz ) and cultured overnight at a cell density of 0.3 10/ml at 37c and 5% co2 in roswell park memorial institute 1640 medium with glutamax ( invitrogen ) , 10% fetal bovine serum , and 50 m -mercaptoethanol . five nanomolar human recombinant sdf-1 ( r&d systems ) was preincubated with nox - a12 in various concentrations in hank 's balanced salt solution ( invitrogen ) containing 1 mg / ml bovine serum albumin ( bsa ) and 20 mm 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid ( hepes ) ( hbh ) in the lower compartments of a 96-transwell plate with 5-m pores ( costar corning , # 3388 ) at 37c for 20 min . added to the upper compartments were 1 10 thp-1 cells in hbh buffer , which were allowed to migrate at 37c for 3 h. migrated cells were quantified by resazurin ( sigma ) fluorescence . relative fluorescence intensity was normalized and plotted against nox - a12 concentrations for determination of half - maximal inhibitory concentration ( ic50 ) using graphpad prism . pathhunter express cxcr7 activated g protein coupled receptor ( gpcr ) internalization cells ( discoverx ) were incubated with 10 nm human recombinant sdf-1 ( r&d systems ) and various concentrations of nox - a12 at 37c for 3 h. cxcr7 internalization was quantified according to the instructions of the supplier , and relative luminescence intensity was normalized and plotted against nox - a12 concentrations for ic50 determination using graphpad prism software . human umbilical vein endothelial cells ( huvecs ) were obtained from lonza and cultured in endothelial cell growth medium2 ( lonza ) at 37c and 5% co2 . one day before the experiment , huvecs were seeded in a 10-cm culture plate at 40% confluency and incubated overnight . cells were washed once with phosphate buffered saline ( pbs ) and incubated with diic12(3 ) fluorescent dye ( bd biosciences ) at a concentration of 5 g / ml in endothelial cell basal medium2 ( lonza ) containing 0.5% bsa and 0.5% fetal bovine serum for 15 min at 37c . cells were washed twice with pbs , mildly trypsinized , washed , and resuspended in dulbecco 's modified eagle 's medium ( dmem ) without phenol red containing 20 mm hepes and 0.1% bsa at a concentration of 3.2 10 cells / ml . cells were incubated at 37c and 5% co2 at least 60 min before adding to the fluoroblok transwell inserts with fluorescent blocking pet membrane and 3-m pores ( bd biosciences , falcon , # 351151 ) . the lower side of the inserts was coated with 5 g / ml human fibronectin ( r&d systems ) at room temperature for 1 h , washed once with pbs , and then incubated with 1 g / ml human sdf-1 ( r&d systems ) and nox - a12 in dmem without phenol red and 20 mm hepes at room temperature for 2 h. the filters were washed once and inserted in a bd falcon 24-well companion plate containing 750 l dmem without phenol red , 20 mm hepes , and 0.1% bsa . two hundred fifty microliters of stained and equilibrated huvecs as we have described were added to the upper compartment . cell migration was quantified using a bottom fluorescence reading plate reader ( synergy2 , biotek ) and plotted against incubation time . injection of 50 mg / kg of the carcinogen enu ( sigma ) into sprague - dawley rats ( taconic farms ) at gestational day 18 . during each experiment , at least one control pregnant rat received saline vehicle under identical conditions . pups were born and weaned at normal age , after which they were monitored at least weekly for signs of illness , which usually developed after 120 days of age , at which time the pups started to show signs of brain lesions ( loss of weight , hind limb paralysis , and lethargy ) . in the first study , designed only to evaluate survival , the rats were assigned randomly to the various groups on day 115 of age , just before any rats started to die . in the second study , designed to evaluate tumor response by mri as well as survival , tumor size was estimated by mri starting on day 130 of age , and the rats were assigned to groups so as to have approximately the same total tumor volume in each group . all animal procedures were approved by stanford university 's administrative panel on laboratory animal care . whole brain irradiation was performed with a phillips x - ray unit operated at 200 kvp with a dose rate of 1.21 gy / min ( 20 ma with added filtration of 0.5 mm copper , distance from x - ray source to the target of 31 cm , and a half value layer of 1.3 mm copper ) . the rats were anesthetized and placed in individual lead boxes with a cutout that allowed the brain to be irradiated tangentially with full shielding of the buccal cavity as well as the rest of the body . to ensure the maximum uniformity of the dose delivered , the animals were turned 180 degrees halfway through each irradiation ( giving the equivalent of parallel opposed fields ) . sdf-1 spiegelmer nox - a12 ( primary sequence : 5-gcguggugugaucuagauguauuggcugauccuagucagguacgc-3 , modified with 40 kda peg at the 5-terminus and synthesized at noxxon pharma , berlin , germany ) ; adequate exposure of the drug was confirmed in a separate pharmacokinetics study ( supplemental fig . the compound was dissolved at a concentration of 10 mg / ml in sterile 5% glucose solution with occasional shaking for up to 30 min at room temperature . for the 5 mg / kg group , the frozen solution was thawed on the day of injection and diluted to 5 mg / ml , and 1 ml / kg was subcutaneously administered every 2 days for 8 weeks ; for the 20 mg / kg group , the 10 mg / ml solution was used directly and 2 ml / kg was administered for 4 or 8 weeks ; for the 10 mg / kg group , the 10 mg / kg solution was used directly and 1 ml / kg was administered for 10 weeks . in the study in which nox - a12 was combined with temozolomide ( tmz ) , the tmz ( 10 mg / kg i.p ; sigma - aldrich ) was given 5 days / week for 3 weeks . mri measurements ( t2-weighted images ) were performed on a 1 t machine ( bruker ) commencing on day 130 after birth and then repeated every 2 weeks until death . rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group . rats were securely fixed to an mri head holder ( stanford center of biomedical imaging ) , and t2-weighted coronal images of 1.5 mm thickness were obtained . images were read by a reviewer who was blinded to treatment group using an osirix computer program . each imaging slice that contained tumor was outlined freehand and the volume calculated ; the sum of all the slice volumes was considered the tumor volume for that subject . brains were removed and positioned for cutting using the same stereotactic readings from the bregma and lambda , so that they were at the precise angle in which they underwent mri and brains removed . fifty - micrometer sections were cut from 2.2 mm anterior to 7.5 mm posterior to the bregma ; all sections were stained with standard hematoxylin and eosin . using the serial imaging for verification statistical analyses were performed by the 2-tailed student 's t - test or 1-way anova to determine statistical significance . meier curves and the log - rank test were used to compare survival times among the groups . thp-1 myelomonocytes ( which highly express cxcr4 but do not express cxcr7 as demonstrated by flow cytometry ) show sdf-1mediated chemotaxis . nox - a12 is able to inhibit a stimulus of 5 nm sdf-1 in a dose - dependent manner with an ic50 of 3.9 0.2 nm ( fig . pathhunter express cxcr7 activated gpcr internalization cells ( discoverx ) were used to demonstrate sdf-1dependent cxcr7 activation . nox - a12 shows inhibition of sdf-1 mediated cxcr7 internalization dose dependently with an ic50 of 3.0 0.9 nm ( fig . huvecs , which are known to express both sdf-1 receptors , cxcr4 and cxcr7 , migrate toward immobilized sdf-1 on fibronectin . 1c ) . in summary , we demonstrated that nox - a12 blocks sdf-1dependent activation of both receptors , cxcr4 and cxcr7 , with high potency . furthermore , nox - a12 inhibits sdf-1dependent chemotaxis of monocytes and endothelial cells , which both play an important role in vasculogenesis of irradiated solid tumors . thp-1 cells are attracted by 5 nm of human sdf-1 ( set to 100% ) . one representative dose - response curve ( mean sd of triplicates ) of 3 independent experiments is shown . nox - a12 inhibits sdf-1mediated chemotaxis of cxcr4-expressing thp-1 cells with an ic50 of 3.9 0.2 nm . one representative dose - response curve ( mean sd of triplicates ) of 4 independent experiments is shown . nox - a12 inhibits sdf-1mediated cxcr7 internalization with an ic50 of 3.0 0.9 nm . ( c ) nox - a12 inhibits sdf-1mediated migration of human ecs . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . sdf-1 immobilized on fibronectin increases migration of huvecs , which is completely inhibited by nox - a12 . each curve reflects the means of duplicates sd from a single experiment and is representative of 5 independent experiments . thp-1 cells are attracted by 5 nm of human sdf-1 ( set to 100% ) . one representative dose - response curve ( mean sd of triplicates ) of 3 independent experiments is shown . nox - a12 inhibits sdf-1mediated chemotaxis of cxcr4-expressing thp-1 cells with an ic50 of 3.9 0.2 nm . one representative dose - response curve ( mean sd of triplicates ) of 4 independent experiments is shown . nox - a12 inhibits sdf-1mediated cxcr7 internalization with an ic50 of 3.0 0.9 nm . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . sdf-1 immobilized on fibronectin increases migration of huvecs , which is completely inhibited by nox - a12 . each curve reflects the means of duplicates sd from a single experiment and is representative of 5 independent experiments . to ensure that the rats entering the first set of studies would have brain tumors of a size close to those producing neurological symptoms and death , we randomized rats born to mothers treated with enu ( 50 mg / kg ) on day 18 of gestation at day 115 of age . this was just before any of the rats began to die from their brain tumors ( fig 2 ) . in this study we included a group of rats given nox - a12 alone and controls given vehicle alone for 28 days . we also tested 2 different concentrations of nox - a12 and 2 different periods of drug administration after irradiation . in order to increase the power of the comparisons , we pooled the data from 2 prior experiments in which identically treated rats were either not irradiated or given 20 gy whole brain irradiation ( wbi ) . as can be seen ( fig . 2 and table 1 ) , the dose of 20 gy wbi extended the median life span of the rats by a small and not quite significant amount of approximately 7 days ( p = .07 vs non - irradiated ) . however , the addition of nox - a12 prolonged the median life span of the irradiated rats by a significant amount : by 95 days for the rats given the 8-week infusion at the low dose ( 5 mg / kg ) and by 153 days for the rats given the higher dose ( 20 mg / kg ) in comparison with the rats receiving 20 gy wbi alone . as can be seen , there was no significant effect of nox - a12 alone . we also gave 20 gy wbi with or without 8 weeks of nox - a12 to rats that had not been given enu in utero ( so did not have brain tumors ) . we saw no deaths in these rats up to the observation period of 418 days ( not shown in fig . 2 ) . the median life spans of the different groups and their significance levels are shown in table 1 . table 1.analysis of survival of enu - treated rats at day 384p valuecontrolnox-20 alone20 gy alone20 gy + nox-5 , 4 wk20 gy + nox-20 , 4 wk20 gy + nox-5 , 8 wk20 gy + nox-20 , 8 wk20 gy + no enuno enu + nox-20 , 8 wkmed . surv189173.5196244.5221291349>384>418vs 20 gy0.07ns0.10ns0.050.003<0.00010.01vs control ns0.070.050.020.0006<0.0001<0.00010.008controls and 20 gy alone were pooled data from 3 studies . fig . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . analysis of survival of enu - treated rats at day 384 controls and 20 gy alone were pooled data from 3 studies . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . survival times are shown in table 1 . in a second set of experiments , our aim was to take the study to a more clinically realistic setting by assessing the effect of the treatments on the brain tumors in real time using mri and to compare the efficacy of sdf-1 blockade with that of the clinically used drug tmz , in potentiating the efficacy of irradiation . to perform the study , we began mri of the rat brains on day 130 after birth and treated only animals that had brain tumors identified by mri . this protocol therefore had a key difference versus the first study in that treatment was started from 2 to 7 weeks later than day 115 used previously . thus the average initial tumor sizes were larger in this study , making it more of a challenge to affect survival . prior to commencing the study , we established the sensitivity of mri to detect brain tumors of various sizes . we imaged rats at various times after day 130 of birth and sacrificed them to relate the mris to tumor brain histology . we performed serial sections on the rats sacrificed and attempted to correlate the histological images ( hematoxylin and eosin staining ) with serial section mri . as shown in fig . 3 , we saw an excellent correlation between the 2 methods except for the smallest tumors , barely detectable in the histological sections . we therefore concluded that mri was measuring all the significant brain tumors induced by enu . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . figure 4 shows the geometric means of the tumors in all the groups as a function of time from the beginning of treatment ( initiated days 132165 ) . all the estimations of tumor size from the mri measurements were made by one individual ( t.j . ) without any knowledge of the treatment that the rat had received . a second individual sorted the data into the treatment groups and calculated the group means . the salient features of the data are as follows : the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . 4).the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in this group continued to be undetectable until the appearance of 2 recurrences 105 days after the initiation of treatment.the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . fig . 4.addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . in utero enu - treated rats were imaged by mr starting on day 130 of age , repeated every 2 weeks until death . rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group at the start of treatment . the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in this group continued to be undetectable until the appearance of 2 recurrences 105 days after the initiation of treatment . the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . in utero enu - treated rats were imaged by mr starting on day 130 of age , repeated every 2 weeks until death . rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group at the start of treatment . though our primary purpose in this study was to measure tumor size following treatment , we also observed that the survival of the rats given nox - a12 following irradiation was significantly extended compared with those rats given irradiation alone or irradiation + tmz . we also in the same experiment assessed the effect of adding tmz to the irradiation + nox - a12 protocol and found that the addition of tmz did not further extend the life span of the rats given irradiation + nox - a12 , further supporting our conclusion that the addition of nox - a12 is superior to the addition of tmz . in order to be sure that the efficacy of nox - a12 was not just confined to this rat model , we performed an experiment with u251 human gbm implanted intracranially into immune deficient nude mice , measuring tumor response using bioluminescent imaging . the results obtained showed that the addition of nox - a12 ( 10 mg / kg 3 per wk ) following 12 gy wbi produced substantial additional tumor shrinkage and growth delay compared with irradiation only ( supplemental fig . thp-1 myelomonocytes ( which highly express cxcr4 but do not express cxcr7 as demonstrated by flow cytometry ) show sdf-1mediated chemotaxis . nox - a12 is able to inhibit a stimulus of 5 nm sdf-1 in a dose - dependent manner with an ic50 of 3.9 0.2 nm ( fig . pathhunter express cxcr7 activated gpcr internalization cells ( discoverx ) were used to demonstrate sdf-1dependent cxcr7 activation . nox - a12 shows inhibition of sdf-1 mediated cxcr7 internalization dose dependently with an ic50 of 3.0 0.9 nm ( fig . huvecs , which are known to express both sdf-1 receptors , cxcr4 and cxcr7 , migrate toward immobilized sdf-1 on fibronectin . 1c ) . in summary , we demonstrated that nox - a12 blocks sdf-1dependent activation of both receptors , cxcr4 and cxcr7 , with high potency . furthermore , nox - a12 inhibits sdf-1dependent chemotaxis of monocytes and endothelial cells , which both play an important role in vasculogenesis of irradiated solid tumors . thp-1 cells are attracted by 5 nm of human sdf-1 ( set to 100% ) . one representative dose - response curve ( mean sd of triplicates ) of 3 independent experiments is shown . nox - a12 inhibits sdf-1mediated chemotaxis of cxcr4-expressing thp-1 cells with an ic50 of 3.9 0.2 nm . one representative dose - response curve ( mean sd of triplicates ) of 4 independent experiments is shown . nox - a12 inhibits sdf-1mediated cxcr7 internalization with an ic50 of 3.0 0.9 nm . ( c ) nox - a12 inhibits sdf-1mediated migration of human ecs . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . sdf-1 immobilized on fibronectin increases migration of huvecs , which is completely inhibited by nox - a12 . each curve reflects the means of duplicates sd from a single experiment and is representative of 5 independent experiments . thp-1 cells are attracted by 5 nm of human sdf-1 ( set to 100% ) . one representative dose - response curve ( mean sd of triplicates ) of 3 independent experiments is shown . nox - a12 inhibits sdf-1mediated chemotaxis of cxcr4-expressing thp-1 cells with an ic50 of 3.9 0.2 nm . one representative dose - response curve ( mean sd of triplicates ) of 4 independent experiments is shown . nox - a12 inhibits sdf-1mediated cxcr7 internalization with an ic50 of 3.0 0.9 nm . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . sdf-1 immobilized on fibronectin increases migration of huvecs , which is completely inhibited by nox - a12 . each curve reflects the means of duplicates sd from a single experiment and is representative of 5 independent experiments . to ensure that the rats entering the first set of studies would have brain tumors of a size close to those producing neurological symptoms and death , we randomized rats born to mothers treated with enu ( 50 mg / kg ) on day 18 of gestation at day 115 of age . this was just before any of the rats began to die from their brain tumors ( fig 2 ) . in this study we included a group of rats given nox - a12 alone and controls given vehicle alone for 28 days . we also tested 2 different concentrations of nox - a12 and 2 different periods of drug administration after irradiation . in order to increase the power of the comparisons , we pooled the data from 2 prior experiments in which identically treated rats were either not irradiated or given 20 gy whole brain irradiation ( wbi ) . as can be seen ( fig . 2 and table 1 ) , the dose of 20 gy wbi extended the median life span of the rats by a small and not quite significant amount of approximately 7 days ( p = .07 vs non - irradiated ) . however , the addition of nox - a12 prolonged the median life span of the irradiated rats by a significant amount : by 95 days for the rats given the 8-week infusion at the low dose ( 5 mg / kg ) and by 153 days for the rats given the higher dose ( 20 mg / kg ) in comparison with the rats receiving 20 gy wbi alone . as can be seen , there was no significant effect of nox - a12 alone . we also gave 20 gy wbi with or without 8 weeks of nox - a12 to rats that had not been given enu in utero ( so did not have brain tumors ) . we saw no deaths in these rats up to the observation period of 418 days ( not shown in fig . 2 ) . the median life spans of the different groups and their significance levels are shown in table 1 . table 1.analysis of survival of enu - treated rats at day 384p valuecontrolnox-20 alone20 gy alone20 gy + nox-5 , 4 wk20 gy + nox-20 , 4 wk20 gy + nox-5 , 8 wk20 gy + nox-20 , 8 wk20 gy + no enuno enu + nox-20 , 8 wkmed . surv189173.5196244.5221291349>384>418vs 20 gy0.07ns0.10ns0.050.003<0.00010.01vs control ns0.070.050.020.0006<0.0001<0.00010.008controls and 20 gy alone were pooled data from 3 studies . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . analysis of survival of enu - treated rats at day 384 controls and 20 gy alone were pooled data from 3 studies . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . in a second set of experiments , our aim was to take the study to a more clinically realistic setting by assessing the effect of the treatments on the brain tumors in real time using mri and to compare the efficacy of sdf-1 blockade with that of the clinically used drug tmz , in potentiating the efficacy of irradiation . to perform the study , we began mri of the rat brains on day 130 after birth and treated only animals that had brain tumors identified by mri . this protocol therefore had a key difference versus the first study in that treatment was started from 2 to 7 weeks later than day 115 used previously . thus the average initial tumor sizes were larger in this study , making it more of a challenge to affect survival . prior to commencing the study , we established the sensitivity of mri to detect brain tumors of various sizes . we imaged rats at various times after day 130 of birth and sacrificed them to relate the mris to tumor brain histology . we performed serial sections on the rats sacrificed and attempted to correlate the histological images ( hematoxylin and eosin staining ) with serial section mri . as shown in fig . 3 , we saw an excellent correlation between the 2 methods except for the smallest tumors , barely detectable in the histological sections . we therefore concluded that mri was measuring all the significant brain tumors induced by enu . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . figure 4 shows the geometric means of the tumors in all the groups as a function of time from the beginning of treatment ( initiated days 132165 ) . all the estimations of tumor size from the mri measurements were made by one individual ( t.j . ) without any knowledge of the treatment that the rat had received . a second individual sorted the data into the treatment groups and calculated the group means . the salient features of the data are as follows : the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . 4).the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in this group continued to be undetectable until the appearance of 2 recurrences 105 days after the initiation of treatment.the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . 4.addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . in utero enu - treated rats were imaged by mr starting on day 130 of age , repeated every 2 weeks until death . rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group at the start of treatment . the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in this group continued to be undetectable until the appearance of 2 recurrences 105 days after the initiation of treatment . the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . in utero enu - treated rats were imaged by mr starting on day 130 of age , repeated every 2 weeks until death . rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group at the start of treatment . though our primary purpose in this study was to measure tumor size following treatment , we also observed that the survival of the rats given nox - a12 following irradiation was significantly extended compared with those rats given irradiation alone or irradiation + tmz . we also in the same experiment assessed the effect of adding tmz to the irradiation + nox - a12 protocol and found that the addition of tmz did not further extend the life span of the rats given irradiation + nox - a12 , further supporting our conclusion that the addition of nox - a12 is superior to the addition of tmz . in order to be sure that the efficacy of nox - a12 was not just confined to this rat model , we performed an experiment with u251 human gbm implanted intracranially into immune deficient nude mice , measuring tumor response using bioluminescent imaging . the results obtained showed that the addition of nox - a12 ( 10 mg / kg 3 per wk ) following 12 gy wbi produced substantial additional tumor shrinkage and growth delay compared with irradiation only ( supplemental fig . in earlier preclinical studies , we have shown that we could inhibit or delay the regrowth of subcutaneously implanted fadu human head and neck cancer or intracranially implanted u251 human gbm following irradiation using a variety of strategies to prevent the radiation - induced influx of cd11b+ myelomonocytes . these included inhibition of the transcription factor hypoxia inducible factor1 , inhibition of cxcr4 ( by the small molecule plerixafor as well as by anti - cxcr4 antibodies ) , depletion of macrophages using carrageenan , and use of cd11b blocking antibodies . although we obtained total abrogation of regrowth in the u251 tumor , we achieved only a modest increase in growth inhibition with the u87 human gbm . this result suggests that some tumors might be more , or could become more , dependent on alternative pathways of vasculogenesis after irradiation . such pathways may also include cxcr7 , the more recently discovered second receptor for sdf-1 . this assumption seems an attractive possibility because cxcr7 is highly expressed on endothelial cells of many tumors , including gbm , and cxcr7 has also been shown to facilitate transendothelial cell migration and vascular tube formation by epcs in vitro . the present study was performed with 2 main objectives : first , to investigate the efficacy of an inhibitor to the chemokine sdf-1 , which blocks the signaling of both known sdf-1 receptors , cxcr4 and cxcr7 ( fig . 1a c ) ; second , to evaluate the strategy of employing a more refractory and naturally occurring tumor model as close as possible to clinically appearing brain tumors . the key advantages of this model are that the tumors arise autochthonously in immune competent hosts and have a genetic diversity and aggressiveness comparable to human brain tumors . furthermore , macroscopic tumors frequently contain high levels of vascular endothelial growth factor , hemorrhage , and focal necrosis , all general characteristics of the most malignant glioblastomas . most importantly , these tumors , similar to human glioblastomas , are very resistant to treatment : maximum tolerated repeated doses of tmz do not affect survival , and high doses of vascular endothelial growth factor inhibitors have only a slight effect on tumor progression ( recht et al , unpublished ) . as is evident from fig . 2 , we confirmed the resistance of these tumors by the fact that a dose of 20 gy wbi resulted in a small and not significant ( p = .07 ) increased survival of the rats . 2 as well , we showed that inhibition of the sdf-1 pathway in combination with a single dose of 20 gy markedly enhanced the survival of the rats . this finding of an enhanced response on tumors was also confirmed by real - time mri measurements ( fig . we also showed that the effect of blocking sdf-1 with nox - a12 in combination with irradiation was significantly greater than the effect of addition of tmz to tumor irradiation . to address the question of whether the in vivo effects of nox - a12 on tumor regrowth are the result of a direct reduction of tumor cell growth by sdf-1 inhibition , we irradiated log phase u251 cells in vitro and maintained similar levels of nox - a12 in the medium following irradiation . we saw no effect of the drug on the survival of u251 human gbm cells to irradiation , nor to the growth of the cells with or without prior treatment with irradiation ( supplemental fig . we therefore conclude that the effect of nox - a12 is not a direct one on the tumor cells but is consistent with inhibition of the post - irradiation recovery of tumor blood flow in line with the observations published earlier with u251 gbm using the cxcr4 inhibitor amd-3100 following irradiation . these data lead to the question on the mechanism by which sdf-1 inhibition prevents the eventual restoration of the tumor vasculature after irradiation . to address this question we first have to ask , how does the tumor restore its vasculature following irradiation ? there are 3 main possibilities : angiogenesis sprouting from nearby unirradiated blood vessels eventually reaches the irradiated tumors . however , we consider this possibility unlikely because the whole brains of the rodents were irradiated and not just the tumors themselves . we note , however , that this is a possibility in the more normal situation in radiotherapy in which the radiation dose is more directly focused on the tumor itself . despite this , there are situations such as with multiple brain metastases in which wbi is delivered.the tumor vasculature regrows from surviving ecs in and around the tumors . they argue that several investigators ( including ourselves ) have failed to find bone marrow derived ( bmd ) ecs in tumors after irradiation and that the radiation - induced influx of pro - inflammatory and pro - angiogenic monocytes / macrophages would protect the ecs from radiation killing . although this is a possibility , the protective effect of the bmd monocytes on the irradiated endothelial cells would have to be beyond what has ever been observed for radiation cell killing to account for the magnitude of the effects we see with inhibition of the sdf-1 pathway . also , increased levels of sdf-1 and bmd monocytes in the tumor occur days to weeks after irradiation and therefore after the completion of cellular dna repair.circulating ecs or epcs and pro - angiogenic monocytes colonize the tumor after irradiation , thereby restoring the vasculature through the process of vasculogenesis . we and others have demonstrated the influx of pro - angiogenic cd11b+ monocytes into solid tumors following irradiation but , as noted earlier , failed to detect bmd ecs in the tumors . however , it was reported that the circulation of ecs or epcs derives from other tissues , including vessel walls , the gastrointestinal tract , and liver . furthermore , several authors , including ourselves , have shown an increase in circulating ecs or epcs after treatment of tumors with vascular disruptive agents , chemotherapy , and irradiation.based on these considerations , we believe that the evidence supports the post - irradiation colonization of the tumors with myelomonocytes and ecs and/or epcs ; these cells seem to play a major role in restoring tumor vasculature after irradiation . several investigators have shown that the recruitment and retention of pro - angiogenic cells from the bone marrow to sites of ischemic tissue damage or to tumors are mediated by the interaction of sdf-1 with cxcr4 . in support of this , 1 ) show that nox - a12 potently inhibits sdf-1dependent chemotaxis of both myelomonocytes and ecs . interestingly , we have previously demonstrated that sdf-1 is induced by hypoxia in vitro in a manner dependent on hypoxia inducible factor1 , that u251 tumors become hypoxic following irradiation , and that sdf-1 is produced in the tumors as they become hypoxic . as noted earlier , we demonstrated with the u251 intracranial model that the post - irradiation recruitment of bmd cells can be prevented using amd3100 ( plerixafor ) , a drug that blocks the interaction of sdf-1 with cxcr4 , and that this delays tumor recurrence following both single and fractionated doses of irradiation . of note , besides its activity to block cxcr4 , amd3100 is an allosteric agonist of cxcr7 that can signal through -arrestin and may activate the endothelium , leading to increased invasiveness of cancer cells . however , we consider this possibility unlikely because the whole brains of the rodents were irradiated and not just the tumors themselves . we note , however , that this is a possibility in the more normal situation in radiotherapy in which the radiation dose is more directly focused on the tumor itself . despite this , there are situations such as with multiple brain metastases in which wbi is delivered . they argue that several investigators ( including ourselves ) have failed to find bone marrow derived ( bmd ) ecs in tumors after irradiation and that the radiation - induced influx of pro - inflammatory and pro - angiogenic monocytes / macrophages would protect the ecs from radiation killing . although this is a possibility , the protective effect of the bmd monocytes on the irradiated endothelial cells would have to be beyond what has ever been observed for radiation cell killing to account for the magnitude of the effects we see with inhibition of the sdf-1 pathway . also , increased levels of sdf-1 and bmd monocytes in the tumor occur days to weeks after irradiation and therefore after the completion of cellular dna repair . circulating ecs or epcs and pro - angiogenic monocytes colonize the tumor after irradiation , thereby restoring the vasculature through the process of vasculogenesis . we and others have demonstrated the influx of pro - angiogenic cd11b+ monocytes into solid tumors following irradiation but , as noted earlier , failed to detect bmd ecs in the tumors . however , it was reported that the circulation of ecs or epcs derives from other tissues , including vessel walls , the gastrointestinal tract , and liver . furthermore , several authors , including ourselves , have shown an increase in circulating ecs or epcs after treatment of tumors with vascular disruptive agents , chemotherapy , and irradiation . one possible extension of our work is with the use of anti - angiogenic therapy that does not involve irradiation . xu and colleagues have demonstrated that anti - angiogenic therapy of patients with rectal cancer with bevacizumab upregulates both sdf-1 and cxcr4 in the tumors . these data raise the possibility that anti - angiogenic therapy may increase vasculogenesis and that blockage of sdf-1 or its receptors may enhance the efficacy of this therapy . in preliminary studies we have in fact shown this to be the case with the c6 tumor implanted intracranially in rats ( liu and brown , unpublished ) . in summary , the present data confirm and extend our earlier findings with cxcr4 blockade with the u251 tumor using a more clinically realistic glioma model in which the tumors develop in an autochthonous manner in immune competent hosts . we further show that inhibition of sdf-1 post - irradiation is superior in shrinking brain tumors after irradiation compared with tmz at doses equivalent to those used with concomitant radiotherapy in treating gbm . a summary of the proposed mechanism of tumor vasculogenesis and the various successful inhibitors of the process is presented in fig 5 . 5.model showing the vasculogenesis pathway that restores tumor vasculature after irradiation and the various inhibitors of this pathway that can improve the radiation response of the tumor . in addition to summarizing the data from the present study , the figure draws data from prior publications . abbreviations : mab , monoclonal antibody ; hif-1 , hypoxia - inducible factor 1 ; vegf , vascular endothelial growth factor ; ir , irradiation . model showing the vasculogenesis pathway that restores tumor vasculature after irradiation and the various inhibitors of this pathway that can improve the radiation response of the tumor . in addition to summarizing the data from the present study , the figure draws data from prior publications . abbreviations : mab , monoclonal antibody ; hif-1 , hypoxia - inducible factor 1 ; vegf , vascular endothelial growth factor ; ir , irradiation . based on the results obtained from this study , we believe that a clinical trial of nox - a12 in combination with standard therapy in first - line glioblastoma patients would be justified . nox - a12 doses and treatment times in these preclinical studies were chosen based on equivalents found to be safe and well tolerated in humans , and the drug is currently in phase ii studies for the treatment of chronic lymphocytic leukemia and multiple myeloma .

backgroundtumor irradiation blocks local angiogenesis , forcing any recurrent tumor to form new vessels from circulating cells . we have previously demonstrated that the post - irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel forming cells by blocking the interaction of cxcr4 with its ligand stromal cell - derived factor ( sdf)1 ( cxcl12 ) . in the present study we test this strategy by directly neutralizing sdf-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts.methodswe used nox - a12 , an l - enantiomeric rna oligonucleotide that binds and inhibits sdf-1 with high affinity . we tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-n-nitrosourea.resultsrats treated in utero with n - ethyl - n - nitrosourea began to die of brain tumors from approximately 120 days of age . we delivered a single dose of whole brain irradiation ( 20 gy ) on day 115 of age , began treatment with nox - a12 immediately following irradiation , and continued with either 5 or 20 mg / kg for 4 or 8 weeks , doses and times equivalent to well - tolerated human exposures . we found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment . in addition we treated tumors only when they were visible by mri and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide.conclusionsinhibition of sdf-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme .

Materials and Methods In vitro Characterization of NOX-A12 Experimental Animals Drug Treatment and Irradiations MRI Measurements of Rat Brain Tumors Histological Examination of the Brains Statistical Analysis Results NOX-A12 Is a Specific Inhibitor of SDF-1 Blocking Interaction With CXCR4 and CXCR7 Blockade of SDF-1 Post-irradiation With NOX-A12 Prolongs Survival of Rats With ENU-induced Brain Cancer Assessment of Rat Brain Tumor Response by MRI Discussion Supplementary Material Funding Supplementary Material

five nanomolar human recombinant sdf-1 ( r&d systems ) was preincubated with nox - a12 in various concentrations in hank 's balanced salt solution ( invitrogen ) containing 1 mg / ml bovine serum albumin ( bsa ) and 20 mm 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid ( hepes ) ( hbh ) in the lower compartments of a 96-transwell plate with 5-m pores ( costar corning , # 3388 ) at 37c for 20 min . the lower side of the inserts was coated with 5 g / ml human fibronectin ( r&d systems ) at room temperature for 1 h , washed once with pbs , and then incubated with 1 g / ml human sdf-1 ( r&d systems ) and nox - a12 in dmem without phenol red and 20 mm hepes at room temperature for 2 h. the filters were washed once and inserted in a bd falcon 24-well companion plate containing 750 l dmem without phenol red , 20 mm hepes , and 0.1% bsa . pups were born and weaned at normal age , after which they were monitored at least weekly for signs of illness , which usually developed after 120 days of age , at which time the pups started to show signs of brain lesions ( loss of weight , hind limb paralysis , and lethargy ) . in the first study , designed only to evaluate survival , the rats were assigned randomly to the various groups on day 115 of age , just before any rats started to die . in the second study , designed to evaluate tumor response by mri as well as survival , tumor size was estimated by mri starting on day 130 of age , and the rats were assigned to groups so as to have approximately the same total tumor volume in each group . for the 5 mg / kg group , the frozen solution was thawed on the day of injection and diluted to 5 mg / ml , and 1 ml / kg was subcutaneously administered every 2 days for 8 weeks ; for the 20 mg / kg group , the 10 mg / ml solution was used directly and 2 ml / kg was administered for 4 or 8 weeks ; for the 10 mg / kg group , the 10 mg / kg solution was used directly and 1 ml / kg was administered for 10 weeks . in the study in which nox - a12 was combined with temozolomide ( tmz ) , the tmz ( 10 mg / kg i.p ; sigma - aldrich ) was given 5 days / week for 3 weeks . five nanomolar human recombinant sdf-1 ( r&d systems ) was preincubated with nox - a12 in various concentrations in hank 's balanced salt solution ( invitrogen ) containing 1 mg / ml bovine serum albumin ( bsa ) and 20 mm 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid ( hepes ) ( hbh ) in the lower compartments of a 96-transwell plate with 5-m pores ( costar corning , # 3388 ) at 37c for 20 min . the lower side of the inserts was coated with 5 g / ml human fibronectin ( r&d systems ) at room temperature for 1 h , washed once with pbs , and then incubated with 1 g / ml human sdf-1 ( r&d systems ) and nox - a12 in dmem without phenol red and 20 mm hepes at room temperature for 2 h. the filters were washed once and inserted in a bd falcon 24-well companion plate containing 750 l dmem without phenol red , 20 mm hepes , and 0.1% bsa . pups were born and weaned at normal age , after which they were monitored at least weekly for signs of illness , which usually developed after 120 days of age , at which time the pups started to show signs of brain lesions ( loss of weight , hind limb paralysis , and lethargy ) . in the first study , designed only to evaluate survival , the rats were assigned randomly to the various groups on day 115 of age , just before any rats started to die . in the second study , designed to evaluate tumor response by mri as well as survival , tumor size was estimated by mri starting on day 130 of age , and the rats were assigned to groups so as to have approximately the same total tumor volume in each group . for the 5 mg / kg group , the frozen solution was thawed on the day of injection and diluted to 5 mg / ml , and 1 ml / kg was subcutaneously administered every 2 days for 8 weeks ; for the 20 mg / kg group , the 10 mg / ml solution was used directly and 2 ml / kg was administered for 4 or 8 weeks ; for the 10 mg / kg group , the 10 mg / kg solution was used directly and 1 ml / kg was administered for 10 weeks . in the study in which nox - a12 was combined with temozolomide ( tmz ) , the tmz ( 10 mg / kg i.p ; sigma - aldrich ) was given 5 days / week for 3 weeks . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . to ensure that the rats entering the first set of studies would have brain tumors of a size close to those producing neurological symptoms and death , we randomized rats born to mothers treated with enu ( 50 mg / kg ) on day 18 of gestation at day 115 of age . however , the addition of nox - a12 prolonged the median life span of the irradiated rats by a significant amount : by 95 days for the rats given the 8-week infusion at the low dose ( 5 mg / kg ) and by 153 days for the rats given the higher dose ( 20 mg / kg ) in comparison with the rats receiving 20 gy wbi alone . we also gave 20 gy wbi with or without 8 weeks of nox - a12 to rats that had not been given enu in utero ( so did not have brain tumors ) . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . in a second set of experiments , our aim was to take the study to a more clinically realistic setting by assessing the effect of the treatments on the brain tumors in real time using mri and to compare the efficacy of sdf-1 blockade with that of the clinically used drug tmz , in potentiating the efficacy of irradiation . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . the salient features of the data are as follows : the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . 4).the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in this group continued to be undetectable until the appearance of 2 recurrences 105 days after the initiation of treatment.the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . 4.addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . though our primary purpose in this study was to measure tumor size following treatment , we also observed that the survival of the rats given nox - a12 following irradiation was significantly extended compared with those rats given irradiation alone or irradiation + tmz . we also in the same experiment assessed the effect of adding tmz to the irradiation + nox - a12 protocol and found that the addition of tmz did not further extend the life span of the rats given irradiation + nox - a12 , further supporting our conclusion that the addition of nox - a12 is superior to the addition of tmz . in order to be sure that the efficacy of nox - a12 was not just confined to this rat model , we performed an experiment with u251 human gbm implanted intracranially into immune deficient nude mice , measuring tumor response using bioluminescent imaging . the results obtained showed that the addition of nox - a12 ( 10 mg / kg 3 per wk ) following 12 gy wbi produced substantial additional tumor shrinkage and growth delay compared with irradiation only ( supplemental fig . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . huvecs were stained with the fluorescent dye diic12(3 ) , and migration through the fluoroblok membrane was quantified in a bottom reading plate reader ; 1 g / ml of human sdf-1 was preincubated either with or without an equimolar concentration of nox - a12 and then added to the bottom of the transwell inserts , which were coated with fibronectin . to ensure that the rats entering the first set of studies would have brain tumors of a size close to those producing neurological symptoms and death , we randomized rats born to mothers treated with enu ( 50 mg / kg ) on day 18 of gestation at day 115 of age . in order to increase the power of the comparisons , we pooled the data from 2 prior experiments in which identically treated rats were either not irradiated or given 20 gy whole brain irradiation ( wbi ) . however , the addition of nox - a12 prolonged the median life span of the irradiated rats by a significant amount : by 95 days for the rats given the 8-week infusion at the low dose ( 5 mg / kg ) and by 153 days for the rats given the higher dose ( 20 mg / kg ) in comparison with the rats receiving 20 gy wbi alone . we also gave 20 gy wbi with or without 8 weeks of nox - a12 to rats that had not been given enu in utero ( so did not have brain tumors ) . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . rats born to mothers treated with a single injection of the carcinogen enu on day 18 of gestation were sham irradiated or given a dose of 20 gy to the whole brain with shielding of the buccal cavity . the rats receiving nox - a12 were injected subcutaneously every 2 days with either 5 or 20 mg / kg starting soon after irradiation and continued for either 4 or 8 weeks . in a second set of experiments , our aim was to take the study to a more clinically realistic setting by assessing the effect of the treatments on the brain tumors in real time using mri and to compare the efficacy of sdf-1 blockade with that of the clinically used drug tmz , in potentiating the efficacy of irradiation . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . rats born to mothers given a single injection of enu on day 18 of gestation were imaged by mri , and when 1 or more tumors per rat were detected , the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the mri scans . the salient features of the data are as follows : the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . 4).the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in this group continued to be undetectable until the appearance of 2 recurrences 105 days after the initiation of treatment.the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . 4.addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . the tumors in the rats treated with nox - a12 alone continued to grow as expected ( fig . the tumors in the rats treated with 20 gy + nox - a12 ( blue line ) disappeared by 28 days after the start of treatment . the tumors in the rats given 20 gy alone or 20 gy + tmz behaved similarly with an initial decrease in volume to day 45 followed by a regrowth . addition of nox - a12 following irradiation of the enu - induced brain tumors produces complete responses by mri . though our primary purpose in this study was to measure tumor size following treatment , we also observed that the survival of the rats given nox - a12 following irradiation was significantly extended compared with those rats given irradiation alone or irradiation + tmz . we also in the same experiment assessed the effect of adding tmz to the irradiation + nox - a12 protocol and found that the addition of tmz did not further extend the life span of the rats given irradiation + nox - a12 , further supporting our conclusion that the addition of nox - a12 is superior to the addition of tmz . in order to be sure that the efficacy of nox - a12 was not just confined to this rat model , we performed an experiment with u251 human gbm implanted intracranially into immune deficient nude mice , measuring tumor response using bioluminescent imaging . the results obtained showed that the addition of nox - a12 ( 10 mg / kg 3 per wk ) following 12 gy wbi produced substantial additional tumor shrinkage and growth delay compared with irradiation only ( supplemental fig . the key advantages of this model are that the tumors arise autochthonously in immune competent hosts and have a genetic diversity and aggressiveness comparable to human brain tumors . we also showed that the effect of blocking sdf-1 with nox - a12 in combination with irradiation was significantly greater than the effect of addition of tmz to tumor irradiation . we saw no effect of the drug on the survival of u251 human gbm cells to irradiation , nor to the growth of the cells with or without prior treatment with irradiation ( supplemental fig . we therefore conclude that the effect of nox - a12 is not a direct one on the tumor cells but is consistent with inhibition of the post - irradiation recovery of tumor blood flow in line with the observations published earlier with u251 gbm using the cxcr4 inhibitor amd-3100 following irradiation . furthermore , several authors , including ourselves , have shown an increase in circulating ecs or epcs after treatment of tumors with vascular disruptive agents , chemotherapy , and irradiation.based on these considerations , we believe that the evidence supports the post - irradiation colonization of the tumors with myelomonocytes and ecs and/or epcs ; these cells seem to play a major role in restoring tumor vasculature after irradiation . several investigators have shown that the recruitment and retention of pro - angiogenic cells from the bone marrow to sites of ischemic tissue damage or to tumors are mediated by the interaction of sdf-1 with cxcr4 . interestingly , we have previously demonstrated that sdf-1 is induced by hypoxia in vitro in a manner dependent on hypoxia inducible factor1 , that u251 tumors become hypoxic following irradiation , and that sdf-1 is produced in the tumors as they become hypoxic . as noted earlier , we demonstrated with the u251 intracranial model that the post - irradiation recruitment of bmd cells can be prevented using amd3100 ( plerixafor ) , a drug that blocks the interaction of sdf-1 with cxcr4 , and that this delays tumor recurrence following both single and fractionated doses of irradiation . in summary , the present data confirm and extend our earlier findings with cxcr4 blockade with the u251 tumor using a more clinically realistic glioma model in which the tumors develop in an autochthonous manner in immune competent hosts . we further show that inhibition of sdf-1 post - irradiation is superior in shrinking brain tumors after irradiation compared with tmz at doses equivalent to those used with concomitant radiotherapy in treating gbm .

production of fgbps fgbp1 , a recombinant form of fgbp that lacks the wall / membrane anchor domain , was produced as described previously ( 7 ) . for generation of fgbpm , a recombinant form of fgbp that lacks the central domain between the a and b repeats ( amino acids 279 - 348 ) and does not bind to equine igg,3 fgbp gene sequences encoding amino acids 37 - 278 and 349 - 470 were amplified and juxtaposed in the vector pqe30 . transformation of escherichia coli xl-1blue with pqe30 and purification of the recombinant protein was carried out as previously described ( 7 , 11 ) . analysis of the interaction of fgbp with recombinant equine igs by elisa interaction of fgbp1 with recombinant forms of the seven equine igg ( reqigg ) subclasses , produced and purified as described previously ( 16 ) , was analyzed by elisa . fgbp1 ( 10 g / ml in carbonate buffer ) was coated overnight at 4 c onto 96-well maxisorp immunoplates ( nunc , roskilde , denmark ) . plates were washed in milli - q water and then blocked for 1 h at room temperature with 5% nonfat milk powder in pbs containing 0.5% tween 20 ( pbs - t ) . plates were then incubated with serial dilutions ( 0 - 20 g / ml in pbs - t ) of one of the reqigg subclasses or recombinant equine monomeric equine iga4 ( eqiga ) for 1 h at room temperature . binding of reqigs to fgbp was detected by incubation for 1 h at room temperature with hrp - conjugated goat anti - mouse light chain antibody ( 0.2 g / ml in pbs - t ) ( bethyl laboratories inc . goat igg does not bind to fgbp ( 9 ) and so was suitable as the detection reagent in these experiments . the ph dependence of the fgbp - igg interaction was analyzed using the same elisa except that reqiggs ( serial dilutions of 0 - 10 g / ml ) were diluted in pbs - t at either ph 5.5 , ph 7.4 , or ph 8.5 , and fgbp1 and fgbpm were used at 5 g / ml . biosensor analysis of fgbp1 binding to reqigg biosensor experiments were carried out using a biacore x instrument ( biacore ab , uppsala , sweden ) . recombinant fgbp1 ( 20 m in 10 mm sodium acetate , ph 5.5 ) was immobilized on a cm5 biosensor chip ( biacore ) using the manufacturer 's instructions for amine coupling chemistry . recombinant fgbpm was coupled under the same conditions to the reference surface of the chip . recombinant eqiggs were diluted in biacore hbs - ep buffer ( 0.01 m hepes , 0.15 m nacl , 3 mm edta , 0.005% surfactant p20 ) across a concentration range of 0 - 150 g / ml and were independently injected over the chip ( 60 l and 30 l / min ) . after each injection the sensor chip was regenerated with 20 l of 10 mm glycine , ph 3 , followed by a 10-l injection of 10 mm glycine , ph 2 , if required . biosensor analysis of recombinant human igg1 ( 18 ) binding to fgbp1 was also carried out as described above . protein g and protein a competition elisas plates were coated with 2.5 g / ml of either reqigg4 or reqigg7 and then washed and blocked as described above . plates were placed on ice , and 100 l / well ice - cold hrp - conjugated protein g ( 0.25 g / ml in pbs - t ) ( sigma ) , and serial dilutions of either fgbp1 or fgbpm ( both 0 - 50g / ml in pbs - t ) were added simultaneously to each well . plates were incubated for 2 h at room temperature and then developed as described above . a similar elisa was used to assess competition between hrp - conjugated protein a ( sigma ) ( 20g / ml ) and fgbp1 or fgbpm ( serial dilutions of 0 - 50g / ml ) using plates coated with 5 g / ml reqigg4 or reqigg7 . it was necessary to use different concentrations of hrp - conjugated protein g and protein a in the assay because of the different binding affinities of the two proteins for igg . binding of recombinant human igs to fgbp two previously described recombinant human igg1/iga1 domain - swap mutants ( 18 ) were assessed for binding to fgbp1 . in the first mutant ( 123 ) , the ch3 domain of human igg1 is replaced by the ch3 domain of human iga1 , whereas in the second ( 123 ) , the ch3 domain of human iga1 is replaced by the ch3 domain of human igg1 . binding of fgbp1 to a recombinant version of the human igg3 allotype , ighg301 or igg3m(5 ) ( 19 ) , purified from transfectant supernatant by affinity chromatography on 3-nitro-4-hydroxy-5-iodophenylacetate - sepharose , was also investigated . this allotype is known to lack the ability to bind protein a ( 20 , 21 ) . binding analysis , using recombinant human igg1 and human iga1 , produced as described previously ( 17 ) , as positive and negative controls , respectively , was carried out by elisa as described above using 5 g / ml fgbp1 to coat and serial dilutions ( up to 10 g / ml ) of recombinant igs . site - directed mutagenesis of eqigg7 heavy ( h ) chain single or double point mutations were introduced into the ch2 or ch3 domains of the eqigg7 h - chain using a quikchange ii xl site - directed mutagenesis kit ( stratagene , la jolla , ca ) , following the manufacturer 's instructions . residues mutated in eqigg7 corresponded to those in human igg1 responsible for binding protein a or protein g ( 22 , 23 ) . selected residues in eqigg7 were exchanged for the amino acid(s ) present in the non - fgbp - binding eqigg subclasses , at positions where a non - conservative amino acid alteration was found . mutant antibodies were purified and analyzed by sds - page and western blotting as previously described ( 16 ) . the fgbp1 binding capacities of mutant reqigg7s were compared with that of wild - type reqigg7 using biosensor analysis as described above except that 100-l injections of antibody were used . table 1names and description of reqigg7 mutantsmutantdescriptionaeqigg subclasses carrying the same residues as those substituted m252k met in ch2 domain substituted with lys igg5 k382q lys in ch3 domain substituted with gln igg1 , igg2 , igg3 , igg5 , igg6 hn433/434eh his in ch3 domain substituted with glu , asn in ch3 domain substituted with his glu in igg5 h433r his in ch3 domain substituted with arg igg6 hy435/436tv his in ch3 domain substituted with thr , tyr in ch3 domain substituted with val thr in igg6 val in igg3 anumbering corresponds to human igg1 numbering . fgbp - mediated inhibition of c1q binding and activation of the classic complement pathway by equine igg was assessed by elisa using reqigg3 , reqigg4 , and reqigg7 ( 10 g / ml ) , as described previously ( 16 ) , with the following modifications . after coating of wells with 3-nitro-4-hydroxy-5-iodophenylacetate - bovine serum albumin and subsequent incubation with recombinant igs , plates were incubated with serial dilutions of fgbp1 or fgbpm ( 0 - 10 g / ml in pbs - t containing 0.5% bovine serum albumin ) for 1 h at room temperature . plates were incubated then with either human c1q protein ( 0.5 g / ml , calbiochem ) or human serum . in the former case , binding of c1q to igg was detected using sheep anti - human c1q - hrp , as previously described , whereas in the latter , activation of the classic complement pathway was detected using a 1/500 dilution of biotin - conjugated mouse anti - human c4c ( quidel , santa clara , ca ) followed by streptavidin - hrp ( 1/500 , dako , ely , cambs , uk ) . c4c generation is a specific marker for activation of the classic complement pathway ( 24 ) . human c1q and human serum ( as a source of complement ) were used rather than their equine equivalents because of the availability of detection reagents for the human but not equine proteins . moreover , cross - species reactivity of igg and complement components has been well demonstrated ( 25 ) . fgbp1 bound strongly to igg4 and igg7 , but the remaining five igg subclasses and iga showed little or no binding . open diamond , reqigg1 ; cross , reqigg2 ; open square , reqigg3 ; open circle , reqigg4 ; open triangle , reqigg5 ; plus sign , reqigg6 ; closed triangle , reqigg7 ; closed square , reqiga . for both equine igg4 ( b ) and igg7 ( c ) , binding to fgbp1 was most efficient at ph 7.4 - 8.5 . closed diamond , ph 5.0 ; open square , ph 7.4 ; closed triangle , ph 8.5 . binding of reqiggs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . fgbp1 bound strongly to igg4 and igg7 , but the remaining five igg subclasses and iga showed little or no binding . open diamond , reqigg1 ; cross , reqigg2 ; open square , reqigg3 ; open circle , reqigg4 ; open triangle , reqigg5 ; plus sign , reqigg6 ; closed triangle , reqigg7 ; closed square , reqiga . b and c , ph dependence of the binding to fgbp1 . for both equine igg4 ( b ) and igg7 ( c ) , binding to fgbp1 was most efficient at ph 7.4 - 8.5 . closed diamond , ph 5.0 ; open square , ph 7.4 ; closed triangle , ph 8.5 . binding of reqiggs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . measurements were carried out at different concentrations ( 0 , 35 , 50 , 75 , 100 , 125 , and 150 g / ml ) for the soluble analyte equine igg4 ( a ) and equine igg7 ( b ) , respectively . measurements for the remaining five subclasses were carried out at 150 g / ml only ( c ) . the response curves shown represent specific binding to fgbp1 ( following subtraction of any nonspecific binding to the fgbpm control ) . measurements were carried out at different concentrations ( 0 , 35 , 50 , 75 , 100 , 125 , and 150 g / ml ) for the soluble analyte equine igg4 ( a ) and equine igg7 ( b ) , respectively . measurements for the remaining five subclasses were carried out at 150 g / ml only ( c ) . the response curves shown represent specific binding to fgbp1 ( following subtraction of any nonspecific binding to the fgbpm control ) . only igg4 and igg7 showed concentration - dependent interaction with immobilized fgbp1 . analysis of fgbp - igg binding binding of recombinant fgbp1 to the seven reqigg subclasses and monomeric reqiga was first analyzed by elisa . fgbp1 bound strongly to reqigg4 and reqigg7 , with little or no binding to the remaining five reqigg subclasses or to reqiga ( fig . because igg binding by the bacterial igg - binding proteins staphylococcal protein a and streptococcal protein g is known to be ph - dependent ( 26 ) , we tested the effect of ph variation on the fgbp1-igg interaction ( fig . 1 , b and c ) . an increase in ph from 7.4 to ph 8.5 had little effect on binding , whereas a decrease in ph to 5.5 reduced binding to roughly a third of that seen at physiological ph . hence , the ph dependence of igg binding by fgbp is more similar to that of protein a ( strongest binding at ph 8) than to that of protein g ( strongest binding at ph 4 - 5 ) ( 26 ) . figure 3.elisa analysis of competition for binding to reqigg4 and reqigg7 between fgbp proteins and protein a and protein g. a , competition of binding of hrp - conjugated protein g ( 0.25 g / ml ) to reqigg4 and reqigg7 coated at 2.5 g / ml . b , competition of binding of hrp - conjugated protein a ( 20 g / ml ) to reqigg4 and reqigg7 coated at 5 g / ml . the figures shows mean ( s.e . ) of three independent experiments . reqigg4 with fgbp1 competitor ( closed circle ) and fgbpm competitor ( open circle ) ; reqigg7 with fgbp1 competitor ( open triangle ) and fgbpm competitor ( closed triangle ) . elisa analysis of competition for binding to reqigg4 and reqigg7 between fgbp proteins and protein a and protein g. a , competition of binding of hrp - conjugated protein g ( 0.25 g / ml ) to reqigg4 and reqigg7 coated at 2.5 g / ml . b , competition of binding of hrp - conjugated protein a ( 20 g / ml ) to reqigg4 and reqigg7 coated at 5 g / ml . the figures shows mean ( s.e . ) of three independent experiments . reqigg4 with fgbp1 competitor ( closed circle ) and fgbpm competitor ( open circle ) ; reqigg7 with fgbp1 competitor ( open triangle ) and fgbpm competitor ( closed triangle ) . figure 4.elisa analysis of fgbp1 binding to human igg1 , human igg1/iga1 domain - swap mutants and human igg3 allotype ighg301 . binding of igs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . open diamond , human igg1 ; open triangle , human iga1 ; closed square , 123 mutant ; open circle , 123 mutant ; cross , human igg3 ighg301 allotype . elisa analysis of fgbp1 binding to human igg1 , human igg1/iga1 domain - swap mutants and human igg3 allotype ighg301 . binding of igs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . open diamond , human igg1 ; open triangle , human iga1 ; closed square , 123 mutant ; open circle , 123 mutant ; cross , human igg3 ighg301 allotype . to establish the affinity of the fgbp - igg interaction and confirm the equine igg subclass specificity we used surface plasmon resonance measurements . these experiments gave identical results with regard to subclass specificity to those obtained by elisa , confirming that reqigg4 and reqigg7 are the predominant subclasses capable of binding fgbp1 ( fig . these subclasses share similar affinity in the micromolar range ( ka values of 6.96 10 m for reqigg7 and 6.79 10 m for reqigg4 ) . hence , binding affinity is much lower than that of protein g for igg ( 26 ) and closer to that of fragment b of protein a ( 27 ) . protein a and protein g are known to bind in the interdomain region of igg fc ( 22 , 23 ) and have both been shown to interact with reqigg4 and reqigg7 , although the binding to protein a is relatively weak ( 16 ) . therefore , we used a competition elisa to see if fgbp could inhibit binding of proteins a and g to reqigg4 and reqigg7 . we found that fgbp1 , but not the non - igg - binding control protein fgbpm , was able to compete with both protein a and protein g for binding to reqigg4 and reqigg7 ( fig . 3 ) . this inhibition was concentration - dependent , and binding of both protein a and protein g was negligible at the maximum concentration of fgbp1 ( 50 g / ml ) . these findings suggest that the fgbp binding site(s ) on reqigg4 and reqigg7 overlap with those of protein a and protein g in the fc interdomain region . to determine whether one or both of the igg fc domains contribute to fgbp binding , we utilized recombinant human igg1 and two human igg1/iga1 domain - swap mutants . using biosensor analysis ( data not shown ) , we found that human igg1 interacted with fgbp1 with an affinity ( ka of 5.71 10 m ) only slightly lower than that of reqigg4 and reqigg7 . a domain - swap mutant lacking the ch2 domain but retaining the ch3 domain of igg1 ( 123 ) was able to bind to fgbp1 , but at a much reduced level compared with that of wild - type human igg1 ( fig . 4 ) . however , fgbp1 binding was completely absent for a mutant retaining the ch2 domain but lacking the ch3 domain of igg1 ( 123 ) ( fig . hence , both fc domains are required for maximal binding , but the ch3 domain appears to be the most critical . in addition , we included in this analysis a recombinant human igg3 antibody of the ighg301 allotype . igg3 allotypes capable of binding protein a possess a his residue at position 435 within the interaction site for protein a. however , the ighg301 allotype carries an arg at this critical ch3 domain residue and is unable to bind protein a ( 20 , 21 ) . we found that the ighg301 allotype did not bind fgbp1 , indicating that residue 435 is critical also for interaction with fgbp ( fig . 4 ) . to further localize the fgbp binding site on equine igg , we generated five igg7 mutants with single or double point mutations in the regions corresponding to the protein a / protein g binding sites ( table 1 ) . analysis of the igg7 mutants by sds - page and western blotting revealed that all the mutant reqigg7s assembled like wild - type reqigg7 and retained the anticipated reactivity with antigen and both polyclonal and monoclonal anti - equine iggb ( i.e. anti - eqigg4 and -eqigg7 ) reagents ( fig . 5 ) . these test outcomes and the fact that the substitutions were based on residues present in other equine igg isotypes suggest that the likelihood of unintended conformational disruption in the mutants is extremely low , allowing us to draw meaningful conclusions on the relative contributions of the mutated residues to fgbp binding . binding of the mutant reqigg7s to fgbp1 was analyzed using surface plasmon resonance experiments ( fig . mutants m252k and k382q both showed decreased fgbp1 binding compared with wild - type reqigg7 . however , the 45-fold decrease in fgbp1 binding by m252k ( ka of 1.53 10 m ) was more dramatic than the 2.4-fold decrease shown by k382q ( ka of 2.91 10 m ) . mutant h433r showed a small ( 1.7-fold , ka of 4 10 m ) reduction in fgbp1 binding capacity compared with wild - type reqigg7 . however , the double point mutants hn433/434eh and hy435/436tv showed drastically impaired abilities to bind fgbp1 , retaining < 5% of the binding capacity of wild - type reqigg7 . the fgbp1 binding affinities and interdomain site sequence alignment of the tested iggs are summarized in fig . 7 . taken together these results suggest that the binding site on igg for fgbp encompasses some of the same interdomain residues as those involved in interaction with protein a and protein g. residues in the leu - ser and the leu - tyr loops are particularly implicated . comparison of the binding affinities of mutants h433r and hn433/434eh suggests that either his is not critical for fgbp binding , or that alteration of this residue to a positively charged amino acid such as arginine ( as in h433r ) can be tolerated without significant impact on the affinity of the interaction . in contrast , residues his and/or tyr appear to be critical for the interaction , because in both instances where these residues are substituted ( with arg and phe in human igg3 allotype ighg301 and with thr and val in reqigg7 mutant hy435/436tv ) binding to fgbp1 is ablated . overall , we can conclude that the binding site for fgbp on reqigg fc appears to center on the interdomain region , utilizing residues common to both the protein a and protein g binding sites . proteins were run under non - reducing ( a ) and reducing ( b and c ) conditions and stained with coomassie brilliant blue ( a and b ) or immunoblotted and probed with polyclonal goat anti - horse iggb which recognizes reqigg4 and reqigg7 ( c ) . lane 1 , wild - type reqigg7 ; lane 2 , m252k ; lane 3 , k382q ; lane 4 , h433r ; lane 5 , hn433/434eh ; lane 6 , hy435/436tv . proteins were run under non - reducing ( a ) and reducing ( b and c ) conditions and stained with coomassie brilliant blue ( a and b ) or immunoblotted and probed with polyclonal goat anti - horse iggb which recognizes reqigg4 and reqigg7 ( c ) . lane 1 , wild - type reqigg7 ; lane 2 , m252k ; lane 3 , k382q ; lane 4 , h433r ; lane 5 , hn433/434eh ; lane 6 , hy435/436tv . injections of 100 l ( 30 l / min ) of reqigg7s at 0 , 5 , 10 , 25 , 35 , 50 , 75 , 100 , 125 , and 150g / ml are shown . a , wild - type reqigg7 ; b , m252k ; c , k382q ; d , h433r ; e , hn433/434eh ; f , hy435/436tv . the response curves shown represent specific binding to fgbp1 ( following subtraction of any nonspecific binding to the fgbpm control ) . injections of 100 l ( 30 l / min ) of reqigg7s at 0 , 5 , 10 , 25 , 35 , 50 , 75 , 100 , 125 , and 150g / ml are shown . a , wild - type reqigg7 ; b , m252k ; c , k382q ; d , h433r ; e , hn433/434eh ; f , hy435/436tv . the response curves shown represent specific binding to fgbp1 ( following subtraction of any nonspecific binding to the fgbpm control ) . figure 7.amino acid alignments of reqigg subclasses and reqigg7 mutants showing only ch2 and ch3 residues critical for interaction with protein a and protein g. residue numbers ( shown above sequences ) correspond to human igg1 numbering . in the corresponding higg1 sequence ( shown for comparison at the top ) , residues implicated in interactions are indicated as follows : protein g , normal text ; protein a , underlined ; both protein a and protein g , box . the corresponding sequence for human igg3 allotype ighg301 ( higg301 ) is also shown . in the middle section , the fgbp - binding subclasses eqigg4 and eqigg7 are shown above the non - binding subclasses . reqigg7 mutants are shown in the lower part of the figure , with substitutions encircled . amino acid alignments of reqigg subclasses and reqigg7 mutants showing only ch2 and ch3 residues critical for interaction with protein a and protein g. residue numbers ( shown above sequences ) correspond to human igg1 numbering . in the corresponding higg1 sequence ( shown for comparison at the top ) , residues implicated in interactions are indicated as follows : protein g , normal text ; protein a , underlined ; both protein a and protein g , box . the corresponding sequence for human igg3 allotype ighg301 ( higg301 ) is also shown . in the middle section , the fgbp - binding subclasses eqigg4 and eqigg7 are shown above the non - binding subclasses . reqigg7 mutants are shown in the lower part of the figure , with substitutions encircled . were able to disrupt c1q binding , detected using anti - c1q antibody ( a ) , and activation of the classical complement pathway , detected using anti - c4c antibody ( b ) , by reqigg4 and reqigg7 but not by reqigg3 , which binds poorly to fgbp1 . closed diamond , reqigg3 ; open square , reqigg4 ; closed triangle , reqigg7 . in each case , c1q binding / activation are shown as a percentage of the binding / activation seen in the absence of fgbp1 . increasing concentrations of fgbp1 were able to disrupt c1q binding , detected using anti - c1q antibody ( a ) , and activation of the classical complement pathway , detected using anti - c4c antibody ( b ) , by reqigg4 and reqigg7 but not by reqigg3 , which binds poorly to fgbp1 . closed diamond , reqigg3 ; open square , reqigg4 ; closed triangle , reqigg7 . in each case , c1q binding / activation are shown as a percentage of the binding / activation seen in the absence of fgbp1 . we have previously demonstrated that reqigg1 , reqigg3 , reqigg4 , and reqigg7 bind strongly to c1q and activate the classical complement pathway ( 16 ) . to gain an insight into the physiological consequences of fgbp binding to eqigg4 and eqigg7 , we investigated whether fgbp1 could inhibit their ability to bind c1q and activate the classical complement pathway . we found that fgbp1 was able to disrupt c1q binding and complement activation by reqigg4 and reqigg7 ( fig . in contrast , fgbp1 was unable to disrupt c1q binding and complement activation by reqigg3 , which we have shown binds poorly or not at all to fgbp1 ( fig . the control protein , fgbpm , had no effect on c1q binding or complement activation . with the highest concentration of fgbp1 used ( 10 g / ml ) , c1q binding and complement activation by reqigg4 and reqigg7 decreased to 50 and 40% , respectively , of that seen in the absence of fgbp1 . this partial rather than complete inhibition of antibody - mediated complement activation may be a consequence of using soluble rather than cell - bound fgbp ( see discussion below ) . ig - binding proteins have been identified in a range of bacteria , and their diversity in ig binding characteristics in terms of ig isotype , subclass , and species has long been recognized . the horse has an unusually large number of igg subclasses , which vary in their structural and functional attributes ( 14 - 16 ) . in this study we addressed the eqigg subclass specificity of fgbp and found that this bacterial protein is highly specific for only two ( eqigg4 and eqigg7 ) of the seven eqigg subclasses . eqigg4 and eqigg7 are 97% identical at the amino acid level , and prior to genomic identification were together thought to comprise a single subclass termed iggb ( 15 , 28 ) . for all the binding characteristics and effector functions thus far tested , these two subclasses share common features , presumably due to their high sequence similarity ( 16 ) . we have demonstrated that fgbp binds to the interdomain region of igg fc and that both of the fc domains are required for full binding . the residues implicated in our mutagenesis studies as critical for binding to fgbp are known to mediate interaction with staphylococcal protein a and streptococcal protein g also ( 22 , 23 ) . indeed , there is a striking overlap of the regions recognized by these three distinct bacterial proteins ( fig . 9 ) , despite the fact that they derive from very different streptococcal and staphylococcal strains and these bacteria are pathogenic in different mammalian species . even more remarkably , the fact that these unrelated igg - binding proteins bind to similar sites in the igg interdomain region parallels the situation for the unrelated bacterial iga binding proteins sir22 from s. pyogenes , protein from group b streptococcus , and ssl7 from staphylococcus aureus , which all bind to the fc domain interface in human iga ( 29 - 31 ) ( fig . 9 ) . although a different immunoglobulin class is involved , again we see the equivalent region being targeted by proteins produced by very different bacterial pathogens . thus , it seems that convergent evolution may have favored the appearance of bacterial proteins that bind to the ch2/ch3 interface in igg and iga . this interdomain region , in igg at least , has been recognized as one of only a limited number of regions on the ig surface that is particularly suited to protein - protein interaction ( 32 , 33 ) . figure 9.interaction sites for streptococcal and staphylococcal ig - binding proteins on igg and iga fc regions . the ch2 and ch3 domains of the heavy chains of igg ( a - c ) ( pdb accession : 1fc1 ) and iga ( d - f ) ( pdb accession : 1ow0 ) are shown in light blue and dark blue , respectively . residues shown to be critical for interaction by mutagenesis studies and/or by x - ray crystallography of complexes are highlighted by spheres . a , fgbp from s. equi in purple ; b , protein g from group c and group g streptococci in red ; c , protein a from s. aureus in yellow ; d , sir22 from s. pyogenes in orange ; e, protein from group b streptococcus in pink ; f , ssl7 from s. aureus in green . remarkably , all the ig - binding proteins target the ch2-ch3 domain interface , regardless of specificity for igg or iga , streptococcal or staphylococcal origin , or host species ( equine or human ) . interaction sites for streptococcal and staphylococcal ig - binding proteins on igg and iga fc regions . the ch2 and ch3 domains of the heavy chains of igg ( a - c ) ( pdb accession : 1fc1 ) and iga ( d - f ) ( pdb accession : 1ow0 ) are shown in light blue and dark blue , respectively . residues shown to be critical for interaction by mutagenesis studies and/or by x - ray crystallography of complexes are highlighted by spheres . a , fgbp from s. equi in purple ; b , protein g from group c and group g streptococci in red ; c , protein a from s. aureus in yellow ; d , sir22 from s. pyogenes in orange ; e, protein from group b streptococcus in pink ; f , ssl7 from s. aureus in green . remarkably , all the ig - binding proteins target the ch2-ch3 domain interface , regardless of specificity for igg or iga , streptococcal or staphylococcal origin , or host species ( equine or human ) . the evolutionary reasons why such sites of relative vulnerability have been retained on the surface of ig fc regions are likely to be complex ( 34 ) but most probably relate to their roles as interaction sites for key host receptors . in igg , for example , the fc interdomain region forms the interaction site for fcrn , the so - called neonatal fc receptor that mediates a number of processes fundamental to igg function , including regulation of igg turnover and transepithelial transfer of igg ( 35 ) . for iga , the ch2/ch3 interdomain region serves as the interaction site for its specific fc receptor , fcri , which triggers efficient elimination mechanisms against iga - coated pathogens ( 18 , 36 ) . despite the marked overlap in their binding sites on igg , the eqigg subclass specificities for fgbp , protein g , and protein a are distinct . although fgbp binds almost exclusively to eqigg4 and eqigg7 , protein g binds strongly to eqigg1 , eqigg4 , and eqigg7 and shows intermediate binding to eqigg3 , low binding to eqigg2 and eqigg6 , and no binding to eqigg5 ( 16 ) . binding of eqiggs to protein a is generally quite low with the subclasses binding in the following order eqigg1 > eqigg3 = eqigg4 = eqigg7 > eqigg2 = eqigg5 > eqigg6 ( 16 ) . his in the ch3 domain of igg , which is critical for binding to protein a , either alone or together with its neighbor tyr , also appears to be a requirement for binding to fgbp . thus deviations from the his - tyr motif may account , at least in part , for the inability of eqigg6 and eqigg2 to bind fgbp . although our mutagenesis experiments have pinpointed amino acid differences that may account for the deficiency of some of the eqigg subclasses in fgbp binding , we predict that there may be additional residues in the eqigg fc outside the protein a and protein g binding sites that make unique contributions to the igg - fgbp interaction . together eqigg4 and eqigg7 are the most prevalent forms of igg in serum and are also found in mucosal secretions ( 37 ) . furthermore , both subclasses are able to stimulate a strong respiratory burst from equine peripheral blood leukocytes and effectively activate complement via the classical pathway ( 16 ) . accumulated evidence has highlighted that antibodies , in particular those that recognize fgbp , play a vital role in protection against strangles ( 38 - 40 ) . furthermore , fgbp - specific iggb ( i.e. eqigg4/eqigg7 ) has been recognized as the predominant igg subclass present in the sera of acute and convalescent strangles cases and in the nasal mucosa during the acute response ( 13 ) . the non - immune binding of antibodies by bacterial immunoglobulin binding proteins is presumed to subvert the host immune response . functionally , this could be through prevention of initial antibody binding to bacterial epitopes , evasion of detection by other components of the immune response by coating of bacteria with host antibody , or by blocking of fc - mediated effector functions such as complement activation or fc - receptor binding . several studies have investigated the ability of bacterial ig binding proteins to interfere with the effector functions of igg . the majority of available evidence appears to argue against the ability of such proteins to inhibit the binding of igg to fcr on phagocytic cells ( 41 ) . however , protein a has been shown to be capable of inhibiting c1q binding to surface - immobilized igg , thereby preventing complement activation ( 42 ) . similarly , the binding of protein h from s. pyogenes to surface - immobilized igg can inhibit c1q binding and partially block complement - mediated lysis ( 42 ) . in the current study , binding of fgbp to surface - immobilized eqigg4 and eqigg7 was clearly able to disrupt the binding of c1q and prevent subsequent activation of the classical complement pathway . our results indicate that the observed inhibition of c1q binding and complement activation depends on the binding of fgbp to eqigg , because when an eqigg subclass that is unable to bind fgbp is used ( eqigg3 ) , no inhibition is seen . because specific antibody and complement have both been shown necessary to combat s. equi ( 43 ) , such targeting of the major complement - activating eqigg subclasses may afford the bacterium an effective means to disrupt antibody - mediated elimination . the observed fgbp - mediated inhibition of both c1q binding to igg and complement activation , although substantial , was not complete . our use of a soluble version of fgbp may have underestimated the potency , in this regard , of fgbp , which in its natural form is an abundant cell wall - associated protein ( 5 - 7 ) . one might speculate that anchoring of igg close to the bacterial surface by cell wall - bound fgbp is likely to increase the chance of steric hindrance . thus the approach and binding of such a large molecule as c1q would be compromised , resulting in more complete inhibition of the complement pathway . support for this notion comes from related studies with protein g. although one study found soluble streptococcal protein g unable to block both c1q binding to igg and complement - mediated lysis of igg - sensitized red blood cells ( 42 ) , another demonstrated that , when igg is bound by bacterial cell - surface protein g , interaction with c1q is completely blocked ( 44 ) . the authors of the second study proposed that the presence of protein g in a defined orientation and spacing on the streptococcal cell surface facilitates inhibition of the interaction of c1q with protein g - bound igg . in a similar fashion , one might argue that the particular spatial arrangement of fgbp on the surface of s. equi cells may effectively prevent the approach and interaction of bulky c1q molecules with any igg that is bound to fgbp . thus we consider that there are good reasons to believe that the fgbp - igg interaction is likely to make an important contribution to the pathophysiology of strangles , at least in part through inhibition of complement - mediated clearance mechanisms . under the physiological conditions of plasma , one might expect binding of both fibrinogen and igg to fgbp , assuming access / steric hindrance is not an issue . further experiments will be necessary to assess the impact that fibrinogen binding might have on the interaction of igg with fgbp and on the prevalent mechanism of inhibition of complement activation ( 45 ) . s. equi has evolved multiple secreted and cell surface - associated factors that interact with host proteins and promote adherence and/or help to evade immune responses ( 1 ) . these include another immunoglobulin - binding protein , the 2-macroglobulin , albumin , and igg - binding protein or eag ( 46 ) . the equine igg subclass specificity and the biological effects of igg binding by eag have not been elucidated and would make an informative comparison with fgbp , particularly because this protein is the subject of vaccine studies ( 47 - 49 ) . zooepidemicus , is reported to bind igg from a broader range of species than fgbp and to bind horse igg ( subclass not specified ) with a much higher affinity ( ka of 1 10 m ) ( 50 ) . in conclusion , we have shown that the fc - mediated binding of equine igg by s. equi fgbp is highly specific for the eqigg4 and eqigg7 subclasses . in common with several other streptococcal and staphylococcal igg- and iga - specific ig - binding proteins , fgbp binds to the domain interface of the ig fc region , illustrating a common mode of interaction that has been adopted by these important virulence factors .

the m protein of streptococcus equi subsp . equi known as fibrinogen - binding protein ( fgbp ) is a cell wall - associated protein with antiphagocytic activity that binds igg . recombinant versions of the seven equine igg subclasses were used to investigate the subclass specificity of fgbp . fgbp bound predominantly to equine igg4 and igg7 , with little or no binding to the other subclasses . competitive binding experiments revealed that fgbp could inhibit the binding of staphylococcal protein a and streptococcal protein g to both igg4 and igg7 , implicating the fc interdomain region in binding to fgbp . to identify which of the two igg fc domains contributed to the interaction with fgbp , we tested two human igg1/iga1 domain swap mutants and found that both domains are required for full binding , with the ch3 domain playing a critical role . the binding site for fgbp was further localized using recombinant equine igg7 antibodies with single or double point mutations to residues lying at the ch2-ch3 interface . we found that interaction of fgbp with equine igg4 and igg7 was able to disrupt c1q binding and antibody - mediated activation of the classical complement pathway , demonstrating an effective means by which s. equi may evade the immune response . the mode of interaction of fgbp with igg fits a common theme for bacterial ig - binding proteins . remarkably , for those interactions studied in detail , it emerges that all the ig - binding proteins target the ch2-ch3 domain interface , regardless of specificity for igg or iga , streptococcal or staphylococcal origin , or host species ( equine or human ) .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION

for generation of fgbpm , a recombinant form of fgbp that lacks the central domain between the a and b repeats ( amino acids 279 - 348 ) and does not bind to equine igg,3 fgbp gene sequences encoding amino acids 37 - 278 and 349 - 470 were amplified and juxtaposed in the vector pqe30 . analysis of the interaction of fgbp with recombinant equine igs by elisa interaction of fgbp1 with recombinant forms of the seven equine igg ( reqigg ) subclasses , produced and purified as described previously ( 16 ) , was analyzed by elisa . the ph dependence of the fgbp - igg interaction was analyzed using the same elisa except that reqiggs ( serial dilutions of 0 - 10 g / ml ) were diluted in pbs - t at either ph 5.5 , ph 7.4 , or ph 8.5 , and fgbp1 and fgbpm were used at 5 g / ml . it was necessary to use different concentrations of hrp - conjugated protein g and protein a in the assay because of the different binding affinities of the two proteins for igg . binding of recombinant human igs to fgbp two previously described recombinant human igg1/iga1 domain - swap mutants ( 18 ) were assessed for binding to fgbp1 . site - directed mutagenesis of eqigg7 heavy ( h ) chain single or double point mutations were introduced into the ch2 or ch3 domains of the eqigg7 h - chain using a quikchange ii xl site - directed mutagenesis kit ( stratagene , la jolla , ca ) , following the manufacturer 's instructions . fgbp - mediated inhibition of c1q binding and activation of the classic complement pathway by equine igg was assessed by elisa using reqigg3 , reqigg4 , and reqigg7 ( 10 g / ml ) , as described previously ( 16 ) , with the following modifications . in the former case , binding of c1q to igg was detected using sheep anti - human c1q - hrp , as previously described , whereas in the latter , activation of the classic complement pathway was detected using a 1/500 dilution of biotin - conjugated mouse anti - human c4c ( quidel , santa clara , ca ) followed by streptavidin - hrp ( 1/500 , dako , ely , cambs , uk ) . c4c generation is a specific marker for activation of the classic complement pathway ( 24 ) . fgbp1 bound strongly to igg4 and igg7 , but the remaining five igg subclasses and iga showed little or no binding . for both equine igg4 ( b ) and igg7 ( c ) , binding to fgbp1 was most efficient at ph 7.4 - 8.5 . binding of reqiggs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . fgbp1 bound strongly to igg4 and igg7 , but the remaining five igg subclasses and iga showed little or no binding . for both equine igg4 ( b ) and igg7 ( c ) , binding to fgbp1 was most efficient at ph 7.4 - 8.5 . binding of reqiggs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . only igg4 and igg7 showed concentration - dependent interaction with immobilized fgbp1 . analysis of fgbp - igg binding binding of recombinant fgbp1 to the seven reqigg subclasses and monomeric reqiga was first analyzed by elisa . fgbp1 bound strongly to reqigg4 and reqigg7 , with little or no binding to the remaining five reqigg subclasses or to reqiga ( fig . because igg binding by the bacterial igg - binding proteins staphylococcal protein a and streptococcal protein g is known to be ph - dependent ( 26 ) , we tested the effect of ph variation on the fgbp1-igg interaction ( fig . figure 3.elisa analysis of competition for binding to reqigg4 and reqigg7 between fgbp proteins and protein a and protein g. a , competition of binding of hrp - conjugated protein g ( 0.25 g / ml ) to reqigg4 and reqigg7 coated at 2.5 g / ml . elisa analysis of competition for binding to reqigg4 and reqigg7 between fgbp proteins and protein a and protein g. a , competition of binding of hrp - conjugated protein g ( 0.25 g / ml ) to reqigg4 and reqigg7 coated at 2.5 g / ml . figure 4.elisa analysis of fgbp1 binding to human igg1 , human igg1/iga1 domain - swap mutants and human igg3 allotype ighg301 . binding of igs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . elisa analysis of fgbp1 binding to human igg1 , human igg1/iga1 domain - swap mutants and human igg3 allotype ighg301 . binding of igs is expressed as absorbance obtained for the binding to fgbp1 minus absorbance for binding to the fgbpm control ( binding to the latter was typically < 0.15 absorbance units ) . to establish the affinity of the fgbp - igg interaction and confirm the equine igg subclass specificity we used surface plasmon resonance measurements . protein a and protein g are known to bind in the interdomain region of igg fc ( 22 , 23 ) and have both been shown to interact with reqigg4 and reqigg7 , although the binding to protein a is relatively weak ( 16 ) . therefore , we used a competition elisa to see if fgbp could inhibit binding of proteins a and g to reqigg4 and reqigg7 . we found that fgbp1 , but not the non - igg - binding control protein fgbpm , was able to compete with both protein a and protein g for binding to reqigg4 and reqigg7 ( fig . this inhibition was concentration - dependent , and binding of both protein a and protein g was negligible at the maximum concentration of fgbp1 ( 50 g / ml ) . these findings suggest that the fgbp binding site(s ) on reqigg4 and reqigg7 overlap with those of protein a and protein g in the fc interdomain region . to determine whether one or both of the igg fc domains contribute to fgbp binding , we utilized recombinant human igg1 and two human igg1/iga1 domain - swap mutants . a domain - swap mutant lacking the ch2 domain but retaining the ch3 domain of igg1 ( 123 ) was able to bind to fgbp1 , but at a much reduced level compared with that of wild - type human igg1 ( fig . hence , both fc domains are required for maximal binding , but the ch3 domain appears to be the most critical . igg3 allotypes capable of binding protein a possess a his residue at position 435 within the interaction site for protein a. however , the ighg301 allotype carries an arg at this critical ch3 domain residue and is unable to bind protein a ( 20 , 21 ) . we found that the ighg301 allotype did not bind fgbp1 , indicating that residue 435 is critical also for interaction with fgbp ( fig . to further localize the fgbp binding site on equine igg , we generated five igg7 mutants with single or double point mutations in the regions corresponding to the protein a / protein g binding sites ( table 1 ) . analysis of the igg7 mutants by sds - page and western blotting revealed that all the mutant reqigg7s assembled like wild - type reqigg7 and retained the anticipated reactivity with antigen and both polyclonal and monoclonal anti - equine iggb ( i.e. these test outcomes and the fact that the substitutions were based on residues present in other equine igg isotypes suggest that the likelihood of unintended conformational disruption in the mutants is extremely low , allowing us to draw meaningful conclusions on the relative contributions of the mutated residues to fgbp binding . however , the double point mutants hn433/434eh and hy435/436tv showed drastically impaired abilities to bind fgbp1 , retaining < 5% of the binding capacity of wild - type reqigg7 . taken together these results suggest that the binding site on igg for fgbp encompasses some of the same interdomain residues as those involved in interaction with protein a and protein g. residues in the leu - ser and the leu - tyr loops are particularly implicated . comparison of the binding affinities of mutants h433r and hn433/434eh suggests that either his is not critical for fgbp binding , or that alteration of this residue to a positively charged amino acid such as arginine ( as in h433r ) can be tolerated without significant impact on the affinity of the interaction . overall , we can conclude that the binding site for fgbp on reqigg fc appears to center on the interdomain region , utilizing residues common to both the protein a and protein g binding sites . figure 7.amino acid alignments of reqigg subclasses and reqigg7 mutants showing only ch2 and ch3 residues critical for interaction with protein a and protein g. residue numbers ( shown above sequences ) correspond to human igg1 numbering . in the corresponding higg1 sequence ( shown for comparison at the top ) , residues implicated in interactions are indicated as follows : protein g , normal text ; protein a , underlined ; both protein a and protein g , box . amino acid alignments of reqigg subclasses and reqigg7 mutants showing only ch2 and ch3 residues critical for interaction with protein a and protein g. residue numbers ( shown above sequences ) correspond to human igg1 numbering . in the corresponding higg1 sequence ( shown for comparison at the top ) , residues implicated in interactions are indicated as follows : protein g , normal text ; protein a , underlined ; both protein a and protein g , box . were able to disrupt c1q binding , detected using anti - c1q antibody ( a ) , and activation of the classical complement pathway , detected using anti - c4c antibody ( b ) , by reqigg4 and reqigg7 but not by reqigg3 , which binds poorly to fgbp1 . in each case , c1q binding / activation are shown as a percentage of the binding / activation seen in the absence of fgbp1 . increasing concentrations of fgbp1 were able to disrupt c1q binding , detected using anti - c1q antibody ( a ) , and activation of the classical complement pathway , detected using anti - c4c antibody ( b ) , by reqigg4 and reqigg7 but not by reqigg3 , which binds poorly to fgbp1 . to gain an insight into the physiological consequences of fgbp binding to eqigg4 and eqigg7 , we investigated whether fgbp1 could inhibit their ability to bind c1q and activate the classical complement pathway . we found that fgbp1 was able to disrupt c1q binding and complement activation by reqigg4 and reqigg7 ( fig . in contrast , fgbp1 was unable to disrupt c1q binding and complement activation by reqigg3 , which we have shown binds poorly or not at all to fgbp1 ( fig . in this study we addressed the eqigg subclass specificity of fgbp and found that this bacterial protein is highly specific for only two ( eqigg4 and eqigg7 ) of the seven eqigg subclasses . we have demonstrated that fgbp binds to the interdomain region of igg fc and that both of the fc domains are required for full binding . the residues implicated in our mutagenesis studies as critical for binding to fgbp are known to mediate interaction with staphylococcal protein a and streptococcal protein g also ( 22 , 23 ) . even more remarkably , the fact that these unrelated igg - binding proteins bind to similar sites in the igg interdomain region parallels the situation for the unrelated bacterial iga binding proteins sir22 from s. pyogenes , protein from group b streptococcus , and ssl7 from staphylococcus aureus , which all bind to the fc domain interface in human iga ( 29 - 31 ) ( fig . figure 9.interaction sites for streptococcal and staphylococcal ig - binding proteins on igg and iga fc regions . remarkably , all the ig - binding proteins target the ch2-ch3 domain interface , regardless of specificity for igg or iga , streptococcal or staphylococcal origin , or host species ( equine or human ) . interaction sites for streptococcal and staphylococcal ig - binding proteins on igg and iga fc regions . a , fgbp from s. equi in purple ; b , protein g from group c and group g streptococci in red ; c , protein a from s. aureus in yellow ; d , sir22 from s. pyogenes in orange ; e, protein from group b streptococcus in pink ; f , ssl7 from s. aureus in green . remarkably , all the ig - binding proteins target the ch2-ch3 domain interface , regardless of specificity for igg or iga , streptococcal or staphylococcal origin , or host species ( equine or human ) . in igg , for example , the fc interdomain region forms the interaction site for fcrn , the so - called neonatal fc receptor that mediates a number of processes fundamental to igg function , including regulation of igg turnover and transepithelial transfer of igg ( 35 ) . for iga , the ch2/ch3 interdomain region serves as the interaction site for its specific fc receptor , fcri , which triggers efficient elimination mechanisms against iga - coated pathogens ( 18 , 36 ) . despite the marked overlap in their binding sites on igg , the eqigg subclass specificities for fgbp , protein g , and protein a are distinct . although fgbp binds almost exclusively to eqigg4 and eqigg7 , protein g binds strongly to eqigg1 , eqigg4 , and eqigg7 and shows intermediate binding to eqigg3 , low binding to eqigg2 and eqigg6 , and no binding to eqigg5 ( 16 ) . binding of eqiggs to protein a is generally quite low with the subclasses binding in the following order eqigg1 > eqigg3 = eqigg4 = eqigg7 > eqigg2 = eqigg5 > eqigg6 ( 16 ) . his in the ch3 domain of igg , which is critical for binding to protein a , either alone or together with its neighbor tyr , also appears to be a requirement for binding to fgbp . although our mutagenesis experiments have pinpointed amino acid differences that may account for the deficiency of some of the eqigg subclasses in fgbp binding , we predict that there may be additional residues in the eqigg fc outside the protein a and protein g binding sites that make unique contributions to the igg - fgbp interaction . functionally , this could be through prevention of initial antibody binding to bacterial epitopes , evasion of detection by other components of the immune response by coating of bacteria with host antibody , or by blocking of fc - mediated effector functions such as complement activation or fc - receptor binding . however , protein a has been shown to be capable of inhibiting c1q binding to surface - immobilized igg , thereby preventing complement activation ( 42 ) . similarly , the binding of protein h from s. pyogenes to surface - immobilized igg can inhibit c1q binding and partially block complement - mediated lysis ( 42 ) . in the current study , binding of fgbp to surface - immobilized eqigg4 and eqigg7 was clearly able to disrupt the binding of c1q and prevent subsequent activation of the classical complement pathway . our results indicate that the observed inhibition of c1q binding and complement activation depends on the binding of fgbp to eqigg , because when an eqigg subclass that is unable to bind fgbp is used ( eqigg3 ) , no inhibition is seen . because specific antibody and complement have both been shown necessary to combat s. equi ( 43 ) , such targeting of the major complement - activating eqigg subclasses may afford the bacterium an effective means to disrupt antibody - mediated elimination . our use of a soluble version of fgbp may have underestimated the potency , in this regard , of fgbp , which in its natural form is an abundant cell wall - associated protein ( 5 - 7 ) . one might speculate that anchoring of igg close to the bacterial surface by cell wall - bound fgbp is likely to increase the chance of steric hindrance . support for this notion comes from related studies with protein g. although one study found soluble streptococcal protein g unable to block both c1q binding to igg and complement - mediated lysis of igg - sensitized red blood cells ( 42 ) , another demonstrated that , when igg is bound by bacterial cell - surface protein g , interaction with c1q is completely blocked ( 44 ) . the authors of the second study proposed that the presence of protein g in a defined orientation and spacing on the streptococcal cell surface facilitates inhibition of the interaction of c1q with protein g - bound igg . in a similar fashion , one might argue that the particular spatial arrangement of fgbp on the surface of s. equi cells may effectively prevent the approach and interaction of bulky c1q molecules with any igg that is bound to fgbp . under the physiological conditions of plasma , one might expect binding of both fibrinogen and igg to fgbp , assuming access / steric hindrance is not an issue . further experiments will be necessary to assess the impact that fibrinogen binding might have on the interaction of igg with fgbp and on the prevalent mechanism of inhibition of complement activation ( 45 ) . the equine igg subclass specificity and the biological effects of igg binding by eag have not been elucidated and would make an informative comparison with fgbp , particularly because this protein is the subject of vaccine studies ( 47 - 49 ) . in conclusion , we have shown that the fc - mediated binding of equine igg by s. equi fgbp is highly specific for the eqigg4 and eqigg7 subclasses . in common with several other streptococcal and staphylococcal igg- and iga - specific ig - binding proteins , fgbp binds to the domain interface of the ig fc region , illustrating a common mode of interaction that has been adopted by these important virulence factors .

the infant mortality rate in zimbabwe which is at 97 deaths per 1,000 is significantly high.[1 , 2 ] paradoxically , exclusive breast feeding ( ebf ) , which is one of the major interventions to this social catastrophe which is recommended , free , accessible , sustainable and safe is not far from the reach of many rural women but is being underutilized . a large body of evidence demonstrates the benefits of ebf infant feeding practice for child survival , growth , and development . this is premised on its high immunological , nutritional and hygienic value as compared to other liquids , solids and bottled infant milk formulas . lack of ebf is associated with high incidences of diarrhoea , pneumonia , bacterial meningitis , bacteraemia , respiratory tract infection , necrotizing enteri colitis and malnutrition . these in turn are responsible for high morbidity and mortality in the lifelong associated with poor school performance , impaired intellectual and social development.[4 , 5 , 6 ] ebf is defined as giving breast milk only to infants from 0 - 6 months , and not feeding any food or liquid , not even water , with the exception of drops or syrups consisting of vitamins , mineral supplements or medicine as only recommended by the health workers a global health journal series on child survival identified the promotion of ebf of infants during the first 6 months of life and continued breastfeeding to 12 months as the single most - effective preventive public health intervention for reducing mortality among children aged , 5 years and below . more recently , the series on maternal and child under - nutrition estimated that 1.06 million child deaths ( 10% of all mortality in children aged 5 years ) are attributable to non - exclusive breastfeeding in the first 6 months of life . according to the national nutrition survey the current national ebf rate in zimbabwe stands at a staggering rate of 5 percent . the multiple indicator monitoring survey provides a higher national rate of 21% , with a higher proportion of 29% in urban areas being exclusively breast fed compared to 26% in rural areas the ministry of health and child care in zimbabwe recommends and promotes ebf as the most appropriate infant feeding practice for children from 0 - 6 months of age . despite efforts by the respective ministries to increase the ebf uptake , its uptake by mothers of infants remain very low.[1 , 2 , 4 ] breastfeeding is a complex process governed by psychological and physiological factors , which in turn are conditioned by a wide spectrum of environmental , socioeconomic and cultural circumstances . in view of the fact that ebf is a physiological process which transpires in a specific socio - economic milieu , the purpose of this paper is to investigate the socio - economic factors that hinders its uptake among rural women in zimbabwe the concurrent nested model of mixed research methodology was employed and was identified by its use of one data collection phase , during which both quantitative and qualitative data are collected simultaneously . the study was carried out in mataga ward , mberengwa district at mataga rural health center , and midlands s province in zimbabwe from april to june 2013 . the target population was mothers who have infants from 0 - 6 months who were accessing neonatal health services during the data collection month . convenience sampling method was utilized to select 20 mothers who took part in the study . purposive sampling was utilized used to select the key informants ( nurses and village health workers ) . the study was reviewed and approved by the university of zimbabwe ethics board as well as the general academic board before implementation . in addition , research participants were asked to give their informed consent through the signing of a consent form before any responses were solicited from them . an interview schedule composed of open - ended and closed questionnaires was used to collect data from mothers and an interview guide was used for the key informants . quantitative data was analysed using the statistical package for social scientists version 17 while qualitative data was analysed through content coding procedures based on a set of predetermined themes of interest . the main research questions were : 1 ) what are the challenges did you / are you experiencing in practicing ebf . 2 ) what are the barriers that prevent or affect you / mothers from practicing ebf ? the concurrent nested model of mixed research methodology was employed and was identified by its use of one data collection phase , during which both quantitative and qualitative data are collected simultaneously . the study was carried out in mataga ward , mberengwa district at mataga rural health center , and midlands s province in zimbabwe from april to june 2013 . the target population was mothers who have infants from 0 - 6 months who were accessing neonatal health services during the data collection month . convenience sampling method was utilized to select 20 mothers who took part in the study . purposive sampling was utilized used to select the key informants ( nurses and village health workers ) . the study was reviewed and approved by the university of zimbabwe ethics board as well as the general academic board before implementation . in addition , research participants were asked to give their informed consent through the signing of a consent form before any responses were solicited from them . an interview schedule composed of open - ended and closed questionnaires was used to collect data from mothers and an interview guide was used for the key informants . quantitative data was analysed using the statistical package for social scientists version 17 while qualitative data was analysed through content coding procedures based on a set of predetermined themes of interest . the main research questions were : 1 ) what are the challenges did you / are you experiencing in practicing ebf . 2 ) what are the barriers that prevent or affect you / mothers from practicing ebf ? out of the 20 mothers interviewed in the study , 45% ( n=9 ) were from the 20 - 25 age range . 15% ( n=3 ) respondents were within 15 - 19 age range , while age groups 26 - 30 and 36 + had 3 respondents each . 10% ( n=2 ) of the respondents belonged to the 31 - 35 age group . the high composition of mothers in the 20 - 25 age groups confirms the findings of the zimbabwe demographic health survey which reported a high fertility rate among women from the age of 20 - 25 . a large proportion of respondents 13 ( 65% ) managed to reach primary level . a total of 7 ( 35% ) mothers managed to reach secondary level . the primary school attendant rate in this study tallies with the findings of the multiple indicator monitoring survey which recorded a 99 % primary education net attendance of the girl child in zimbabwe . education levels influence the uptake of ebf . among the 20 ( 100% ) mothers who were interviewed , five ( 25% ) had an income estimate of $ 5 , four ( 20% ) mothers earned an estimate of $ 20 , four ( 20% ) mothers earned an estimate of $ 50 , three ( 15% ) mothers earned $ 100 , while a paltry two ( 10% ) mothers earned more than $ 100 . eighteen ( 90% ) mothers and 13(65 ) fathers reported that they were not employed . many respondents exhibited extensive knowledge and understanding of the meaning and benefits of exclusive breast feeding . fifteen ( 70% ) mothers indicated that they understand the meaning of ebf and its benefits while five ( 25% ) mothers indicated knowledge of the meaning of ebf but not its benefits . there was no significant correlation between this knowledge of the meaning of ebf and the actual practice . for example , out of the 70% ( n=14 ) of the mothers who reported knowledge of ebf and its benefits , only 25 % ( n=5 ) of them managed to practice ebf . this state of affairs is a clear pointer to the fact that knowledge of ebf does not translate to the active uptake of the same . these results can be elucidated by the theory of planned behaviour which states that knowledge alone is not sufficient to change behaviour but attitudes , subjective norms and perceived behavioural control regarding behaviour , all influence the intention to perform certain behaviour . the discrepancy between knowledge and behaviour could also well be traced back to the influence of culture in rewarding and punishing whatever is deemed acceptable and unacceptable behaviour in the zimbabwean society . to this end , respondents testified that their culture expects and demands that babies be fed on solids at least within a week of their birth . failure to comply with such an expectation could be misconstrued for infidelity in a family where tradition holds that feeding on solids is a sign of genetic connection of the baby with its ancestors . five of the 7 mothers ( 71% ) who had secondary education managed to practice ebf . only two mothers ( 14% ) with primary education managed to practice exclusive breast feeding . these results show a higher prevalence of ebf among respondents with secondary education ( 71% ) than with primary education ( 14% ) . such a nexus between level of education and ebf stands as a clear testimony that education , like in other parts of the world , remains a vital weapon to fight against the power of some retrogressive traditional beliefs in zimbabwean society . the influence of education on the uptake of ebf was also recognized by all the nurses ( key informants ) who registered a high prevalence among mothers who have secondary education . implicit in these results is that educating a girl child is likely to promote and sustain positive health behavior in traditional and rural communities . zimbabwe s tradition demands that traditional medicine be administered on babies to try and avert the death caused by the condition that culminates in the subsiding of the fontanel in babies due mainly to severe dehydration ( a condition known as nhova in vernacular ) . this practice is also common in kenya where infants are given various herbs to treat the condition known in kenya as ndebele . traditionally , this condition is attributed to evil spells . out of the 70% ( n=14 ) respondents not practicing ebf , 12 of them reported that they had treated nhova on their babies through administering different foods and medicines such as barks , juices , roots , herbs , cooking oil and wild fruits . once more , such a traditional practice militates against the core tenets of exclusive breast feeding that prohibit the intake of any solids let alone medicine unless prescribed by a qualified medical practitioner . to sustain ebf , sight should not be lost of these traditional practices and safeguards that have tended to supersede modern medicine as guarantees of survival in resource poor communities . significant others such as grandmothers , mothers in - law and husbands influence the infant feeding practices pursued by mothers . respondents identified mothers - in - law as key authority figures regarding the uptake of ebf . for example , 70% ( n=14 ) of respondents indicated that mothers in law were key decision makers with regard to infant feeding . in an african culture , older persons are regarded as a fountain of knowledge and younger persons tap knowledge from them . most of the advice that came from the mothers - in - law discourage exclusive breast feeding in favor of mixed infant feeding practice . the mother - in - law is usually powerful and influential in major decisions concerning the behavior and conduct of her daughter - in - law . according to one respondent , the mother in law gave the infant porridge a day after delivery arguing that the family tradition requires that the baby be fed on porridge the day she gets home from the hospital by its grandmother . the respondents confess that their in - laws admonish that their clan is so unique that any of its offspring is born hungry . on the same note , four respondents indicated that on leaving their babies with their grandmothers , they were automatically fed on porridge on account of cultural norms and traditions . it is worth noting that such a scenario is in no way peculiar to zimbabwe as it corresponds with findings in malawi that the influence of the mother - in - law and husband was one of the greatest barriers to ebf uptake in malawi . this state of affairs has its explanation in the theory of planned behavior which envisages that people s behaviors are influenced by normative beliefs , which are beliefs about the normative expectations of others and motivation to comply with those expectations . implicit in this scenario is that sometimes one s level of education in a rural environment becomes subservient to the expectations and dictates of culture and tradition . in such a situation , it becomes important for any health promotion strategy to be ingrained within the cultural expectations of the particular locality within which it is conducted . power dynamics within the family was cited as a key factor exerting its influence with regard to the feeding of the babies . in an african rural milieu , eighteen ( 90% ) respondents noted that they had been advised by their husbands on infant feeding . indications are that fathers of children of 65% ( n=13 ) of the 18 respondents were against ebf as they argued that the child should be given porridge as breast milk was not enough for the baby and that was why the baby is always crying . another respondent reported that the husband is the one who bought peanut butter and margarine so that she can prepare porridge for the infant . one respondent confirmed having been confronted with what has tended to be the greatest question of the day each time ebf was discussed in a traditional african setup namely : implicit in such a scenario is that gender roles cut across all the facts of life , undermining even those aspects of human existence ordinarily thought to belong to the wife in a matrimonial relationship . this makes it inevitable for health promotion practitioners to look at husbands as key stakeholders if ever such programs as ebf are to be successful . poverty and unemployment were cited as some of the impediments against ebf by many respondents . respondents contend that they find it difficult to practice ebf given that inadequate food with low nutritional values exacerbate the low uptake of ebf . this study observed that women in zimbabwe have limited income and economic their status is low . among the 100% ( n=20 ) respondents interviewed 25% ( n=5 ) had an income estimate of $ 5 , 20% ( n=4 ) respondents earn an estimate of $ 20 , 20% ( n=4 ) respondents earn an estimate of $ 50 , 15% ( n=3 ) respondents earn $ 100 , while a paltry 10% ( n=2 ) respondents earns more than $ 100 . prior study has shown that wealth quintile or income influences the uptake of exclusive breast feeding . low amounts of milk from ill - fed and malnourished mothers have come as an unfortunate deterrent against ebf . the study noted a high prevalence of ebf among mothers whose incomes were above $ 100 . for example , 66% ( n=4 ) of the 100% ( n=6 ) respondents actively participating in ebf had an income above $ 100 . the three nurses and two village health workers in the study reported that most mothers are not adhering to ebf because they were not producing enough milk . the above findings are in sync with those from the zimbabwe vulnerability assessment committee which found out that 39 - 45 percent of the people in meringa were food insecure . the findings therefore draw the attention of all players in the promotion of ebf program as well as those involved in hiv prevention ( especially the prevention of mother to child transmission of hiv ) to seriously take poverty , unemployment and malnutrition as obstacles against the achievement of set objectives . informed by such a reality on the ground , the success of these programs rests on the adoption of a holistic approach to programming . such programs as supplementary feeding of lactating mothers as well as social cash transfers can go a long way in fostering and sustaining ebf . this study could not directly establish the influence of the gender of the child on ebf as the number of the male child was significantly very low to the number of girls . 13 ( 65% ) mothers confirmed that they had a girl child while 35% ( n=7 ) mothers confirmed that they had a boy child . however , six mothers reported the challenges they faced in practicing ebf , adding that the boy child eats more than the girl child and hence mother s milk is not enough for the male infants . one of the village health workers respondent # 2 reported that boys eat very much and that mother s milk might not be enough for the boy child . however , nurse respondent # 1 and village health worker respondent # 3 denied that boys eat more than girls at this tender age . it was raised that these are some of the beliefs that are held by communities that impede the ebf uptake . all of the village health workers ( n=4 ; 100% ) confirmed that this was a wide belief that boys eat very much and it is impossible for them to be satisfied by mother s milk alone . this sends a clear message to public health promoters that they need to know and address the misconceptions and myths held by communities that militate against the uptake of ebf especially for boy - children . out of the 20 mothers interviewed in the study , 45% ( n=9 ) were from the 20 - 25 age range . 15% ( n=3 ) respondents were within 15 - 19 age range , while age groups 26 - 30 and 36 + had 3 respondents each . 10% ( n=2 ) of the respondents belonged to the 31 - 35 age group . the high composition of mothers in the 20 - 25 age groups confirms the findings of the zimbabwe demographic health survey which reported a high fertility rate among women from the age of 20 - 25 . a total of 7 ( 35% ) mothers managed to reach secondary level . the primary school attendant rate in this study tallies with the findings of the multiple indicator monitoring survey which recorded a 99 % primary education net attendance of the girl child in zimbabwe . among the 20 ( 100% ) mothers who were interviewed , five ( 25% ) had an income estimate of $ 5 , four ( 20% ) mothers earned an estimate of $ 20 , four ( 20% ) mothers earned an estimate of $ 50 , three ( 15% ) mothers earned $ 100 , while a paltry two ( 10% ) mothers earned more than $ 100 . eighteen ( 90% ) mothers and 13(65 ) fathers reported that they were not employed . many respondents exhibited extensive knowledge and understanding of the meaning and benefits of exclusive breast feeding . fifteen ( 70% ) mothers indicated that they understand the meaning of ebf and its benefits while five ( 25% ) mothers indicated knowledge of the meaning of ebf but not its benefits . there was no significant correlation between this knowledge of the meaning of ebf and the actual practice . for example , out of the 70% ( n=14 ) of the mothers who reported knowledge of ebf and its benefits , only 25 % ( n=5 ) of them managed to practice ebf . this state of affairs is a clear pointer to the fact that knowledge of ebf does not translate to the active uptake of the same . these results can be elucidated by the theory of planned behaviour which states that knowledge alone is not sufficient to change behaviour but attitudes , subjective norms and perceived behavioural control regarding behaviour , all influence the intention to perform certain behaviour . the discrepancy between knowledge and behaviour could also well be traced back to the influence of culture in rewarding and punishing whatever is deemed acceptable and unacceptable behaviour in the zimbabwean society . to this end , respondents testified that their culture expects and demands that babies be fed on solids at least within a week of their birth . failure to comply with such an expectation could be misconstrued for infidelity in a family where tradition holds that feeding on solids is a sign of genetic connection of the baby with its ancestors . five of the 7 mothers ( 71% ) who had secondary education managed to practice ebf . eleven only two mothers ( 14% ) with primary education managed to practice exclusive breast feeding . these results show a higher prevalence of ebf among respondents with secondary education ( 71% ) than with primary education ( 14% ) . such a nexus between level of education and ebf stands as a clear testimony that education , like in other parts of the world , remains a vital weapon to fight against the power of some retrogressive traditional beliefs in zimbabwean society . the influence of education on the uptake of ebf was also recognized by all the nurses ( key informants ) who registered a high prevalence among mothers who have secondary education . implicit in these results is that educating a girl child is likely to promote and sustain positive health behavior in traditional and rural communities . zimbabwe s tradition demands that traditional medicine be administered on babies to try and avert the death caused by the condition that culminates in the subsiding of the fontanel in babies due mainly to severe dehydration ( a condition known as nhova in vernacular ) . this practice is also common in kenya where infants are given various herbs to treat the condition known in kenya as ndebele . traditionally , this condition is attributed to evil spells . out of the 70% ( n=14 ) respondents not practicing ebf , 12 of them reported that they had treated nhova on their babies through administering different foods and medicines such as barks , juices , roots , herbs , cooking oil and wild fruits . once more , such a traditional practice militates against the core tenets of exclusive breast feeding that prohibit the intake of any solids let alone medicine unless prescribed by a qualified medical practitioner . to sustain ebf , sight should not be lost of these traditional practices and safeguards that have tended to supersede modern medicine as guarantees of survival in resource poor communities . significant others such as grandmothers , mothers in - law and husbands influence the infant feeding practices pursued by mothers . respondents identified mothers - in - law as key authority figures regarding the uptake of ebf . for example , 70% ( n=14 ) of respondents indicated that mothers in law were key decision makers with regard to infant feeding . in an african culture , older persons are regarded as a fountain of knowledge and younger persons tap knowledge from them . most of the advice that came from the mothers - in - law discourage exclusive breast feeding in favor of mixed infant feeding practice . the mother - in - law is usually powerful and influential in major decisions concerning the behavior and conduct of her daughter - in - law . according to one respondent , the mother in law gave the infant porridge a day after delivery arguing that the family tradition requires that the baby be fed on porridge the day she gets home from the hospital by its grandmother . the respondents confess that their in - laws admonish that their clan is so unique that any of its offspring is born hungry . on the same note , four respondents indicated that on leaving their babies with their grandmothers , they were automatically fed on porridge on account of cultural norms and traditions . it is worth noting that such a scenario is in no way peculiar to zimbabwe as it corresponds with findings in malawi that the influence of the mother - in - law and husband was one of the greatest barriers to ebf uptake in malawi . this state of affairs has its explanation in the theory of planned behavior which envisages that people s behaviors are influenced by normative beliefs , which are beliefs about the normative expectations of others and motivation to comply with those expectations . implicit in this scenario is that sometimes one s level of education in a rural environment becomes subservient to the expectations and dictates of culture and tradition . in such a situation , it becomes important for any health promotion strategy to be ingrained within the cultural expectations of the particular locality within which it is conducted . power dynamics within the family was cited as a key factor exerting its influence with regard to the feeding of the babies . in an african rural milieu , eighteen ( 90% ) respondents noted that they had been advised by their husbands on infant feeding . indications are that fathers of children of 65% ( n=13 ) of the 18 respondents were against ebf as they argued that the child should be given porridge as breast milk was not enough for the baby and that was why the baby is always crying . another respondent reported that the husband is the one who bought peanut butter and margarine so that she can prepare porridge for the infant . one respondent confirmed having been confronted with what has tended to be the greatest question of the day each time ebf was discussed in a traditional african setup namely : implicit in such a scenario is that gender roles cut across all the facts of life , undermining even those aspects of human existence ordinarily thought to belong to the wife in a matrimonial relationship . this makes it inevitable for health promotion practitioners to look at husbands as key stakeholders if ever such programs as ebf are to be successful . poverty and unemployment were cited as some of the impediments against ebf by many respondents . respondents contend that they find it difficult to practice ebf given that inadequate food with low nutritional values exacerbate the low uptake of ebf . this study observed that women in zimbabwe have limited income and economic their status is low . among the 100% ( n=20 ) respondents interviewed 25% ( n=5 ) had an income estimate of $ 5 , 20% ( n=4 ) respondents earn an estimate of $ 20 , 20% ( n=4 ) respondents earn an estimate of $ 50 , 15% ( n=3 ) respondents earn $ 100 , while a paltry 10% ( n=2 ) respondents earns more than $ 100 . prior study has shown that wealth quintile or income influences the uptake of exclusive breast feeding . low amounts of milk from ill - fed and malnourished mothers have come as an unfortunate deterrent against ebf . the study noted a high prevalence of ebf among mothers whose incomes were above $ 100 . for example , 66% ( n=4 ) of the 100% ( n=6 ) respondents actively participating in ebf had an income above $ 100 . the three nurses and two village health workers in the study reported that most mothers are not adhering to ebf because they were not producing enough milk . the above findings are in sync with those from the zimbabwe vulnerability assessment committee which found out that 39 - 45 percent of the people in meringa were food insecure . the findings therefore draw the attention of all players in the promotion of ebf program as well as those involved in hiv prevention ( especially the prevention of mother to child transmission of hiv ) to seriously take poverty , unemployment and malnutrition as obstacles against the achievement of set objectives . informed by such a reality on the ground , the success of these programs rests on the adoption of a holistic approach to programming . such programs as supplementary feeding of lactating mothers as well as social cash transfers can go a long way in fostering and sustaining ebf . this study could not directly establish the influence of the gender of the child on ebf as the number of the male child was significantly very low to the number of girls . 13 ( 65% ) mothers confirmed that they had a girl child while 35% ( n=7 ) mothers confirmed that they had a boy child . however , six mothers reported the challenges they faced in practicing ebf , adding that the boy child eats more than the girl child and hence mother s milk is not enough for the male infants . one of the village health workers respondent # 2 reported that boys eat very much and that mother s milk might not be enough for the boy child . however , nurse respondent # 1 and village health worker respondent # 3 denied that boys eat more than girls at this tender age . it was raised that these are some of the beliefs that are held by communities that impede the ebf uptake . all of the village health workers ( n=4 ; 100% ) confirmed that this was a wide belief that boys eat very much and it is impossible for them to be satisfied by mother s milk alone . this sends a clear message to public health promoters that they need to know and address the misconceptions and myths held by communities that militate against the uptake of ebf especially for boy - children . this study has revealed many impediments that confront the rural zimbabwean mothers against adopting ebf . it is therefore pertinent that efforts by the ministry of health and child care to upscale ebf be holistic so that it reaches all areas that determine the mothers to practice ebf . the respective government ministries should ensure increased male involvement in health programs that target families . male influence poses a barrier to the practice of ebf , hence it is imperative that they accompany their wives to clinics so that they are educated on the recommended infant feeding practices . community awareness and behavior change community mobilization on ebf should be scaled up and should not only target mothers but should be extended to members of the extended family such as grandmothers and mothers - in - law . the government needs to avail food to members of the community that are in hunger . mothers in rural areas are unable to practice ebf because they were producing little milk owing to lack of food and starvation.lack of education is associated with low ebf uptake . education of women has far greater values not just for ebf practice but for many health outcomes.societal norms , peer pressure , and traditional practices play significant roles in ebf practices among rural women . the government needs to avail food to members of the community that are in hunger . mothers in rural areas are unable to practice ebf because they were producing little milk owing to lack of food and starvation . education of women has far greater values not just for ebf practice but for many health outcomes . societal norms , peer pressure , and traditional practices play significant roles in ebf practices among rural women .

objectives : exclusive breast feeding ( ebf ) uptake in zimbabwe is very low . given that ebf is a physiological process which transpires in a specific socio - economic milieu , this study investigates the socio - economic factors militating against its uptake.methods:the study used a mixed research methodology . the concurrent nested model of mixed methods was utilized using one data collection phase , during which both quantitative and qualitative data were collected simultaneously.results:the research noted that factors such as low education , low income , gender inequalities , social influence , and traditional practices were hindering the uptake of exclusive breast feeding.conclusions and global health implications : the study envisages that it is pertinent for infant feeding programs to address socio - economic barriers to ebf in order to influence a positive uptake . the potential interventions include increasing men s involvement , raising awareness on ebf , and strengthening the village health worker program .

Introduction Methodology Study Design, Target Population and Sampling Data Collection and Analysis Results Demographic characteristics - age distribution) Respondents level of education Income of the respondents (mothers) Knowledge and understanding of EBF Level of education and EBF practice Traditional practices hindering EBF (Nhova Treatment) Pressure from in-laws Gender inequality and EBF Income and EBF Gender of the child Conclusions and Public Health Implications

the infant mortality rate in zimbabwe which is at 97 deaths per 1,000 is significantly high. [1 , 2 ] paradoxically , exclusive breast feeding ( ebf ) , which is one of the major interventions to this social catastrophe which is recommended , free , accessible , sustainable and safe is not far from the reach of many rural women but is being underutilized . a large body of evidence demonstrates the benefits of ebf infant feeding practice for child survival , growth , and development . lack of ebf is associated with high incidences of diarrhoea , pneumonia , bacterial meningitis , bacteraemia , respiratory tract infection , necrotizing enteri colitis and malnutrition . [4 , 5 , 6 ] ebf is defined as giving breast milk only to infants from 0 - 6 months , and not feeding any food or liquid , not even water , with the exception of drops or syrups consisting of vitamins , mineral supplements or medicine as only recommended by the health workers a global health journal series on child survival identified the promotion of ebf of infants during the first 6 months of life and continued breastfeeding to 12 months as the single most - effective preventive public health intervention for reducing mortality among children aged , 5 years and below . according to the national nutrition survey the current national ebf rate in zimbabwe stands at a staggering rate of 5 percent . the multiple indicator monitoring survey provides a higher national rate of 21% , with a higher proportion of 29% in urban areas being exclusively breast fed compared to 26% in rural areas the ministry of health and child care in zimbabwe recommends and promotes ebf as the most appropriate infant feeding practice for children from 0 - 6 months of age . despite efforts by the respective ministries to increase the ebf uptake , its uptake by mothers of infants remain very low. [1 , 2 , 4 ] breastfeeding is a complex process governed by psychological and physiological factors , which in turn are conditioned by a wide spectrum of environmental , socioeconomic and cultural circumstances . in view of the fact that ebf is a physiological process which transpires in a specific socio - economic milieu , the purpose of this paper is to investigate the socio - economic factors that hinders its uptake among rural women in zimbabwe the concurrent nested model of mixed research methodology was employed and was identified by its use of one data collection phase , during which both quantitative and qualitative data are collected simultaneously . the study was carried out in mataga ward , mberengwa district at mataga rural health center , and midlands s province in zimbabwe from april to june 2013 . the target population was mothers who have infants from 0 - 6 months who were accessing neonatal health services during the data collection month . convenience sampling method was utilized to select 20 mothers who took part in the study . purposive sampling was utilized used to select the key informants ( nurses and village health workers ) . the study was reviewed and approved by the university of zimbabwe ethics board as well as the general academic board before implementation . quantitative data was analysed using the statistical package for social scientists version 17 while qualitative data was analysed through content coding procedures based on a set of predetermined themes of interest . the concurrent nested model of mixed research methodology was employed and was identified by its use of one data collection phase , during which both quantitative and qualitative data are collected simultaneously . the study was carried out in mataga ward , mberengwa district at mataga rural health center , and midlands s province in zimbabwe from april to june 2013 . the target population was mothers who have infants from 0 - 6 months who were accessing neonatal health services during the data collection month . convenience sampling method was utilized to select 20 mothers who took part in the study . purposive sampling was utilized used to select the key informants ( nurses and village health workers ) . the study was reviewed and approved by the university of zimbabwe ethics board as well as the general academic board before implementation . quantitative data was analysed using the statistical package for social scientists version 17 while qualitative data was analysed through content coding procedures based on a set of predetermined themes of interest . 2 ) what are the barriers that prevent or affect you / mothers from practicing ebf ? out of the 20 mothers interviewed in the study , 45% ( n=9 ) were from the 20 - 25 age range . 15% ( n=3 ) respondents were within 15 - 19 age range , while age groups 26 - 30 and 36 + had 3 respondents each . the primary school attendant rate in this study tallies with the findings of the multiple indicator monitoring survey which recorded a 99 % primary education net attendance of the girl child in zimbabwe . education levels influence the uptake of ebf . many respondents exhibited extensive knowledge and understanding of the meaning and benefits of exclusive breast feeding . fifteen ( 70% ) mothers indicated that they understand the meaning of ebf and its benefits while five ( 25% ) mothers indicated knowledge of the meaning of ebf but not its benefits . for example , out of the 70% ( n=14 ) of the mothers who reported knowledge of ebf and its benefits , only 25 % ( n=5 ) of them managed to practice ebf . this state of affairs is a clear pointer to the fact that knowledge of ebf does not translate to the active uptake of the same . failure to comply with such an expectation could be misconstrued for infidelity in a family where tradition holds that feeding on solids is a sign of genetic connection of the baby with its ancestors . only two mothers ( 14% ) with primary education managed to practice exclusive breast feeding . such a nexus between level of education and ebf stands as a clear testimony that education , like in other parts of the world , remains a vital weapon to fight against the power of some retrogressive traditional beliefs in zimbabwean society . the influence of education on the uptake of ebf was also recognized by all the nurses ( key informants ) who registered a high prevalence among mothers who have secondary education . zimbabwe s tradition demands that traditional medicine be administered on babies to try and avert the death caused by the condition that culminates in the subsiding of the fontanel in babies due mainly to severe dehydration ( a condition known as nhova in vernacular ) . this practice is also common in kenya where infants are given various herbs to treat the condition known in kenya as ndebele . traditionally , this condition is attributed to evil spells . out of the 70% ( n=14 ) respondents not practicing ebf , 12 of them reported that they had treated nhova on their babies through administering different foods and medicines such as barks , juices , roots , herbs , cooking oil and wild fruits . once more , such a traditional practice militates against the core tenets of exclusive breast feeding that prohibit the intake of any solids let alone medicine unless prescribed by a qualified medical practitioner . to sustain ebf , sight should not be lost of these traditional practices and safeguards that have tended to supersede modern medicine as guarantees of survival in resource poor communities . significant others such as grandmothers , mothers in - law and husbands influence the infant feeding practices pursued by mothers . respondents identified mothers - in - law as key authority figures regarding the uptake of ebf . for example , 70% ( n=14 ) of respondents indicated that mothers in law were key decision makers with regard to infant feeding . in an african culture , older persons are regarded as a fountain of knowledge and younger persons tap knowledge from them . most of the advice that came from the mothers - in - law discourage exclusive breast feeding in favor of mixed infant feeding practice . the mother - in - law is usually powerful and influential in major decisions concerning the behavior and conduct of her daughter - in - law . according to one respondent , the mother in law gave the infant porridge a day after delivery arguing that the family tradition requires that the baby be fed on porridge the day she gets home from the hospital by its grandmother . it is worth noting that such a scenario is in no way peculiar to zimbabwe as it corresponds with findings in malawi that the influence of the mother - in - law and husband was one of the greatest barriers to ebf uptake in malawi . this state of affairs has its explanation in the theory of planned behavior which envisages that people s behaviors are influenced by normative beliefs , which are beliefs about the normative expectations of others and motivation to comply with those expectations . implicit in this scenario is that sometimes one s level of education in a rural environment becomes subservient to the expectations and dictates of culture and tradition . in such a situation , it becomes important for any health promotion strategy to be ingrained within the cultural expectations of the particular locality within which it is conducted . in an african rural milieu , eighteen ( 90% ) respondents noted that they had been advised by their husbands on infant feeding . one respondent confirmed having been confronted with what has tended to be the greatest question of the day each time ebf was discussed in a traditional african setup namely : implicit in such a scenario is that gender roles cut across all the facts of life , undermining even those aspects of human existence ordinarily thought to belong to the wife in a matrimonial relationship . respondents contend that they find it difficult to practice ebf given that inadequate food with low nutritional values exacerbate the low uptake of ebf . this study observed that women in zimbabwe have limited income and economic their status is low . prior study has shown that wealth quintile or income influences the uptake of exclusive breast feeding . low amounts of milk from ill - fed and malnourished mothers have come as an unfortunate deterrent against ebf . the study noted a high prevalence of ebf among mothers whose incomes were above $ 100 . the three nurses and two village health workers in the study reported that most mothers are not adhering to ebf because they were not producing enough milk . the findings therefore draw the attention of all players in the promotion of ebf program as well as those involved in hiv prevention ( especially the prevention of mother to child transmission of hiv ) to seriously take poverty , unemployment and malnutrition as obstacles against the achievement of set objectives . informed by such a reality on the ground , the success of these programs rests on the adoption of a holistic approach to programming . this study could not directly establish the influence of the gender of the child on ebf as the number of the male child was significantly very low to the number of girls . however , six mothers reported the challenges they faced in practicing ebf , adding that the boy child eats more than the girl child and hence mother s milk is not enough for the male infants . one of the village health workers respondent # 2 reported that boys eat very much and that mother s milk might not be enough for the boy child . however , nurse respondent # 1 and village health worker respondent # 3 denied that boys eat more than girls at this tender age . all of the village health workers ( n=4 ; 100% ) confirmed that this was a wide belief that boys eat very much and it is impossible for them to be satisfied by mother s milk alone . this sends a clear message to public health promoters that they need to know and address the misconceptions and myths held by communities that militate against the uptake of ebf especially for boy - children . out of the 20 mothers interviewed in the study , 45% ( n=9 ) were from the 20 - 25 age range . 15% ( n=3 ) respondents were within 15 - 19 age range , while age groups 26 - 30 and 36 + had 3 respondents each . the primary school attendant rate in this study tallies with the findings of the multiple indicator monitoring survey which recorded a 99 % primary education net attendance of the girl child in zimbabwe . among the 20 ( 100% ) mothers who were interviewed , five ( 25% ) had an income estimate of $ 5 , four ( 20% ) mothers earned an estimate of $ 20 , four ( 20% ) mothers earned an estimate of $ 50 , three ( 15% ) mothers earned $ 100 , while a paltry two ( 10% ) mothers earned more than $ 100 . many respondents exhibited extensive knowledge and understanding of the meaning and benefits of exclusive breast feeding . there was no significant correlation between this knowledge of the meaning of ebf and the actual practice . this state of affairs is a clear pointer to the fact that knowledge of ebf does not translate to the active uptake of the same . the discrepancy between knowledge and behaviour could also well be traced back to the influence of culture in rewarding and punishing whatever is deemed acceptable and unacceptable behaviour in the zimbabwean society . failure to comply with such an expectation could be misconstrued for infidelity in a family where tradition holds that feeding on solids is a sign of genetic connection of the baby with its ancestors . eleven only two mothers ( 14% ) with primary education managed to practice exclusive breast feeding . such a nexus between level of education and ebf stands as a clear testimony that education , like in other parts of the world , remains a vital weapon to fight against the power of some retrogressive traditional beliefs in zimbabwean society . the influence of education on the uptake of ebf was also recognized by all the nurses ( key informants ) who registered a high prevalence among mothers who have secondary education . traditionally , this condition is attributed to evil spells . out of the 70% ( n=14 ) respondents not practicing ebf , 12 of them reported that they had treated nhova on their babies through administering different foods and medicines such as barks , juices , roots , herbs , cooking oil and wild fruits . once more , such a traditional practice militates against the core tenets of exclusive breast feeding that prohibit the intake of any solids let alone medicine unless prescribed by a qualified medical practitioner . to sustain ebf , sight should not be lost of these traditional practices and safeguards that have tended to supersede modern medicine as guarantees of survival in resource poor communities . significant others such as grandmothers , mothers in - law and husbands influence the infant feeding practices pursued by mothers . respondents identified mothers - in - law as key authority figures regarding the uptake of ebf . for example , 70% ( n=14 ) of respondents indicated that mothers in law were key decision makers with regard to infant feeding . most of the advice that came from the mothers - in - law discourage exclusive breast feeding in favor of mixed infant feeding practice . the respondents confess that their in - laws admonish that their clan is so unique that any of its offspring is born hungry . it is worth noting that such a scenario is in no way peculiar to zimbabwe as it corresponds with findings in malawi that the influence of the mother - in - law and husband was one of the greatest barriers to ebf uptake in malawi . this state of affairs has its explanation in the theory of planned behavior which envisages that people s behaviors are influenced by normative beliefs , which are beliefs about the normative expectations of others and motivation to comply with those expectations . implicit in this scenario is that sometimes one s level of education in a rural environment becomes subservient to the expectations and dictates of culture and tradition . in such a situation , it becomes important for any health promotion strategy to be ingrained within the cultural expectations of the particular locality within which it is conducted . in an african rural milieu , eighteen ( 90% ) respondents noted that they had been advised by their husbands on infant feeding . indications are that fathers of children of 65% ( n=13 ) of the 18 respondents were against ebf as they argued that the child should be given porridge as breast milk was not enough for the baby and that was why the baby is always crying . one respondent confirmed having been confronted with what has tended to be the greatest question of the day each time ebf was discussed in a traditional african setup namely : implicit in such a scenario is that gender roles cut across all the facts of life , undermining even those aspects of human existence ordinarily thought to belong to the wife in a matrimonial relationship . respondents contend that they find it difficult to practice ebf given that inadequate food with low nutritional values exacerbate the low uptake of ebf . this study observed that women in zimbabwe have limited income and economic their status is low . among the 100% ( n=20 ) respondents interviewed 25% ( n=5 ) had an income estimate of $ 5 , 20% ( n=4 ) respondents earn an estimate of $ 20 , 20% ( n=4 ) respondents earn an estimate of $ 50 , 15% ( n=3 ) respondents earn $ 100 , while a paltry 10% ( n=2 ) respondents earns more than $ 100 . prior study has shown that wealth quintile or income influences the uptake of exclusive breast feeding . low amounts of milk from ill - fed and malnourished mothers have come as an unfortunate deterrent against ebf . the study noted a high prevalence of ebf among mothers whose incomes were above $ 100 . for example , 66% ( n=4 ) of the 100% ( n=6 ) respondents actively participating in ebf had an income above $ 100 . the three nurses and two village health workers in the study reported that most mothers are not adhering to ebf because they were not producing enough milk . the above findings are in sync with those from the zimbabwe vulnerability assessment committee which found out that 39 - 45 percent of the people in meringa were food insecure . such programs as supplementary feeding of lactating mothers as well as social cash transfers can go a long way in fostering and sustaining ebf . this study could not directly establish the influence of the gender of the child on ebf as the number of the male child was significantly very low to the number of girls . 13 ( 65% ) mothers confirmed that they had a girl child while 35% ( n=7 ) mothers confirmed that they had a boy child . however , six mothers reported the challenges they faced in practicing ebf , adding that the boy child eats more than the girl child and hence mother s milk is not enough for the male infants . one of the village health workers respondent # 2 reported that boys eat very much and that mother s milk might not be enough for the boy child . however , nurse respondent # 1 and village health worker respondent # 3 denied that boys eat more than girls at this tender age . all of the village health workers ( n=4 ; 100% ) confirmed that this was a wide belief that boys eat very much and it is impossible for them to be satisfied by mother s milk alone . this sends a clear message to public health promoters that they need to know and address the misconceptions and myths held by communities that militate against the uptake of ebf especially for boy - children . this study has revealed many impediments that confront the rural zimbabwean mothers against adopting ebf . it is therefore pertinent that efforts by the ministry of health and child care to upscale ebf be holistic so that it reaches all areas that determine the mothers to practice ebf . male influence poses a barrier to the practice of ebf , hence it is imperative that they accompany their wives to clinics so that they are educated on the recommended infant feeding practices . community awareness and behavior change community mobilization on ebf should be scaled up and should not only target mothers but should be extended to members of the extended family such as grandmothers and mothers - in - law . the government needs to avail food to members of the community that are in hunger . mothers in rural areas are unable to practice ebf because they were producing little milk owing to lack of food and starvation.lack of education is associated with low ebf uptake . education of women has far greater values not just for ebf practice but for many health outcomes.societal norms , peer pressure , and traditional practices play significant roles in ebf practices among rural women . mothers in rural areas are unable to practice ebf because they were producing little milk owing to lack of food and starvation . societal norms , peer pressure , and traditional practices play significant roles in ebf practices among rural women .

the design of translating research into action for diabetes ( triad ) has been previously described ( 12 ) . the primary objective of triad was to determine how structural and organizational characteristics of health systems and health care provider groups influence the processes and outcomes of diabetes care . six translational research centers collaborated with 10 health plans and 68 provider groups that served 180,000 diabetes patients . health plans from california , hawaii , indiana , michigan , new jersey , new york , pennsylvania , and texas participated and included a racially and ethnically diverse membership . the institutional review boards at all translational research centers reviewed and approved the triad study protocol , and all participants provided informed consent . the triad study population was a random sample of adult enrollees with diabetes from participating health plans . patients were eligible to participate if they were aged 18 years , community dwelling , spoke english or spanish , not pregnant , continuously enrolled in the health plan for at least 18 months , had diabetes for 1 year , and used services during their health plan enrollment . in addition , eligibility required at least two outpatient visits or one inpatient stay with a diagnostic code for diabetes ( icd-9 250.xx ) , laboratory tests or values suggesting diabetes ( at least two hba1c tests ordered or a diagnostic hba1c or fasting blood glucose level ) , or a prescription for medications for diabetes ( e.g. , insulin or an oral antidiabetic agent ) . at the time of the survey , patients who met these criteria were included only if they confirmed that they had diabetes and received most of their diabetes care through the participating triad health plan . type 2 diabetes was defined as diabetes that was not currently treated with insulin or that was diagnosed after age 30 years , with or without current insulin treatment . patient surveys were completed between july 2000 and october 2001 . of the 13,086 individuals who were contacted and eligible , 11,927 ( 91% ) completed the survey . survey variables included age , sex , race / ethnicity , education , income , bmi , smoking , time since diagnosis of diabetes , treatment for diabetes , and hrqol as measured by the eq-5d . of the patients completing a survey , the participants whose records were reviewed were similar to the overall study population ( 13 ) . centrally trained reviewers used standardized data collection forms to abstract each patient s records of medical treatment and prescribed medications during the 12 months before the survey date ; of these , 5% were abstracted by two abstractors . inter - rater reliability ( ) ranged from 0.86 to 0.94 for the main quality measures derived from the medical record data . variables that were obtained from the record review were diabetes - related micro- and macrovascular complications and comorbid conditions . we chose the eq-5d over other health utility measures , such as the short - form health survey-6d , because of its simplicity , sufficient responsiveness to changes in health states , relatively wide range of utility scores generated by the instrument , and availability of preference weight for our study population ( 11,14 ) . briefly , the eq-5d consists of two parts : five questions relating to distinct dimensions of a person s functional capacity ( mobility , self - care , usual activity , pain / discomfort , and anxiety / depression ) and a visual analog scale . there are three responses for each domain of the questionnaire . because the baseline survey was conducted primarily by phone , the visual analog scale of the eq-5d was not implemented . the responses from the eq-5d were combined with preference weights derived from a sample of the u.s . we constructed categories within each type of complication based on clinically defined disease states and data availability and ordered the individual disease categories based on disease severity . the specific disease categories for each of the five diabetes - related complications include diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , stroke , and cardiovascular disease ( table 2 ) . within diabetic retinopathy , individuals with laser treatment were considered to have proliferative diabetic retinopathy . within cardiovascular disease , the category of other coronary heart diseases was defined as having indicators of coronary heart disease or coronary artery disease or undergoing procedures to treat coronary heart disease such as coronary angioplasty and coronary bypass . peripheral vascular disease was categorized as having or not having the disease . a person had peripheral vascular disease if he or she had a record of peripheral vascular disease or had undergone procedures used to treat the disease such as peripheral vascular angioplasty or bypass . means and standard errors are reported for continuous variables ( except age ) and proportions for categoric variables . tests for differences between categories in eq-5d scores by patient level variables were tested using a test . the eq-5d utility scores were modeled by a multivariate linear regression model adjusting for demographic and clinical characteristics and for disease states . missing values for independent variables were imputed five times using a multiple imputation method ( 15 ) . when estimates of adjacent response categories for any diabetes - related complication in the model were not significantly different from each other or were inconsistent with the order of increasing severity , the two response categories were combined and the model was rerun . only those variables that had statistically significant estimated coefficients were kept in the final model . the estimated coefficients of the indicator variables represent the penalty or deficit from optimal health associated with each variable . subtracting penalty functions from the health utility scores for the healthy reference group created an additive model from which we derived the eq-5d utility score for any combination of treatments , complications , and comorbidities . after combining some of the categoric variables , the reference group was defined as a nonobese man with type 2 diabetes who was not asian or hispanic , not using insulin treatment , and who had an annual household income of more than $ 40,000 with no diabetes - related complications , risk factors for cardiovascular disease , or comorbidities . all statistical analyses were performed using sas 9.2 software ( sas institute inc . , cary , nc ) . patient surveys were completed between july 2000 and october 2001 . of the 13,086 individuals who were contacted and eligible , 11,927 ( 91% ) completed the survey . survey variables included age , sex , race / ethnicity , education , income , bmi , smoking , time since diagnosis of diabetes , treatment for diabetes , and hrqol as measured by the eq-5d . of the patients completing a survey , the participants whose records were reviewed were similar to the overall study population ( 13 ) . centrally trained reviewers used standardized data collection forms to abstract each patient s records of medical treatment and prescribed medications during the 12 months before the survey date ; of these , 5% were abstracted by two abstractors . inter - rater reliability ( ) ranged from 0.86 to 0.94 for the main quality measures derived from the medical record data . variables that were obtained from the record review were diabetes - related micro- and macrovascular complications and comorbid conditions . we used the eq-5d to derive health utility scores . the validity and reliability of the eq-5d we chose the eq-5d over other health utility measures , such as the short - form health survey-6d , because of its simplicity , sufficient responsiveness to changes in health states , relatively wide range of utility scores generated by the instrument , and availability of preference weight for our study population ( 11,14 ) . briefly , the eq-5d consists of two parts : five questions relating to distinct dimensions of a person s functional capacity ( mobility , self - care , usual activity , pain / discomfort , and anxiety / depression ) and a visual analog scale . there are three responses for each domain of the questionnaire . because the baseline survey was conducted primarily by phone , the visual analog scale of the eq-5d was not implemented . the responses from the eq-5d were combined with preference weights derived from a sample of the u.s . we constructed categories within each type of complication based on clinically defined disease states and data availability and ordered the individual disease categories based on disease severity . the specific disease categories for each of the five diabetes - related complications include diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , stroke , and cardiovascular disease ( table 2 ) . within diabetic retinopathy , individuals with laser treatment were considered to have proliferative diabetic retinopathy . within cardiovascular disease , the category of other coronary heart diseases was defined as having indicators of coronary heart disease or coronary artery disease or undergoing procedures to treat coronary heart disease such as coronary angioplasty and coronary bypass . peripheral vascular disease was categorized as having or not having the disease . a person had peripheral vascular disease if he or she had a record of peripheral vascular disease or had undergone procedures used to treat the disease such as peripheral vascular angioplasty or bypass . means and standard errors are reported for continuous variables ( except age ) and proportions for categoric variables . tests for differences between categories in eq-5d scores by patient level variables were tested using a test . the eq-5d utility scores were modeled by a multivariate linear regression model adjusting for demographic and clinical characteristics and for disease states . missing values for independent variables were imputed five times using a multiple imputation method ( 15 ) . the initial model was developed using all variables . when estimates of adjacent response categories for any diabetes - related complication in the model were not significantly different from each other or were inconsistent with the order of increasing severity , the two response categories were combined and the model was rerun . only those variables that had statistically significant estimated coefficients were kept in the final model . the estimated coefficients of the indicator variables represent the penalty or deficit from optimal health associated with each variable . subtracting penalty functions from the health utility scores for the healthy reference group created an additive model from which we derived the eq-5d utility score for any combination of treatments , complications , and comorbidities . after combining some of the categoric variables , the reference group was defined as a nonobese man with type 2 diabetes who was not asian or hispanic , not using insulin treatment , and who had an annual household income of more than $ 40,000 with no diabetes - related complications , risk factors for cardiovascular disease , or comorbidities . all statistical analyses were performed using sas 9.2 software ( sas institute inc . , cary , nc ) . , they reflect the characteristics of the population with type 2 diabetes in the u.s . patients with type 2 diabetes tend to be older , to be more racially diverse , to have higher bmis , and to have a higher proportion of individuals with low income and education levels compared with the general u.s . the average hba1c level in the triad population with type 2 diabetes ( 8.0% ) was similar to that for the u.s . general population with type 2 diabetes during the same period ( 7.5% ) ( 17 ) . ore than 90% of patients were treated with oral antidiabetic agents , insulin monotherapy , or a combination of the two . the mean charlson index , a weighted measure that incorporates 19 diseases to predict 10-year mortality risk according to severity of comorbidities , was 2.3 . for reference , the predicted 10-year mortality risk is 48% for individuals with a charlson index score of 2.0 ( 18 ) . table 2 presents the eq-5d scores by demographic and clinical characteristics of the study population . the eq-5d scores differed across subgroups by age , sex , race , level of education , and household income . the scores also differed by time since diagnosis of diabetes , bmi , smoking status , and diabetes treatment . in general , patients with diabetes - related complications or comorbidities had lower health utility scores than those without . among those with the lowest eq-5d scores were patients who required dialysis , who had hemiplegia , or who had experienced the amputation of both feet . unadjusted eq-5d health utility score by patient characteristics the penalty functions associated with each characteristic are presented in table 3 . the intercept value of 0.920 was the mean health utility score in the reference group . being a woman , being obese , smoking , using insulin , or having an annual household income of less than $ 40,000 were associated with a lower health utility score . the most severe microvascular complications , painful neuropathy and amputations , were associated with a decrement of more than 0.10 in the health utility score . complications associated with a reduction of more than 0.03 in the health utility score were hemiplegia , nonpainful diabetic neuropathy , cerebral vascular accident , congestive heart failure , and dialysis . other complications , such as other coronary heart disease , transient ischemic attack , and peripheral vascular disease , were associated with relatively small reductions in eq-5d scores ( < 0.03 ) . nonproliferative and proliferative diabetic retinopathy did not independently reduce health utility scores and were dropped from the final model . similarly , age , education , time since diagnosis of diabetes , and hypertension were not associated with significant independent reductions in health utility scores and were excluded from the final model . estimated coefficients for the multiple regression model we derived health utility scores for adults with diabetes , its complications , and its comorbidities by using data collected in a large multicenter , prospective observational study of managed care patients from seven u.s . regions . as one might expect , diabetes - related complications were associated with lower utility score , and the magnitude of the impact varied by the complication . in increasing order of deficit , the complications were peripheral vascular disease , other heart diseases , transient ischemic attack , cerebral vascular accident , nonpainful diabetic neuropathy , congestive heart failure , dialysis , hemiplegia , painful neuropathy , and amputation . the variations in the effect of different diabetes complications on hrqol were as much as 10-fold . however , it is not clear whether the lower health utility scores associated with insulin treatment were because insulin treatment did not improve functional capacity or because insulin treatment served as a surrogate for greater diabetes severity . those treated with insulin have been diagnosed with diabetes longer and have experienced more diabetes - related complications than those who are treated with diet or oral antidiabetic agents alone . in our study , insulin treatment was still associated with a lower utility score after adjusting duration of diabetes and diabetes - related complications , implying insulin treatment lowered health utility scores independently . studies that have assessed insulin use as part of overall quality - of - life have shown a decrease in quality - of - life as treatment moved from diet only to oral agents to insulin ( 19 ) . however , studies comparing quality - of - life with specific treatment regimens and ongoing support strategies generally reported no decline , or even an improvement , in quality - of - life with insulin use ( 19 ) . on the one hand , using insulin could lower quality - of - life directly because of inconvenience , adverse effects of insulin injections , and hypoglycemia , or indirectly by stigmatizing a patient as being unable to control his or her diabetes adequately ( 20 ) . on the other hand , insulin use could also improve quality - of - life by leading to better glycemic control , which is positively associated with a high level of perceived quality - of - life ( 21 ) . obesity , especially morbid obesity ( bmi 35 kg / m ) , was associated with a large reduction in health utility scores . ( 20 ) found a negative relationship between excess body weight and hrqol among type 2 diabetic patients , even after adjusting for their demographic characteristics , diabetes duration , diabetes treatment mode , and presence of cardiovascular diseases and other comorbid conditions . obesity impairs physical functioning , decreases energy levels , increases health distress , and decreases self - rated health . our model found a higher health utility score for asians and hispanics and a lower score for women and low - income individuals . these differences in health utility scores by race , sex , or income level are consistent with findings from previous studies ( 21 ) . however , these results do not imply that a diabetes intervention is more cost - effective for asians or hispanics compared with other races , for men compared with women , or for those of lower income compared with those of middle income , because no interactions were found among any of the three variables for type of diabetes treatment or diabetes - related complications ( data not shown ) . coffey et al . ( 2 ) estimated health utility scores associated with different diabetic treatments , complications , and comorbidities among individuals with type 2 diabetes . their study used a self - administered version of the quality of well being index to survey 1,257 people with type 2 diabetes who were patients at endocrinology , diabetes , and ophthalmology clinics at the university of michigan health system . the study estimated the mean health utility score for a diet - controlled nonobese diabetic man without any complications , comorbidities , or cardiovascular risk factors to be 0.687 . the closest comparable utility value in our study was the mean utility value for our reference group , 0.920 . the difference in these values is likely attributable to the hrqol instruments used in the two studies . health utility scores based on the eq-5d tend to be higher than those from other hrqol instruments . in fact , about a quarter of our subjects had a utility value of 1.00 . eq-5d can not distinguish small differences in physical or mental health status in relatively healthy populations . this ceiling effect of eq-5d has been documented in other studies ( 3,4 ) . the estimated range and level of penalty scores associated with individual complications between the coffey et al . they estimated that the penalty score , excluding blindness ( we did not collect information on blindness ) , varied from 0.011 to 0.105 , whereas our estimates varied from 0.012 to 0.108 . clarke et al . ( 4 ) estimated health utility scores in 3,192 type 2 diabetic subjects who participated in the united kingdom prospective diabetes study ( ukpds ) using the eq-5d . they estimated that a man with type 2 diabetes and no complications would have a utility score of 0.8500.962 , depending on the specific regression model . their estimated range of penalty scores for individual complications was much larger ( 0.0550.280 ) . several factors could have contributed to the different results : first , subjects in the ukpds represented a relatively healthy and homogenous newly diagnosed type 2 cohort . our population included type 2 diabetic patients with a wide range of treatments , complications , comorbidities , and duration of diagnosed diabetes . second , in calculating the utility score , we used the preference weights from the u.s . finally , because of data availability , the variables included in the regression models were different . having fewer variables in their regression model may have added weight to the variables they included . ( 22 ) assessed health utility scores using the eq-5d in a sample of 1,136 dutch type 2 diabetic subjects who participated in the code-2 study . older age , obesity , woman , insulin therapy , and presence of complications were associated with lower health utility scores . their relatively small study sample may have limited their ability to assess the effect of each individual complication on hrqol . bagust and beale ( 3 ) also used data collected in the code-2 study to estimate utility scores but combined the dutch data used by redekop et al . because of the much larger sample size of 4,641 , this study was able to estimate eq-5d utility scores associated with specific diabetes complications . except for coronary heart disease , our estimated penalty scores associated with individual complications were lower than those in the bagust and beale regression model ( 3 ) . for example , the estimated penalty score for amputations was 0.108 in our study and 0.272 in the bagust and beale study . the estimated penalty score for coronary heart disease was 0.042 in our study and 0.028 in the bagust and beale study . the use of dissimilar preference weights in deriving the eq-5d score and dissimilar demographic variables and definitions of complications may have contributed to differences in estimated health utility scores . the limited comparability of the estimated health utility scores between our study and the studies cited above might imply that penalty scores reported by different studies should be interpreted in the broad context of differences in study populations and quality - of - life instruments used and of the completeness and types of variables used in the regression models . ( 14 ) estimated the health utility scores associated with diabetes and its complications for patients enrolled in a clinical trial in australia . the health utility score for those who had no any diabetes - related complication was 0.88 , a bit lower than the score in our study . reduction in health utility scores by complications in decreasing order resulted in the following : stroke and/or transient ischemic attack , peripheral revascularization and/or amputation , unstable angina , myocardial infarction , coronary artery bypass graft , and eye disease . differences in study population and definition of complications may have contributed to the different utility scores between the two studies . some limitations of our study should be noted . health unity scores associated with diabetes and its complication can change due to improvements in physical , mental , and social functions of diabetes patients over time . our study focused on assessing the relative decrement utility associated with each diabetes - related complication and its progression rather than the absolute level of health utility scores for those complications . little evidence exists on the change in the relative function levels associated with different complications in the last 10 years . thus , our estimates are very likely still applicable to the current diabetes population . because our income level was measured in 2001 u.s . dollars , the income variable should be adjusted using an inflation calculator ( 23 ) if income is expressed in other years . patients with no health insurance tend to have a lower quality - of - life than those with health insurance ( 24 ) . however , it is less clear that disutility associated with diabetes treatment mode and diabetes - related complications differ between the two groups . in addition , our study population may not represent the entire managed care population with diabetes , and individuals in managed care might not be representative of the entire diabetic population ; for example , poorer , sicker , or older individuals with type 2 diabetes may have been less likely to participate in the triad survey . finally , our study was cross - sectional , and variation in responses could occur if hrqol was measured at multiple points in time . cost - utility analysis , the type of economic evaluation most often used to assess interventions used for the prevention and control of type 2 diabetes , requires valid and accurate health utility estimates by patient characteristics , treatment modalities , and the different clinical states of each diabetes - related complication . we derived a set of such health utility estimates using data collected in a large multicenter diabetes study using u.s .- based preference weights . our empirically derived health utility scores will allow researchers to calculate qalys for studies involving individuals living in the u.s . with type 2 diabetes and representing a wide variety of demographic characteristics , treatments , complications , and comorbidities . the health utility scores provided should facilitate studies of the health burden of diabetes and the cost - utility analysis of alternative strategies for the prevention and treatment of diabetes in the u.s .

objectiveto estimate the health utility scores associated with type 2 diabetes , its treatments , complications , and comorbidities.research design and methodswe analyzed health - related quality - of - life data , collected at baseline during translating research into action for diabetes , a multicenter , prospective , observational study of diabetes care in managed care , for 7,327 individuals with type 2 diabetes . we measured quality - of - life using the euroqol ( eq)-5d , a standardized instrument for which 1.00 indicates perfect health . we used multivariable regression to estimate the independent impact of demographic characteristics , diabetes treatments , complications , and comorbidities on health - related quality-of-life.resultsthe mean eq-5d derived health utility score for those individuals with diabetes was 0.80 . the modeled utility score for a nonobese , non insulin - treated , non - asian , non - hispanic man with type 2 diabetes , with an annual household income of more than $ 40,000 , and with no diabetes complications , risk factors for cardiovascular disease , or comorbidities , was 0.92 . being a woman , being obese , smoking , and having a lower household income were associated with lower utility scores . arranging complications from least to most severe according to the reduction in health utility scores resulted in the following order : peripheral vascular disease , other heart diseases , transient ischemic attack , cerebral vascular accident , nonpainful diabetic neuropathy , congestive heart failure , dialysis , hemiplegia , painful neuropathy , and amputation.conclusionsmajor diabetes complications and comorbidities are associated with decreased health - related quality - of - life . utility estimates from our study can be used to assess the impact of diabetes on quality - of - life and conduct cost - utility analyses .

RESEARCH DESIGN AND METHODS Data sources Health utility measure Diabetes-related complications Statistical analysis RESULTS CONCLUSIONS

the design of translating research into action for diabetes ( triad ) has been previously described ( 12 ) . the primary objective of triad was to determine how structural and organizational characteristics of health systems and health care provider groups influence the processes and outcomes of diabetes care . health plans from california , hawaii , indiana , michigan , new jersey , new york , pennsylvania , and texas participated and included a racially and ethnically diverse membership . the institutional review boards at all translational research centers reviewed and approved the triad study protocol , and all participants provided informed consent . the triad study population was a random sample of adult enrollees with diabetes from participating health plans . patients were eligible to participate if they were aged 18 years , community dwelling , spoke english or spanish , not pregnant , continuously enrolled in the health plan for at least 18 months , had diabetes for 1 year , and used services during their health plan enrollment . in addition , eligibility required at least two outpatient visits or one inpatient stay with a diagnostic code for diabetes ( icd-9 250.xx ) , laboratory tests or values suggesting diabetes ( at least two hba1c tests ordered or a diagnostic hba1c or fasting blood glucose level ) , or a prescription for medications for diabetes ( e.g. , insulin or an oral antidiabetic agent ) . at the time of the survey , patients who met these criteria were included only if they confirmed that they had diabetes and received most of their diabetes care through the participating triad health plan . type 2 diabetes was defined as diabetes that was not currently treated with insulin or that was diagnosed after age 30 years , with or without current insulin treatment . survey variables included age , sex , race / ethnicity , education , income , bmi , smoking , time since diagnosis of diabetes , treatment for diabetes , and hrqol as measured by the eq-5d . of the patients completing a survey , the participants whose records were reviewed were similar to the overall study population ( 13 ) . variables that were obtained from the record review were diabetes - related micro- and macrovascular complications and comorbid conditions . we chose the eq-5d over other health utility measures , such as the short - form health survey-6d , because of its simplicity , sufficient responsiveness to changes in health states , relatively wide range of utility scores generated by the instrument , and availability of preference weight for our study population ( 11,14 ) . briefly , the eq-5d consists of two parts : five questions relating to distinct dimensions of a person s functional capacity ( mobility , self - care , usual activity , pain / discomfort , and anxiety / depression ) and a visual analog scale . the specific disease categories for each of the five diabetes - related complications include diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , stroke , and cardiovascular disease ( table 2 ) . within diabetic retinopathy , individuals with laser treatment were considered to have proliferative diabetic retinopathy . within cardiovascular disease , the category of other coronary heart diseases was defined as having indicators of coronary heart disease or coronary artery disease or undergoing procedures to treat coronary heart disease such as coronary angioplasty and coronary bypass . peripheral vascular disease was categorized as having or not having the disease . a person had peripheral vascular disease if he or she had a record of peripheral vascular disease or had undergone procedures used to treat the disease such as peripheral vascular angioplasty or bypass . the eq-5d utility scores were modeled by a multivariate linear regression model adjusting for demographic and clinical characteristics and for disease states . missing values for independent variables were imputed five times using a multiple imputation method ( 15 ) . when estimates of adjacent response categories for any diabetes - related complication in the model were not significantly different from each other or were inconsistent with the order of increasing severity , the two response categories were combined and the model was rerun . only those variables that had statistically significant estimated coefficients were kept in the final model . the estimated coefficients of the indicator variables represent the penalty or deficit from optimal health associated with each variable . subtracting penalty functions from the health utility scores for the healthy reference group created an additive model from which we derived the eq-5d utility score for any combination of treatments , complications , and comorbidities . after combining some of the categoric variables , the reference group was defined as a nonobese man with type 2 diabetes who was not asian or hispanic , not using insulin treatment , and who had an annual household income of more than $ 40,000 with no diabetes - related complications , risk factors for cardiovascular disease , or comorbidities . survey variables included age , sex , race / ethnicity , education , income , bmi , smoking , time since diagnosis of diabetes , treatment for diabetes , and hrqol as measured by the eq-5d . of the patients completing a survey , the participants whose records were reviewed were similar to the overall study population ( 13 ) . variables that were obtained from the record review were diabetes - related micro- and macrovascular complications and comorbid conditions . we used the eq-5d to derive health utility scores . the validity and reliability of the eq-5d we chose the eq-5d over other health utility measures , such as the short - form health survey-6d , because of its simplicity , sufficient responsiveness to changes in health states , relatively wide range of utility scores generated by the instrument , and availability of preference weight for our study population ( 11,14 ) . briefly , the eq-5d consists of two parts : five questions relating to distinct dimensions of a person s functional capacity ( mobility , self - care , usual activity , pain / discomfort , and anxiety / depression ) and a visual analog scale . the specific disease categories for each of the five diabetes - related complications include diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , stroke , and cardiovascular disease ( table 2 ) . within diabetic retinopathy , individuals with laser treatment were considered to have proliferative diabetic retinopathy . within cardiovascular disease , the category of other coronary heart diseases was defined as having indicators of coronary heart disease or coronary artery disease or undergoing procedures to treat coronary heart disease such as coronary angioplasty and coronary bypass . peripheral vascular disease was categorized as having or not having the disease . a person had peripheral vascular disease if he or she had a record of peripheral vascular disease or had undergone procedures used to treat the disease such as peripheral vascular angioplasty or bypass . the eq-5d utility scores were modeled by a multivariate linear regression model adjusting for demographic and clinical characteristics and for disease states . when estimates of adjacent response categories for any diabetes - related complication in the model were not significantly different from each other or were inconsistent with the order of increasing severity , the two response categories were combined and the model was rerun . only those variables that had statistically significant estimated coefficients were kept in the final model . the estimated coefficients of the indicator variables represent the penalty or deficit from optimal health associated with each variable . subtracting penalty functions from the health utility scores for the healthy reference group created an additive model from which we derived the eq-5d utility score for any combination of treatments , complications , and comorbidities . after combining some of the categoric variables , the reference group was defined as a nonobese man with type 2 diabetes who was not asian or hispanic , not using insulin treatment , and who had an annual household income of more than $ 40,000 with no diabetes - related complications , risk factors for cardiovascular disease , or comorbidities . , cary , nc ) . , they reflect the characteristics of the population with type 2 diabetes in the u.s . patients with type 2 diabetes tend to be older , to be more racially diverse , to have higher bmis , and to have a higher proportion of individuals with low income and education levels compared with the general u.s . the average hba1c level in the triad population with type 2 diabetes ( 8.0% ) was similar to that for the u.s . general population with type 2 diabetes during the same period ( 7.5% ) ( 17 ) . ore than 90% of patients were treated with oral antidiabetic agents , insulin monotherapy , or a combination of the two . the mean charlson index , a weighted measure that incorporates 19 diseases to predict 10-year mortality risk according to severity of comorbidities , was 2.3 . for reference , the predicted 10-year mortality risk is 48% for individuals with a charlson index score of 2.0 ( 18 ) . the eq-5d scores differed across subgroups by age , sex , race , level of education , and household income . the scores also differed by time since diagnosis of diabetes , bmi , smoking status , and diabetes treatment . in general , patients with diabetes - related complications or comorbidities had lower health utility scores than those without . among those with the lowest eq-5d scores were patients who required dialysis , who had hemiplegia , or who had experienced the amputation of both feet . unadjusted eq-5d health utility score by patient characteristics the penalty functions associated with each characteristic are presented in table 3 . the intercept value of 0.920 was the mean health utility score in the reference group . being a woman , being obese , smoking , using insulin , or having an annual household income of less than $ 40,000 were associated with a lower health utility score . the most severe microvascular complications , painful neuropathy and amputations , were associated with a decrement of more than 0.10 in the health utility score . complications associated with a reduction of more than 0.03 in the health utility score were hemiplegia , nonpainful diabetic neuropathy , cerebral vascular accident , congestive heart failure , and dialysis . other complications , such as other coronary heart disease , transient ischemic attack , and peripheral vascular disease , were associated with relatively small reductions in eq-5d scores ( < 0.03 ) . nonproliferative and proliferative diabetic retinopathy did not independently reduce health utility scores and were dropped from the final model . similarly , age , education , time since diagnosis of diabetes , and hypertension were not associated with significant independent reductions in health utility scores and were excluded from the final model . estimated coefficients for the multiple regression model we derived health utility scores for adults with diabetes , its complications , and its comorbidities by using data collected in a large multicenter , prospective observational study of managed care patients from seven u.s . as one might expect , diabetes - related complications were associated with lower utility score , and the magnitude of the impact varied by the complication . in increasing order of deficit , the complications were peripheral vascular disease , other heart diseases , transient ischemic attack , cerebral vascular accident , nonpainful diabetic neuropathy , congestive heart failure , dialysis , hemiplegia , painful neuropathy , and amputation . the variations in the effect of different diabetes complications on hrqol were as much as 10-fold . however , it is not clear whether the lower health utility scores associated with insulin treatment were because insulin treatment did not improve functional capacity or because insulin treatment served as a surrogate for greater diabetes severity . those treated with insulin have been diagnosed with diabetes longer and have experienced more diabetes - related complications than those who are treated with diet or oral antidiabetic agents alone . in our study , insulin treatment was still associated with a lower utility score after adjusting duration of diabetes and diabetes - related complications , implying insulin treatment lowered health utility scores independently . studies that have assessed insulin use as part of overall quality - of - life have shown a decrease in quality - of - life as treatment moved from diet only to oral agents to insulin ( 19 ) . however , studies comparing quality - of - life with specific treatment regimens and ongoing support strategies generally reported no decline , or even an improvement , in quality - of - life with insulin use ( 19 ) . on the one hand , using insulin could lower quality - of - life directly because of inconvenience , adverse effects of insulin injections , and hypoglycemia , or indirectly by stigmatizing a patient as being unable to control his or her diabetes adequately ( 20 ) . on the other hand , insulin use could also improve quality - of - life by leading to better glycemic control , which is positively associated with a high level of perceived quality - of - life ( 21 ) . obesity , especially morbid obesity ( bmi 35 kg / m ) , was associated with a large reduction in health utility scores . ( 20 ) found a negative relationship between excess body weight and hrqol among type 2 diabetic patients , even after adjusting for their demographic characteristics , diabetes duration , diabetes treatment mode , and presence of cardiovascular diseases and other comorbid conditions . obesity impairs physical functioning , decreases energy levels , increases health distress , and decreases self - rated health . our model found a higher health utility score for asians and hispanics and a lower score for women and low - income individuals . these differences in health utility scores by race , sex , or income level are consistent with findings from previous studies ( 21 ) . however , these results do not imply that a diabetes intervention is more cost - effective for asians or hispanics compared with other races , for men compared with women , or for those of lower income compared with those of middle income , because no interactions were found among any of the three variables for type of diabetes treatment or diabetes - related complications ( data not shown ) . ( 2 ) estimated health utility scores associated with different diabetic treatments , complications , and comorbidities among individuals with type 2 diabetes . their study used a self - administered version of the quality of well being index to survey 1,257 people with type 2 diabetes who were patients at endocrinology , diabetes , and ophthalmology clinics at the university of michigan health system . the study estimated the mean health utility score for a diet - controlled nonobese diabetic man without any complications , comorbidities , or cardiovascular risk factors to be 0.687 . the closest comparable utility value in our study was the mean utility value for our reference group , 0.920 . the difference in these values is likely attributable to the hrqol instruments used in the two studies . health utility scores based on the eq-5d tend to be higher than those from other hrqol instruments . in fact , about a quarter of our subjects had a utility value of 1.00 . the estimated range and level of penalty scores associated with individual complications between the coffey et al . ( 4 ) estimated health utility scores in 3,192 type 2 diabetic subjects who participated in the united kingdom prospective diabetes study ( ukpds ) using the eq-5d . they estimated that a man with type 2 diabetes and no complications would have a utility score of 0.8500.962 , depending on the specific regression model . several factors could have contributed to the different results : first , subjects in the ukpds represented a relatively healthy and homogenous newly diagnosed type 2 cohort . our population included type 2 diabetic patients with a wide range of treatments , complications , comorbidities , and duration of diagnosed diabetes . second , in calculating the utility score , we used the preference weights from the u.s . finally , because of data availability , the variables included in the regression models were different . having fewer variables in their regression model may have added weight to the variables they included . ( 22 ) assessed health utility scores using the eq-5d in a sample of 1,136 dutch type 2 diabetic subjects who participated in the code-2 study . older age , obesity , woman , insulin therapy , and presence of complications were associated with lower health utility scores . their relatively small study sample may have limited their ability to assess the effect of each individual complication on hrqol . bagust and beale ( 3 ) also used data collected in the code-2 study to estimate utility scores but combined the dutch data used by redekop et al . because of the much larger sample size of 4,641 , this study was able to estimate eq-5d utility scores associated with specific diabetes complications . except for coronary heart disease , our estimated penalty scores associated with individual complications were lower than those in the bagust and beale regression model ( 3 ) . for example , the estimated penalty score for amputations was 0.108 in our study and 0.272 in the bagust and beale study . the estimated penalty score for coronary heart disease was 0.042 in our study and 0.028 in the bagust and beale study . the use of dissimilar preference weights in deriving the eq-5d score and dissimilar demographic variables and definitions of complications may have contributed to differences in estimated health utility scores . the limited comparability of the estimated health utility scores between our study and the studies cited above might imply that penalty scores reported by different studies should be interpreted in the broad context of differences in study populations and quality - of - life instruments used and of the completeness and types of variables used in the regression models . ( 14 ) estimated the health utility scores associated with diabetes and its complications for patients enrolled in a clinical trial in australia . the health utility score for those who had no any diabetes - related complication was 0.88 , a bit lower than the score in our study . reduction in health utility scores by complications in decreasing order resulted in the following : stroke and/or transient ischemic attack , peripheral revascularization and/or amputation , unstable angina , myocardial infarction , coronary artery bypass graft , and eye disease . differences in study population and definition of complications may have contributed to the different utility scores between the two studies . some limitations of our study should be noted . health unity scores associated with diabetes and its complication can change due to improvements in physical , mental , and social functions of diabetes patients over time . our study focused on assessing the relative decrement utility associated with each diabetes - related complication and its progression rather than the absolute level of health utility scores for those complications . little evidence exists on the change in the relative function levels associated with different complications in the last 10 years . thus , our estimates are very likely still applicable to the current diabetes population . patients with no health insurance tend to have a lower quality - of - life than those with health insurance ( 24 ) . however , it is less clear that disutility associated with diabetes treatment mode and diabetes - related complications differ between the two groups . in addition , our study population may not represent the entire managed care population with diabetes , and individuals in managed care might not be representative of the entire diabetic population ; for example , poorer , sicker , or older individuals with type 2 diabetes may have been less likely to participate in the triad survey . finally , our study was cross - sectional , and variation in responses could occur if hrqol was measured at multiple points in time . cost - utility analysis , the type of economic evaluation most often used to assess interventions used for the prevention and control of type 2 diabetes , requires valid and accurate health utility estimates by patient characteristics , treatment modalities , and the different clinical states of each diabetes - related complication . we derived a set of such health utility estimates using data collected in a large multicenter diabetes study using u.s .- based preference weights . our empirically derived health utility scores will allow researchers to calculate qalys for studies involving individuals living in the u.s . with type 2 diabetes and representing a wide variety of demographic characteristics , treatments , complications , and comorbidities . the health utility scores provided should facilitate studies of the health burden of diabetes and the cost - utility analysis of alternative strategies for the prevention and treatment of diabetes in the u.s .

wistar rats ( male , 200 g ) were made diabetic by streptozotocin ( 55 mg / kg body weight [ bw ] , intraperitoneally ) , and soon after establishment of hyperglycemia ( 34 days after induction of diabetes ) , the rats were divided into four groups : rats in group 1 remained in poor glycemic control for 11 to 12 months ( diab group , glycated hemoglobin [ ghb ] > 10% ) and rats in group 2 were in poor control for 6 months followed by good control for 6 additional months ( pc - rev group ) . in group 3 , rats were maintained in good control ( gc group , ghb approximately 6.0% ) , and rats in group 4 received diet supplemented with lipoic acid ( 400 mg / kg bw ) for the entire duration of the experiment ( d+la group ) . rats diabetic for 11 to 12 months had slightly higher serum triglycerides compared with their age - matched normal rats ( 350475 mg / dl in diabetes versus 250325 mg / dl in normal ) . as a type 2 diabetes model , 12-week and 40-week - old male zdf rats ( charles river laboratories , wilmington , ma , usa ) were used , and age - matched lean rats served as controls . compared with lean rats , zdf rats ( both 12 and 40 weeks old ) were severely hyperglycemic ( ghb values approximately 6% versus approximately 12% ) , and hyperlipidemic ( serum triglycerides 300400 mg / dl in lean versus 8001600 mg / dl in zdf rats ) . the entire animal colony was weighed two times a week , and their blood glucose was monitored once every week and ghb every 8 to 9 weeks , according to the procedures reported previously . glycemic control in rats mice , hemizygous overexpressing mnsod ( mnsod - tg , c57bl/6 background ) , mmp-9 gene - knockout ( mmp-9-ko , b6.fvb ( cg)-mmp9tm1tvu / j ) , and wild - type c57bl/6 ( wt ) were made diabetic by streptozotocin injection ( 55 mg / kg bw ) using the methods described previously . after approximately 10 months of diabetes , treatment of animals conformed to the arvo resolution on the use of animals in research , and was approved by the wayne state university 's animal care and use committee . diabetic and nondiabetic patients at the kresge eye institute , or department of medicine , wayne state university , detroit , mi , usa , were recruited either before scheduled retinal surgery or during an appointment for routine eye care or their routine appointment . diabetic patients with retinal changes were graded using the international clinical disease severity scale for diabetic retinopathy . the study was approved by the wayne state university institutional review board , and written informed consent was received from the patients before obtaining their blood sample . the protocol complied with all aspects of the health insurance portability and accountability act , and was conducted in accordance with the tenets of the declaration of helsinki . blood was collected in tubes containing edta as an anticoagulant , and was used for dna and rna isolation . clinical characteristics of human subjects ( diabetic and nondiabetic ) are included in table 2 . patient characteristics dna from blood was isolated using the protocol specified in qiagen kit for blood dna isolation ( qiagen , valencia , ca , usa ) , with minor modifications . briefly , blood was digested in lysis buffer for 1 hour at 56c , and this was followed by addition of 100% ethanol . the samples were loaded onto qiagen columns , and the dna was eluted in ae buffer . mitochondrial dna damage was performed in 10 ng dna by quantitative extended length pcr using elongase enzyme mix pcr kit ( invitrogen , carlsbad , ca , usa ) and sequence - specific primers , following the method routinely used in our laboratory ; table 3 shows the primer sequences . long region of mtdna ( 913 kb ) and also the d - loop region ( 8001400 bp ) were amplified in a reaction mixture containing 1 pcr buffer a and b , 200 mm dntp , 1 unit elongase , and 0.1 mm primers using an initial incubation cycle of 1 minute at 75c , 1 minute at 94c , and 24 cycles of 15 seconds at 94c and 12 minutes at 65c . the short region of mtdna ( 115220 bp ) and that of the d - loop ( 90150 bp ) were amplified using specific pcr primers . the thermal cycling conditions included 94c for 5 minutes , 28 cycles of 1 minute at 94c , 30 seconds at 60c , 1 minute at 72c , and the final extension was for 10 minutes at 72c . the pcr products were separated on 1.2% and 2.0% agarose gel for long and short amplification products , respectively . the intensity of pcr products was quantified using carestream digital software ( rochester , ny , usa ) , and relative amplification was calculated by normalizing the intensity of the long pcr product to the short pcr product ; decrease in the amplification ratio was indicative of an increase in dna damage . mitochondrial copy numbers were quantified in genomic dna using a sybr green based quantitative pcr ( qpcr ) and primers for cytochrome b ( cytb ) as mtdna marker and -actin as nuclear dna marker . mitochondrial dna transcription was performed in rna extracted from blood by trizol reagent ( invitrogen ) . complementary dna ( cdna ) was synthesized by high - capacity cdna reverse transcription kit ( applied biosystems , carlsbad , ca , usa ) , and transcripts of mtdna - encoded cytb of complex iii and nadph dehydrogenase 6 ( nd6 ) of complex i of the electron transport chain were quantified by sybr green based qpcr using -actin as a housekeeping gene . the retina from formalin - fixed eyes of zdf and lean rats was rinsed overnight with running water , digested with 3% crude trypsin ( invitrogen - gibco , grand island , ny , usa ) containing 200 mm sodium fluoride . trypsin - digested microvasculature was mounted on a glass slide , and stained with periodic acid schiff - hematoxylin to count the degenerative capillaries . the data obtained from rodents were statistically analyzed using sigma stat software ( spss , chicago , il , usa ) . for multiple group comparison , 1-way anova followed by student - newman - keuls test was used for data with normal distribution , whereas kruskal - wallis 1-way analysis followed by dunn 's test was performed for data that did not present normal distribution . a 2-tailed t - test was used for comparison between the data obtained from two groups of patients . data are expressed as mean sd , and p < 0.05 was considered statistically significant . wistar rats ( male , 200 g ) were made diabetic by streptozotocin ( 55 mg / kg body weight [ bw ] , intraperitoneally ) , and soon after establishment of hyperglycemia ( 34 days after induction of diabetes ) , the rats were divided into four groups : rats in group 1 remained in poor glycemic control for 11 to 12 months ( diab group , glycated hemoglobin [ ghb ] > 10% ) and rats in group 2 were in poor control for 6 months followed by good control for 6 additional months ( pc - rev group ) . in group 3 , rats were maintained in good control ( gc group , ghb approximately 6.0% ) , and rats in group 4 received diet supplemented with lipoic acid ( 400 mg / kg bw ) for the entire duration of the experiment ( d+la group ) . rats diabetic for 11 to 12 months had slightly higher serum triglycerides compared with their age - matched normal rats ( 350475 mg / dl in diabetes versus 250325 mg / dl in normal ) . as a type 2 diabetes model , 12-week and 40-week - old male zdf rats ( charles river laboratories , wilmington , ma , usa ) were used , and age - matched lean rats served as controls . compared with lean rats , zdf rats ( both 12 and 40 weeks old ) were severely hyperglycemic ( ghb values approximately 6% versus approximately 12% ) , and hyperlipidemic ( serum triglycerides 300400 mg / dl in lean versus 8001600 mg / dl in zdf rats ) . the entire animal colony was weighed two times a week , and their blood glucose was monitored once every week and ghb every 8 to 9 weeks , according to the procedures reported previously . glycemic control in rats mice , hemizygous overexpressing mnsod ( mnsod - tg , c57bl/6 background ) , mmp-9 gene - knockout ( mmp-9-ko , b6.fvb ( cg)-mmp9tm1tvu / j ) , and wild - type c57bl/6 ( wt ) were made diabetic by streptozotocin injection ( 55 mg / kg bw ) using the methods described previously . after approximately 10 months of diabetes , treatment of animals conformed to the arvo resolution on the use of animals in research , and was approved by the wayne state university 's animal care and use committee . diabetic and nondiabetic patients at the kresge eye institute , or department of medicine , wayne state university , detroit , mi , usa , were recruited either before scheduled retinal surgery or during an appointment for routine eye care or their routine appointment . diabetic patients with retinal changes were graded using the international clinical disease severity scale for diabetic retinopathy . the study was approved by the wayne state university institutional review board , and written informed consent was received from the patients before obtaining their blood sample . the protocol complied with all aspects of the health insurance portability and accountability act , and was conducted in accordance with the tenets of the declaration of helsinki blood was collected in tubes containing edta as an anticoagulant , and was used for dna and rna isolation . clinical characteristics of human subjects ( diabetic and nondiabetic ) are included in table 2 . dna from blood was isolated using the protocol specified in qiagen kit for blood dna isolation ( qiagen , valencia , ca , usa ) , with minor modifications . briefly , blood was digested in lysis buffer for 1 hour at 56c , and this was followed by addition of 100% ethanol . the samples were loaded onto qiagen columns , and the dna was eluted in ae buffer . mitochondrial dna damage was performed in 10 ng dna by quantitative extended length pcr using elongase enzyme mix pcr kit ( invitrogen , carlsbad , ca , usa ) and sequence - specific primers , following the method routinely used in our laboratory ; table 3 shows the primer sequences . long region of mtdna ( 913 kb ) and also the d - loop region ( 8001400 bp ) were amplified in a reaction mixture containing 1 pcr buffer a and b , 200 mm dntp , 1 unit elongase , and 0.1 mm primers using an initial incubation cycle of 1 minute at 75c , 1 minute at 94c , and 24 cycles of 15 seconds at 94c and 12 minutes at 65c . the short region of mtdna ( 115220 bp ) and that of the d - loop ( 90150 bp ) were amplified using specific pcr primers . the thermal cycling conditions included 94c for 5 minutes , 28 cycles of 1 minute at 94c , 30 seconds at 60c , 1 minute at 72c , and the final extension was for 10 minutes at 72c . the pcr products were separated on 1.2% and 2.0% agarose gel for long and short amplification products , respectively . the intensity of pcr products was quantified using carestream digital software ( rochester , ny , usa ) , and relative amplification was calculated by normalizing the intensity of the long pcr product to the short pcr product ; decrease in the amplification ratio was indicative of an increase in dna damage . mitochondrial copy numbers were quantified in genomic dna using a sybr green based quantitative pcr ( qpcr ) and primers for cytochrome b ( cytb ) as mtdna marker and -actin as nuclear dna marker . mitochondrial dna transcription was performed in rna extracted from blood by trizol reagent ( invitrogen ) . complementary dna ( cdna ) was synthesized by high - capacity cdna reverse transcription kit ( applied biosystems , carlsbad , ca , usa ) , and transcripts of mtdna - encoded cytb of complex iii and nadph dehydrogenase 6 ( nd6 ) of complex i of the electron transport chain were quantified by sybr green based qpcr using -actin as a housekeeping gene . the retina from formalin - fixed eyes of zdf and lean rats was rinsed overnight with running water , digested with 3% crude trypsin ( invitrogen - gibco , grand island , ny , usa ) containing 200 mm sodium fluoride . trypsin - digested microvasculature was mounted on a glass slide , and stained with periodic acid schiff - hematoxylin to count the degenerative capillaries . the data obtained from rodents were statistically analyzed using sigma stat software ( spss , chicago , il , usa ) . for multiple group comparison , 1-way anova followed by student - newman - keuls test was used for data with normal distribution , whereas kruskal - wallis 1-way analysis followed by dunn 's test was performed for data that did not present normal distribution . a 2-tailed t - test was used for comparison between the data obtained from two groups of patients . data are expressed as mean sd , and p < 0.05 was considered statistically significant . damage of retinal mtdna in diabetes is well documented ; to evaluate if mtdna damage is also observed in the peripheral blood , extended - length pcr was performed . as shown in figure 1a , blood from diabetic rats had significantly reduced ratio of long to short amplicons of mtdna , compared with the blood from age - matched normal rats , suggesting higher mtdna damage in diabetes . consistent with mtdna damage as a whole , its d - loop region in the same diabetic rats was also significantly damaged ( fig . dna damage was determined by amplifying the ( a ) 13.4-kb and 210-bp amplicons of the mtdna , and ( b ) 819-bp and 148-bp amplicons in the d - loop region of the mtdna . the relative amplification was quantified by normalizing the intensity of the long pcr product to the short pcr product . ( c ) copy numbers of mtdna were determined in the genomic dna by qpcr using cytb as mtdna marker and -actin as a nuclear dna marker . transcripts of mtdna - encoded ( d ) cytb and ( e ) nd6 were quantified by sybr green based qpcr using -actin as a housekeeping gene . each measurement was made in duplicate in five to seven rats in each group , and the values are represented as mean sd . diab , diabetes ; pc - rev , poor control for 6 months followed by good control for 6 months ; gc , good glycemic control . * p versus normal and # p < 0.05 versus diabetes . damaged mtdna results in decreased mtdna copy numbers ; to investigate if decrease in mtdna copy numbers is also seen in the peripheral blood , genomic dna from blood was analyzed . figure 1c clearly shows a 30% to 40% decrease in the ratio of cytb to -actin in blood genomic dna in diabetic rats compared with age - matched normal rats . in the same animals , the transcription of mtdna , as determined by the gene transcripts of mtdna - encoded cytb or nd6 , were also decreased by more than 50% ( figs . e ) , further suggesting that the blood could mimic similar to diabetes - induced mtdna damage as seen in the retina . administration of lipoic acid prevents retinal oxidative stress , mtdna damage , and the development of retinopathy in diabetic rats . to investigate if lipoic acid also ameliorates blood mtdna damage , damage to mtdna and its d - loop region was determined in the peripheral blood from the diabetic rats treated with lipoic acid . as shown in figures 1a and 1b , administration of lipoic acid ameliorated diabetes - induced damage to the peripheral blood mtdna and the d - loop region . clinical studies have shown that the re - institution of good glycemic control in the early stages of diabetes prevents the development of diabetic retinopathy , but if this glycemic control is instituted after a period of hyperglycemia , retinopathy continues to progress and this metabolic memory phenomenon is also duplicated in diabetic rodents . consistent with this , peripheral blood from the rats that were maintained in good control for the entire duration ( gc group ) did not show significant damage to the mtdna or d - loop region , but that from the rats maintained in poor glycemic control for 6 months followed by 6 months of good control ( pc - rev group ) , had significantly higher damage in both mtdna and d - loop region compared with the rats that remained normal . similarly , diabetes - induced decrease in mtdna copy numbers and transcription was also not reversed by 6 months of good control that had followed 6 months of poor control . the values obtained from the rats in pc - rev group were similar to those obtained from the rats in the diabetes group ( fig . 1 ) . to further confirm the utility of peripheral blood mtdna damage as a possible biomarker of diabetic retinopathy , blood from mice overexpressing sod2 or from mice with the mmp-9 gene suppressed was analyzed ; these genetically modulated mice are protected from the development of diabetic retinopathy . as with the rats , blood from wt diabetic mice had significantly higher degree of mtdna and d - loop damage than the blood from age - matched normal mice ( fig . 2 ) ; however , overexpression of sod2 , or suppression of mmp-9 , also prevented diabetes - induced mtdna and d - loop damage in the peripheral blood of these mice ( fig . peripheral blood mtdna damage in diabetic mice , and effect of genetic manipulation of sod2 or mmp-9 . blood from wt mice , or mice overexpressing sod2 or knocked out for mmp-9 , and diabetic for 10 months was analyzed for damage by amplifying ( a ) 10.0 kb and 116-bp amplicons of the mtdna , and ( b ) 830-bp and 103-bp amplicons of the d - loop region . the intensity of the long pcr product was normalized to that of the short pcr product . wt - n , wild - type normal mice ; wt - d , wild - type mice diabetic for approximately 10 months ; sod2-d and mmp9-d , diabetic mice overexpressing sod2 or being knocked out for mmp-9 respectively . * p and # p < 0.05 compared with wt - n and wt - d , respectively . more than 90% of diabetic patients have type 2 diabetes , and they also have high circulating lipids . in addition to hyperglycemia , hyperlipidemia is also now considered as a major component associated with the development of diabetic retinopathy . peripheral blood from 12- and 40-week - old zdf rats was analyzed . despite severe hyperglycemic / hyperlipidemia in both 12- and 40-week - old zdf rats , 40-week - old zdf rats had significant damage to mtdna and its d - loop in the peripheral blood , but 12-week - old rats showed no change compared with their age - matched lean rats ( figs . furthermore , in the same peripheral blood samples , mtdna copy numbers and transcription were also significantly decreased in 40-week - old zdf rats compared with no change in 12-week - old rats ( figs . retinal vasculature from 12-week - old zdf rats did not show any pathology associated with diabetic retinopathy ; zdf rats and their age - matched lean rats had three to six degenerative capillaries , but these numbers were approximately 4-fold higher in 40-week - old zdf rats ( fig . consistent with this , although retina from 12-week - old zdf rats did not present any increase in mtdna damage , 40-week - old zdf rats showed significant increase in mtdna damage ( fig . 4b ) . damage of mtdna and its transcription in the peripheral blood from type 2 diabetic rats . peripheral blood from zdf and age - matched lean rats ( ln ) , 12 weeks and 40 weeks of age , was analyzed for ( a ) mtdna and ( b ) d - loop damage by extended - length pcr . the relative amplification was quantified by normalizing the intensity of the long pcr product to the short pcr product . ( c ) copy numbers of mtdna were determined in the genomic dna by qpcr using cytb as mtdna marker and -actin as a nuclear dna marker . ( d ) transcripts of mtdna - encoded cytb and nd6 were quantified by sybr green based qpcr using -actin as a housekeeping gene . each measurement was made in duplicate in five to seven rats in each group , and the values are represented as mean sd . * p versus age - matched lean rat . retinal histopathology and mtdna damage in zdf rats : ( a ) trypsin - digested retinal microvessels from 12- and 40-week - old zdf rats were stained with periodic acid schiff - hematoxylin for acellular capillaries and the arrow indicates an acellular capillary ; the picture shows microvasculature from 12- and 40-week - old zdf rats and 40-week - old lean rats ; ( b ) mtdna damage in the retina was determined by extended - length pcr . values are represented as mean sd from five to seven rats per group ; * p < 0.05 compared with age - matched lean rats . concomitant with increased peripheral blood mtdna damage in the animal models at a stage when histopathology associated with diabetic retinopathy can be observed , mtdna damage was significantly higher in the blood from patients with documented diabetic retinopathy compared with their age - matched nondiabetic counterparts . however , diabetic patients without retinopathy did not present mtdna damage , and the values obtained from diabetic patients with and without documented retinopathy were significantly different from each other ( p < 0.05 ; fig . similar differences among these two groups of diabetic patients were observed in the d - loop damage ( fig . furthermore , although increased mtdna / d - loop damage in the peripheral blood from patients with diabetic retinopathy was accompanied by decreased mtdna copy numbers ( ratio of cytb and -actin expression in the blood genomic dna ; fig . 6a ) and subnormal mtdna transcription ( mrna levels of mtdna - encoded cytb ; fig . 6b ) , such impairments were not detectable in the peripheral blood from diabetic patients without retinopathy ; the values obtained from nondiabetic patients and diabetic patients without retinopathy were not significantly different from each other . damage was determined by amplifying ( a ) 8.8-kb and 223-bp amplicons in the mtdna , and ( b ) 1.4-kb and 90-bp amplicons in the d - loop region . each measurement was made in duplicate in blood from five to six diabetic patients each with retinopathy ( diab - ret ) or without retinopathy ( diab - no ret ) , and 7 to 10 nondiabetic subjects ( norm ) , and the values are represented as mean sd . * p < 0.05 versus normal . copy number of mtdna and its transcription in the peripheral blood from diabetic subjects . ( a ) copy number was quantified in the genomic dna by qpcr using cytb as mtdna - encoded and -actin as a nuclear dna - encoded gene . ( b ) transcripts of mtdna - encoded cytb were quantified in the blood cdna by qpcr using -actin as a housekeeping gene . the values are represented as mean sd obtained from five to six diabetic patients each with retinopathy ( diab - ret ) or without retinopathy ( diab - no ret ) , and 7 to 10 nondiabetic subjects ( norm ) . damage of retinal mtdna in diabetes is well documented ; to evaluate if mtdna damage is also observed in the peripheral blood , extended - length pcr was performed . as shown in figure 1a , blood from diabetic rats had significantly reduced ratio of long to short amplicons of mtdna , compared with the blood from age - matched normal rats , suggesting higher mtdna damage in diabetes . consistent with mtdna damage as a whole , its d - loop region in the same diabetic rats was also significantly damaged ( fig . dna damage was determined by amplifying the ( a ) 13.4-kb and 210-bp amplicons of the mtdna , and ( b ) 819-bp and 148-bp amplicons in the d - loop region of the mtdna . the relative amplification was quantified by normalizing the intensity of the long pcr product to the short pcr product . ( c ) copy numbers of mtdna were determined in the genomic dna by qpcr using cytb as mtdna marker and -actin as a nuclear dna marker . transcripts of mtdna - encoded ( d ) cytb and ( e ) nd6 were quantified by sybr green based qpcr using -actin as a housekeeping gene . each measurement was made in duplicate in five to seven rats in each group , and the values are represented as mean sd . diab , diabetes ; pc - rev , poor control for 6 months followed by good control for 6 months ; gc , good glycemic control . * p versus normal and # p < 0.05 versus diabetes . damaged mtdna results in decreased mtdna copy numbers ; to investigate if decrease in mtdna copy numbers is also seen in the peripheral blood , genomic dna from blood was analyzed . figure 1c clearly shows a 30% to 40% decrease in the ratio of cytb to -actin in blood genomic dna in diabetic rats compared with age - matched normal rats . in the same animals , the transcription of mtdna , as determined by the gene transcripts of mtdna - encoded cytb or nd6 , were also decreased by more than 50% ( figs . e ) , further suggesting that the blood could mimic similar to diabetes - induced mtdna damage as seen in the retina . administration of lipoic acid prevents retinal oxidative stress , mtdna damage , and the development of retinopathy in diabetic rats . to investigate if lipoic acid also ameliorates blood mtdna damage , damage to mtdna and its d - loop region was determined in the peripheral blood from the diabetic rats treated with lipoic acid . as shown in figures 1a and 1b , administration of lipoic acid ameliorated diabetes - induced damage to the peripheral blood mtdna and the d - loop region . clinical studies have shown that the re - institution of good glycemic control in the early stages of diabetes prevents the development of diabetic retinopathy , but if this glycemic control is instituted after a period of hyperglycemia , retinopathy continues to progress and this metabolic memory phenomenon is also duplicated in diabetic rodents . consistent with this , peripheral blood from the rats that were maintained in good control for the entire duration ( gc group ) did not show significant damage to the mtdna or d - loop region , but that from the rats maintained in poor glycemic control for 6 months followed by 6 months of good control ( pc - rev group ) , had significantly higher damage in both mtdna and d - loop region compared with the rats that remained normal . similarly , diabetes - induced decrease in mtdna copy numbers and transcription was also not reversed by 6 months of good control that had followed 6 months of poor control . the values obtained from the rats in pc - rev group were similar to those obtained from the rats in the diabetes group ( fig . to further confirm the utility of peripheral blood mtdna damage as a possible biomarker of diabetic retinopathy , blood from mice overexpressing sod2 or from mice with the mmp-9 gene suppressed was analyzed ; these genetically modulated mice are protected from the development of diabetic retinopathy . as with the rats , blood from wt diabetic mice had significantly higher degree of mtdna and d - loop damage than the blood from age - matched normal mice ( fig . 2 ) ; however , overexpression of sod2 , or suppression of mmp-9 , also prevented diabetes - induced mtdna and d - loop damage in the peripheral blood of these mice ( fig . 2 ) . peripheral blood mtdna damage in diabetic mice , and effect of genetic manipulation of sod2 or mmp-9 . blood from wt mice , or mice overexpressing sod2 or knocked out for mmp-9 , and diabetic for 10 months was analyzed for damage by amplifying ( a ) 10.0 kb and 116-bp amplicons of the mtdna , and ( b ) 830-bp and 103-bp amplicons of the d - loop region . the intensity of the long pcr product was normalized to that of the short pcr product . wt - n , wild - type normal mice ; wt - d , wild - type mice diabetic for approximately 10 months ; sod2-d and mmp9-d , diabetic mice overexpressing sod2 or being knocked out for mmp-9 respectively . * p and # p < 0.05 compared with wt - n and wt - d , respectively . more than 90% of diabetic patients have type 2 diabetes , and they also have high circulating lipids . in addition to hyperglycemia , hyperlipidemia is also now considered as a major component associated with the development of diabetic retinopathy . peripheral blood from 12- and 40-week - old zdf rats was analyzed . despite severe hyperglycemic / hyperlipidemia in both 12- and 40-week - old zdf rats , 40-week - old zdf rats had significant damage to mtdna and its d - loop in the peripheral blood , but 12-week - old rats showed no change compared with their age - matched lean rats ( figs . furthermore , in the same peripheral blood samples , mtdna copy numbers and transcription were also significantly decreased in 40-week - old zdf rats compared with no change in 12-week - old rats ( figs . retinal vasculature from 12-week - old zdf rats did not show any pathology associated with diabetic retinopathy ; zdf rats and their age - matched lean rats had three to six degenerative capillaries , but these numbers were approximately 4-fold higher in 40-week - old zdf rats ( fig . 4a ) . consistent with this , although retina from 12-week - old zdf rats did not present any increase in mtdna damage , 40-week - old zdf rats showed significant increase in mtdna damage ( fig . 4b ) . damage of mtdna and its transcription in the peripheral blood from type 2 diabetic rats . peripheral blood from zdf and age - matched lean rats ( ln ) , 12 weeks and 40 weeks of age , was analyzed for ( a ) mtdna and ( b ) d - loop damage by extended - length pcr . the relative amplification was quantified by normalizing the intensity of the long pcr product to the short pcr product . ( c ) copy numbers of mtdna were determined in the genomic dna by qpcr using cytb as mtdna marker and -actin as a nuclear dna marker . ( d ) transcripts of mtdna - encoded cytb and nd6 were quantified by sybr green based qpcr using -actin as a housekeeping gene . each measurement was made in duplicate in five to seven rats in each group , and the values are represented as mean sd . retinal histopathology and mtdna damage in zdf rats : ( a ) trypsin - digested retinal microvessels from 12- and 40-week - old zdf rats were stained with periodic acid schiff - hematoxylin for acellular capillaries and the arrow indicates an acellular capillary ; the picture shows microvasculature from 12- and 40-week - old zdf rats and 40-week - old lean rats ; ( b ) mtdna damage in the retina was determined by extended - length pcr . values are represented as mean sd from five to seven rats per group ; * p < 0.05 compared with age - matched lean rats . concomitant with increased peripheral blood mtdna damage in the animal models at a stage when histopathology associated with diabetic retinopathy can be observed , mtdna damage was significantly higher in the blood from patients with documented diabetic retinopathy compared with their age - matched nondiabetic counterparts . however , diabetic patients without retinopathy did not present mtdna damage , and the values obtained from diabetic patients with and without documented retinopathy were significantly different from each other ( p < 0.05 ; fig . similar differences among these two groups of diabetic patients were observed in the d - loop damage ( fig . 5b ) . furthermore , although increased mtdna / d - loop damage in the peripheral blood from patients with diabetic retinopathy was accompanied by decreased mtdna copy numbers ( ratio of cytb and -actin expression in the blood genomic dna ; fig . 6a ) and subnormal mtdna transcription ( mrna levels of mtdna - encoded cytb ; fig . 6b ) , such impairments were not detectable in the peripheral blood from diabetic patients without retinopathy ; the values obtained from nondiabetic patients and diabetic patients without retinopathy were not significantly different from each other . damage was determined by amplifying ( a ) 8.8-kb and 223-bp amplicons in the mtdna , and ( b ) 1.4-kb and 90-bp amplicons in the d - loop region . each measurement was made in duplicate in blood from five to six diabetic patients each with retinopathy ( diab - ret ) or without retinopathy ( diab - no ret ) , and 7 to 10 nondiabetic subjects ( norm ) , and the values are represented as mean sd . * p < 0.05 versus normal . copy number of mtdna and its transcription in the peripheral blood from diabetic subjects . ( a ) copy number was quantified in the genomic dna by qpcr using cytb as mtdna - encoded and -actin as a nuclear dna - encoded gene . ( b ) transcripts of mtdna - encoded cytb were quantified in the blood cdna by qpcr using -actin as a housekeeping gene . the values are represented as mean sd obtained from five to six diabetic patients each with retinopathy ( diab - ret ) or without retinopathy ( diab - no ret ) , and 7 to 10 nondiabetic subjects ( norm ) . duration of diabetes is considered as one of the strongest predictors of the development and progression of diabetic retinopathy . during the initial stages of this progressing disease , patients often do not present any noticeable symptoms , but by the time some vascular lesions are detected in the retina , their vision loss could become irreversible . our previous work has shown that mtdna damage in the retina and its vasculature has an important role in the development of diabetic retinopathy , and this damage is observed before the pathology associated with diabetic retinopathy can be noticed in diabetic rodents . here , we present novel data from animal models of type 1 diabetes showing that mtdna is damaged in the peripheral blood of diabetic rodents , this damage is not detectable during the duration of hyperglycemia when retinal histopathology is not observed , and pharmacologic / genetic manipulations that prevent the development of diabetic retinopathy prevent blood mtdna damage . re - institution of good control in diabetic rats , after a period of poor glycemic control , which fails to reverse retinal mtdna damage and prevent the progression of diabetic retinopathy , also fails to prevent blood mtdna damage . in the type 2 diabetes model , peripheral blood mtdna damage is not observed during the age when retinopathy is not detectable . these results clearly suggest that the damage in peripheral blood mtdna could serve as a noninvasive biomarker of the development of diabetic retinopathy . as a proof of principle , we have provided data from diabetic patients with and without retinopathy documenting increased blood mtdna damage and decreased copy numbers only in the patients with retinopathy compared with their age - matched nondiabetic individuals . mammalian mitochondria have their own double - stranded small dna ( 16 kb ) , which encodes genes for proteins that are critical in normal mitochondrial homeostasis . due to close proximity to the free radical producing electron transport system , and lack of protective histones , mtdna is highly susceptible to oxidative damage . in the pathogenesis of diabetic retinopathy , mtdna is damaged , and the damage is more extensive at the noncoding region , the d - loop region with essential transcription / replication elements ; here we show that the diabetic rodents present similar d - loop damage in their peripheral blood . the number of mitochondria vary depending on the energetic requirements , the environment , and the redox balance , and brain / retina cell with high metabolic rate may have more than 2000 mitochondria , compared with less than 100 mitochondria in a white blood cell . although initial oxidative stress could increase mitochondria numbers , chronic oxidative stress decreases their numbers and increases mtdna damage . our previous study showed decreased mitochondria numbers in the retina and its endothelium in diabetes , and here we show the similar phenomenon in the blood . blood from diabetic rodents , at a stage of diabetes when retinopathy can be detected , have lower mtdna copy numbers , and the therapies that prevent the development of diabetic retinopathy in these rodents also prevent increase in mtdna damage and decrease in mtdna copy numbers in their blood . in support , depletion in mtdna content in lymphocytes is associated with poor glycemic control , and other chronic diseases . consistent with the decreased retinal mtdna transcription , the levels of mtdna - encoded genes cytb and nd6 are also decreased in the blood of diabetic rodents , further supporting the validity of blood mtdna . in diabetes , regulation of oxidative stress via pharmacologic or genetic manipulations protects retinal mitochondria damage and the development of diabetic retinopathy . here , our results demonstrate that the same manipulations also prevent mtdna damage in peripheral blood and ameliorate decrease in copy numbers and transcription . the two genetic manipulations associated with the prevention of the development of retinopathy in diabetic mice , the suppression of mmp-9 , an enzyme that is implicated in increasing mitochondrial membrane permeability and activating the pro - apoptotic machinery , and overexpression of sod2 , also prevent increased mtdna damage in the blood . in addition , administration of lipoic acid , an antioxidant that ameliorates retinal mtdna damage and the development of diabetic retinopathy in rats , also attenuates diabetes - induced mtdna damage in the blood . pioneering dcct - edic studies have clearly shown that the progression of retinopathy continues to benefit from the prior intensive control , and the same metabolic memory phenomenon is also observed in experimental models of diabetic retinopathy . rodent models have shown that the retinal mitochondrial structure , function , and its dna remain damaged , which generates a vicious cycle of superoxide , and the retinopathy continues to progress . convincing data presented here demonstrate that peripheral blood mtdna damage also embodies the same phenomenon , further strengthening an association between the blood mtdna damage and the progression of diabetic retinopathy . the validity of peripheral mtdna damage as a possible biomarker of diabetic retinopathy is further supported by our results from type 2 diabetic animal models ; although mtdna damage and its transcription are not observed in peripheral blood of zdf rats at 12 weeks of age , an age when these animals show no signs of retinopathy and retinal mtdna damage , at 40 weeks , an age when retinopathy and mtdna damage are detectable in the retina and its vasculature , mtdna damage is also observed in the blood . these results clearly suggest that despite severe hyperglycemia / hyperlipidemia , during the earlier stages of the disease , peripheral blood mtdna is not altered , but with the extending duration of hyperglycemia / hyperlipidemia ( 40 weeks of age ) , blood also shows mtdna damage . these results also document that type 1 and type 2 diabetes share similar phenomena . as with experimental models , mtdna damage and decrease in its transcription and copy numbers have been observed in the retinal microvasculature from human donors with diabetic retinopathy . the results presented here clearly demonstrate that the peripheral blood from patients with diabetic retinopathy also has increased mtdna damage and decreased copy numbers compared with their age - matched nondiabetic counterparts ; however , such damage is not detected in the blood from diabetic patients without retinopathy . these results support a close association between peripheral blood mtdna damage and diabetic retinopathy , and confirm the validity of peripheral blood mtdna damage as a possible biomarker of diabetic retinopathy . consistent with our results , others have shown a decrease in mtdna copy numbers and increase in mtdna damage in the peripheral blood from diabetic patients with proliferative retinopathy and neuropathy . we recognize that the number of patients used in the present study is limited , and we do not have details about the severity of hyperglycemia ; additional blood samples will be required to confirm relationship between mtdna damage in the blood and the severity of retinopathy , blood sugar control , and other systemic factors . in addition , we can not rule out the role of other systemic conditions , including blood pressure and other diabetic complications , in mtdna damage in the blood of these patients . however , data from diabetic patients with / without retinopathy is consistent with our results from animal models of type 1 diabetes with pharmacologic / genetic therapies to inhibit the development of diabetic retinopathy , and zdf rats with / without retinopathy , and support the possibility of blood mtdna damage as a potential biomarker of diabetic retinopathy . furthermore , a similar metabolic phenomenon is observed in the progression of diabetic retinopathy , and , here our results showing the failure of mtdna damage in blood to benefit from reversal of hyperglycemia imply a strong relationship between blood mtdna damage and diabetic retinopathy . in conclusion , we show a direct relation between diabetic retinopathy and mtdna damage in the peripheral blood . this is supported by our convincing results from type 1 diabetic rodent models with and without modulating their metabolic abnormalities by either a pharmacologic or by a genetic approach , type 2 diabetic model with severe hyperlipidemia / hyperglycemia at an age when retinopathy is not detectable to the age of animals when retinal mtdna and retinopathy are present , and peripheral blood from patients with and without diabetic retinopathy . damaged mtdna in peripheral blood is now being considered as one of the biomarkers of the obstructive sleep apnea syndrome and also for traumatic brain injury ; the results presented here clearly suggest its use as a possible biomarker of diabetic retinopathy .

purposein the development of diabetic retinopathy , retinal mitochondria become dysfunctional , and mitochondrial dna ( mtdna ) is damaged . because retinopathy is a progressive disease , and circulating glucose levels are high in diabetes , our aim was to investigate if peripheral blood mtdna damage can serve as a potential biomarker of diabetic retinopathy.methodsperipheral blood mtdna damage was investigated by extended - length pcr in rats and mice , diabetic for 10 to 12 months ( streptozotocin - induced , type 1 model ) , and in 12- and 40-week - old zucker diabetic fatty rats ( zdf , type 2 ) . mitochondrial copy number ( in gdna ) and transcription ( in cdna ) were quantified by qpcr . similar parameters were measured in blood from diabetic patients with / without retinopathy.resultsperipheral blood from diabetic rodents had significantly increased mtdna damage and decreased copy numbers and transcription . lipoic acid administration in diabetic rats , or sod2 overexpression or mmp-9 knockdown in mice , the therapies that prevent diabetic retinopathy , also ameliorated blood mtdna damage and restored copy numbers and transcription . although blood from 40-week - old zdf rats had significant mtdna damage , 12-week - old rats had normal mtdna . diabetic patients with retinopathy had increased blood mtdna damage , and decreased transcription and copy numbers compared with diabetic patients without retinopathy and nondiabetic individuals.conclusionstype 1 diabetic rodents with oxidative stress modulated by pharmacologic / genetic means , and type 2 animal model and patients with / without diabetic retinopathy , demonstrate a strong relation between peripheral blood mtdna damage and diabetic retinopathy , and suggest the possibility of use of peripheral blood mtdna as a noninvasive biomarker of diabetic retinopathy .

Methods Animals Human Samples DNA Damage Copy Numbers Transcription Retinal Histopathology Statistics Analysis Results Type 1 Diabetes Amelioration of Blood mtDNA Damage by Regulation of Oxidative Stress Reversal of Hyperglycemia and Blood mtDNA Damage Genetic Manipulations Inhibit the Development of Diabetic Retinopathy and Blood mtDNA Damage Type 2 Diabetes Human Discussion

wistar rats ( male , 200 g ) were made diabetic by streptozotocin ( 55 mg / kg body weight [ bw ] , intraperitoneally ) , and soon after establishment of hyperglycemia ( 34 days after induction of diabetes ) , the rats were divided into four groups : rats in group 1 remained in poor glycemic control for 11 to 12 months ( diab group , glycated hemoglobin [ ghb ] > 10% ) and rats in group 2 were in poor control for 6 months followed by good control for 6 additional months ( pc - rev group ) . rats diabetic for 11 to 12 months had slightly higher serum triglycerides compared with their age - matched normal rats ( 350475 mg / dl in diabetes versus 250325 mg / dl in normal ) . as a type 2 diabetes model , 12-week and 40-week - old male zdf rats ( charles river laboratories , wilmington , ma , usa ) were used , and age - matched lean rats served as controls . compared with lean rats , zdf rats ( both 12 and 40 weeks old ) were severely hyperglycemic ( ghb values approximately 6% versus approximately 12% ) , and hyperlipidemic ( serum triglycerides 300400 mg / dl in lean versus 8001600 mg / dl in zdf rats ) . complementary dna ( cdna ) was synthesized by high - capacity cdna reverse transcription kit ( applied biosystems , carlsbad , ca , usa ) , and transcripts of mtdna - encoded cytb of complex iii and nadph dehydrogenase 6 ( nd6 ) of complex i of the electron transport chain were quantified by sybr green based qpcr using -actin as a housekeeping gene . wistar rats ( male , 200 g ) were made diabetic by streptozotocin ( 55 mg / kg body weight [ bw ] , intraperitoneally ) , and soon after establishment of hyperglycemia ( 34 days after induction of diabetes ) , the rats were divided into four groups : rats in group 1 remained in poor glycemic control for 11 to 12 months ( diab group , glycated hemoglobin [ ghb ] > 10% ) and rats in group 2 were in poor control for 6 months followed by good control for 6 additional months ( pc - rev group ) . rats diabetic for 11 to 12 months had slightly higher serum triglycerides compared with their age - matched normal rats ( 350475 mg / dl in diabetes versus 250325 mg / dl in normal ) . as a type 2 diabetes model , 12-week and 40-week - old male zdf rats ( charles river laboratories , wilmington , ma , usa ) were used , and age - matched lean rats served as controls . compared with lean rats , zdf rats ( both 12 and 40 weeks old ) were severely hyperglycemic ( ghb values approximately 6% versus approximately 12% ) , and hyperlipidemic ( serum triglycerides 300400 mg / dl in lean versus 8001600 mg / dl in zdf rats ) . complementary dna ( cdna ) was synthesized by high - capacity cdna reverse transcription kit ( applied biosystems , carlsbad , ca , usa ) , and transcripts of mtdna - encoded cytb of complex iii and nadph dehydrogenase 6 ( nd6 ) of complex i of the electron transport chain were quantified by sybr green based qpcr using -actin as a housekeeping gene . damage of retinal mtdna in diabetes is well documented ; to evaluate if mtdna damage is also observed in the peripheral blood , extended - length pcr was performed . as shown in figure 1a , blood from diabetic rats had significantly reduced ratio of long to short amplicons of mtdna , compared with the blood from age - matched normal rats , suggesting higher mtdna damage in diabetes . administration of lipoic acid prevents retinal oxidative stress , mtdna damage , and the development of retinopathy in diabetic rats . to investigate if lipoic acid also ameliorates blood mtdna damage , damage to mtdna and its d - loop region was determined in the peripheral blood from the diabetic rats treated with lipoic acid . clinical studies have shown that the re - institution of good glycemic control in the early stages of diabetes prevents the development of diabetic retinopathy , but if this glycemic control is instituted after a period of hyperglycemia , retinopathy continues to progress and this metabolic memory phenomenon is also duplicated in diabetic rodents . to further confirm the utility of peripheral blood mtdna damage as a possible biomarker of diabetic retinopathy , blood from mice overexpressing sod2 or from mice with the mmp-9 gene suppressed was analyzed ; these genetically modulated mice are protected from the development of diabetic retinopathy . peripheral blood mtdna damage in diabetic mice , and effect of genetic manipulation of sod2 or mmp-9 . blood from wt mice , or mice overexpressing sod2 or knocked out for mmp-9 , and diabetic for 10 months was analyzed for damage by amplifying ( a ) 10.0 kb and 116-bp amplicons of the mtdna , and ( b ) 830-bp and 103-bp amplicons of the d - loop region . peripheral blood from 12- and 40-week - old zdf rats was analyzed . despite severe hyperglycemic / hyperlipidemia in both 12- and 40-week - old zdf rats , 40-week - old zdf rats had significant damage to mtdna and its d - loop in the peripheral blood , but 12-week - old rats showed no change compared with their age - matched lean rats ( figs . furthermore , in the same peripheral blood samples , mtdna copy numbers and transcription were also significantly decreased in 40-week - old zdf rats compared with no change in 12-week - old rats ( figs . retinal vasculature from 12-week - old zdf rats did not show any pathology associated with diabetic retinopathy ; zdf rats and their age - matched lean rats had three to six degenerative capillaries , but these numbers were approximately 4-fold higher in 40-week - old zdf rats ( fig . consistent with this , although retina from 12-week - old zdf rats did not present any increase in mtdna damage , 40-week - old zdf rats showed significant increase in mtdna damage ( fig . peripheral blood from zdf and age - matched lean rats ( ln ) , 12 weeks and 40 weeks of age , was analyzed for ( a ) mtdna and ( b ) d - loop damage by extended - length pcr . retinal histopathology and mtdna damage in zdf rats : ( a ) trypsin - digested retinal microvessels from 12- and 40-week - old zdf rats were stained with periodic acid schiff - hematoxylin for acellular capillaries and the arrow indicates an acellular capillary ; the picture shows microvasculature from 12- and 40-week - old zdf rats and 40-week - old lean rats ; ( b ) mtdna damage in the retina was determined by extended - length pcr . concomitant with increased peripheral blood mtdna damage in the animal models at a stage when histopathology associated with diabetic retinopathy can be observed , mtdna damage was significantly higher in the blood from patients with documented diabetic retinopathy compared with their age - matched nondiabetic counterparts . however , diabetic patients without retinopathy did not present mtdna damage , and the values obtained from diabetic patients with and without documented retinopathy were significantly different from each other ( p < 0.05 ; fig . furthermore , although increased mtdna / d - loop damage in the peripheral blood from patients with diabetic retinopathy was accompanied by decreased mtdna copy numbers ( ratio of cytb and -actin expression in the blood genomic dna ; fig . 6b ) , such impairments were not detectable in the peripheral blood from diabetic patients without retinopathy ; the values obtained from nondiabetic patients and diabetic patients without retinopathy were not significantly different from each other . each measurement was made in duplicate in blood from five to six diabetic patients each with retinopathy ( diab - ret ) or without retinopathy ( diab - no ret ) , and 7 to 10 nondiabetic subjects ( norm ) , and the values are represented as mean sd . damage of retinal mtdna in diabetes is well documented ; to evaluate if mtdna damage is also observed in the peripheral blood , extended - length pcr was performed . as shown in figure 1a , blood from diabetic rats had significantly reduced ratio of long to short amplicons of mtdna , compared with the blood from age - matched normal rats , suggesting higher mtdna damage in diabetes . administration of lipoic acid prevents retinal oxidative stress , mtdna damage , and the development of retinopathy in diabetic rats . to investigate if lipoic acid also ameliorates blood mtdna damage , damage to mtdna and its d - loop region was determined in the peripheral blood from the diabetic rats treated with lipoic acid . clinical studies have shown that the re - institution of good glycemic control in the early stages of diabetes prevents the development of diabetic retinopathy , but if this glycemic control is instituted after a period of hyperglycemia , retinopathy continues to progress and this metabolic memory phenomenon is also duplicated in diabetic rodents . to further confirm the utility of peripheral blood mtdna damage as a possible biomarker of diabetic retinopathy , blood from mice overexpressing sod2 or from mice with the mmp-9 gene suppressed was analyzed ; these genetically modulated mice are protected from the development of diabetic retinopathy . peripheral blood mtdna damage in diabetic mice , and effect of genetic manipulation of sod2 or mmp-9 . blood from wt mice , or mice overexpressing sod2 or knocked out for mmp-9 , and diabetic for 10 months was analyzed for damage by amplifying ( a ) 10.0 kb and 116-bp amplicons of the mtdna , and ( b ) 830-bp and 103-bp amplicons of the d - loop region . in addition to hyperglycemia , hyperlipidemia is also now considered as a major component associated with the development of diabetic retinopathy . peripheral blood from 12- and 40-week - old zdf rats was analyzed . despite severe hyperglycemic / hyperlipidemia in both 12- and 40-week - old zdf rats , 40-week - old zdf rats had significant damage to mtdna and its d - loop in the peripheral blood , but 12-week - old rats showed no change compared with their age - matched lean rats ( figs . furthermore , in the same peripheral blood samples , mtdna copy numbers and transcription were also significantly decreased in 40-week - old zdf rats compared with no change in 12-week - old rats ( figs . retinal vasculature from 12-week - old zdf rats did not show any pathology associated with diabetic retinopathy ; zdf rats and their age - matched lean rats had three to six degenerative capillaries , but these numbers were approximately 4-fold higher in 40-week - old zdf rats ( fig . consistent with this , although retina from 12-week - old zdf rats did not present any increase in mtdna damage , 40-week - old zdf rats showed significant increase in mtdna damage ( fig . peripheral blood from zdf and age - matched lean rats ( ln ) , 12 weeks and 40 weeks of age , was analyzed for ( a ) mtdna and ( b ) d - loop damage by extended - length pcr . retinal histopathology and mtdna damage in zdf rats : ( a ) trypsin - digested retinal microvessels from 12- and 40-week - old zdf rats were stained with periodic acid schiff - hematoxylin for acellular capillaries and the arrow indicates an acellular capillary ; the picture shows microvasculature from 12- and 40-week - old zdf rats and 40-week - old lean rats ; ( b ) mtdna damage in the retina was determined by extended - length pcr . concomitant with increased peripheral blood mtdna damage in the animal models at a stage when histopathology associated with diabetic retinopathy can be observed , mtdna damage was significantly higher in the blood from patients with documented diabetic retinopathy compared with their age - matched nondiabetic counterparts . however , diabetic patients without retinopathy did not present mtdna damage , and the values obtained from diabetic patients with and without documented retinopathy were significantly different from each other ( p < 0.05 ; fig . furthermore , although increased mtdna / d - loop damage in the peripheral blood from patients with diabetic retinopathy was accompanied by decreased mtdna copy numbers ( ratio of cytb and -actin expression in the blood genomic dna ; fig . 6b ) , such impairments were not detectable in the peripheral blood from diabetic patients without retinopathy ; the values obtained from nondiabetic patients and diabetic patients without retinopathy were not significantly different from each other . each measurement was made in duplicate in blood from five to six diabetic patients each with retinopathy ( diab - ret ) or without retinopathy ( diab - no ret ) , and 7 to 10 nondiabetic subjects ( norm ) , and the values are represented as mean sd . our previous work has shown that mtdna damage in the retina and its vasculature has an important role in the development of diabetic retinopathy , and this damage is observed before the pathology associated with diabetic retinopathy can be noticed in diabetic rodents . here , we present novel data from animal models of type 1 diabetes showing that mtdna is damaged in the peripheral blood of diabetic rodents , this damage is not detectable during the duration of hyperglycemia when retinal histopathology is not observed , and pharmacologic / genetic manipulations that prevent the development of diabetic retinopathy prevent blood mtdna damage . re - institution of good control in diabetic rats , after a period of poor glycemic control , which fails to reverse retinal mtdna damage and prevent the progression of diabetic retinopathy , also fails to prevent blood mtdna damage . these results clearly suggest that the damage in peripheral blood mtdna could serve as a noninvasive biomarker of the development of diabetic retinopathy . as a proof of principle , we have provided data from diabetic patients with and without retinopathy documenting increased blood mtdna damage and decreased copy numbers only in the patients with retinopathy compared with their age - matched nondiabetic individuals . in the pathogenesis of diabetic retinopathy , mtdna is damaged , and the damage is more extensive at the noncoding region , the d - loop region with essential transcription / replication elements ; here we show that the diabetic rodents present similar d - loop damage in their peripheral blood . blood from diabetic rodents , at a stage of diabetes when retinopathy can be detected , have lower mtdna copy numbers , and the therapies that prevent the development of diabetic retinopathy in these rodents also prevent increase in mtdna damage and decrease in mtdna copy numbers in their blood . in diabetes , regulation of oxidative stress via pharmacologic or genetic manipulations protects retinal mitochondria damage and the development of diabetic retinopathy . here , our results demonstrate that the same manipulations also prevent mtdna damage in peripheral blood and ameliorate decrease in copy numbers and transcription . the two genetic manipulations associated with the prevention of the development of retinopathy in diabetic mice , the suppression of mmp-9 , an enzyme that is implicated in increasing mitochondrial membrane permeability and activating the pro - apoptotic machinery , and overexpression of sod2 , also prevent increased mtdna damage in the blood . in addition , administration of lipoic acid , an antioxidant that ameliorates retinal mtdna damage and the development of diabetic retinopathy in rats , also attenuates diabetes - induced mtdna damage in the blood . convincing data presented here demonstrate that peripheral blood mtdna damage also embodies the same phenomenon , further strengthening an association between the blood mtdna damage and the progression of diabetic retinopathy . the validity of peripheral mtdna damage as a possible biomarker of diabetic retinopathy is further supported by our results from type 2 diabetic animal models ; although mtdna damage and its transcription are not observed in peripheral blood of zdf rats at 12 weeks of age , an age when these animals show no signs of retinopathy and retinal mtdna damage , at 40 weeks , an age when retinopathy and mtdna damage are detectable in the retina and its vasculature , mtdna damage is also observed in the blood . these results clearly suggest that despite severe hyperglycemia / hyperlipidemia , during the earlier stages of the disease , peripheral blood mtdna is not altered , but with the extending duration of hyperglycemia / hyperlipidemia ( 40 weeks of age ) , blood also shows mtdna damage . as with experimental models , mtdna damage and decrease in its transcription and copy numbers have been observed in the retinal microvasculature from human donors with diabetic retinopathy . the results presented here clearly demonstrate that the peripheral blood from patients with diabetic retinopathy also has increased mtdna damage and decreased copy numbers compared with their age - matched nondiabetic counterparts ; however , such damage is not detected in the blood from diabetic patients without retinopathy . these results support a close association between peripheral blood mtdna damage and diabetic retinopathy , and confirm the validity of peripheral blood mtdna damage as a possible biomarker of diabetic retinopathy . consistent with our results , others have shown a decrease in mtdna copy numbers and increase in mtdna damage in the peripheral blood from diabetic patients with proliferative retinopathy and neuropathy . however , data from diabetic patients with / without retinopathy is consistent with our results from animal models of type 1 diabetes with pharmacologic / genetic therapies to inhibit the development of diabetic retinopathy , and zdf rats with / without retinopathy , and support the possibility of blood mtdna damage as a potential biomarker of diabetic retinopathy . furthermore , a similar metabolic phenomenon is observed in the progression of diabetic retinopathy , and , here our results showing the failure of mtdna damage in blood to benefit from reversal of hyperglycemia imply a strong relationship between blood mtdna damage and diabetic retinopathy . this is supported by our convincing results from type 1 diabetic rodent models with and without modulating their metabolic abnormalities by either a pharmacologic or by a genetic approach , type 2 diabetic model with severe hyperlipidemia / hyperglycemia at an age when retinopathy is not detectable to the age of animals when retinal mtdna and retinopathy are present , and peripheral blood from patients with and without diabetic retinopathy . damaged mtdna in peripheral blood is now being considered as one of the biomarkers of the obstructive sleep apnea syndrome and also for traumatic brain injury ; the results presented here clearly suggest its use as a possible biomarker of diabetic retinopathy .

direct reprogramming of somatic cells into pluripotent stem cells ( termed induced pluripotent stem cells ( ipscs ) ) has been achieved by inducing the expression of transcription factors associated with pluripotent cell states and cell transformation . the first successful attempt used over - expression of four transcription factors , oct4 , sox2 , klf4 , and c - myc , to convert somatic fibroblasts to ipscs [ 24 ] . in subsequent studies , ipscs were generated by overexpressing only the first three of these factors , and combinations using other transcription factors ( e.g. , nanog , lin28 , nr5a2 , esrrb ) were also successful [ 58 ] . progress in ipsc technology development has faced two main problems : ( 1 ) risk of cell transformation and mutation by transduced gene expression constructs and ( 2 ) poor efficiency . transduction of somatic cells with synthetic mrnas or proteins has addressed the gene transfer problem [ 911 ] . other strategies have been the use of one or two of the transcription factors in combination with small molecules to achieve reprogramming [ 1214 ] . on the other hand , the low efficiency of reprogramming continues to present a major challenge , especially with gene - free strategies . with the stochastic model for reprogramming now generally accepted , one approach to increasing reprogramming efficiency might be the use of cell populations whose active genetic networks inherently share similarities with those of pluripotent stem cells . previous ipsc studies have focused mainly on transcription factor combinations necessary for the induction and maintenance of a pluripotent state . to the authors ' knowledge , the possibility of tissue stem cells being involved as a predisposed population , though considered , previously has not been investigated specifically . in the closest example on the topic , cultures enriched for neural stem cells were shown to be more efficiently reprogrammed than routinely used fibroblast populations [ 1618 ] . however , this property was attributed to the expression of complementing factors in these cells , instead of their tissue stem cell phenotype per se . other specific investigations of the role of tissue stem cells may have been discouraged by the well - known difficulty of obtaining quantities of these cells sufficient for informative experimental analyses . here , we report the investigation of readily available adult mouse pancreatic stem cell strains for predisposition for pluripotency reprogramming . these clonal tissue stem cells were expanded by the method of suppression of asymmetric cell kinetics ( sack ) . the sack method is based on the concept that tissue stem cells are difficult to expand ex vivo because of their inherent asymmetric self - renewal kinetics [ 19 , 20 ] . in vivo , asymmetric self - renewal allows tissue stem cells to maintain their own number while continuously replenishing differentiated tissue cells . however , in vitro , the same process leads to the dilution and loss of tissue stem cells among their accumulating lineage - committed progeny cells . the sack method uses specific guanine ribonucleotide precursors to shift tissue stem cells reversibly from asymmetric self - renewal kinetics to symmetric self - renewal kinetics , which promotes their exponential expansion in culture . we found that , when simply transferred to culture conditions used for pluripotent stem cells , sack - expanded pancreatic stem cells acquired properties of pluripotent stem cells . moreover , inclusion of xanthine ( xn ) , the sack agent used for their derivation , significantly increased their acquisition of properties characteristic of pluripotency . these findings suggest that targeting symmetrically self - renewing tissue stem cells could significantly increase the efficiency of pluripotency reprogramming when combined with other reprogramming strategies . mouse pancreatic cell strain ( mpanstra ) cells ; ( see section 3 for detailed description ) were maintained in high - glucose dulbecco 's modified eagle 's medium ( dmem ) supplemented with 10% dialyzed fetal bovine serum ( dfbs ; gibco , invitrogen , carlsbad , calif , usa ) , 100 u / ml penicillin , 100 g / ml streptomycin , and 1 mm xanthine ( xn , sigma chemical co. , st . louis , mo , usa ) , at 37c in a humidified incubator with 5% co2 . sto fibroblasts ( american type culture collection , manassas , va , usa ) were maintained in the same media , except the dfbs which was replaced with fetal bovine serum ( fbs ; sigma chemical co. ) , and no xn was added . mouse embryonic stem cells ( mescs ) were cultured , on sto feeder layers inactivated with mitomycin c ( roche applied science , indianapolis , ind , usa ) , in high - glucose dmem supplemented with 20% heat - inactivated fbs ( sigma ) , 100 u / ml penicillin , 100 g / ml streptomycin , 2 mm l - glutamine , nonessential amino acids ( gibco ) , 50 m -mercaptoethanol , and 10 ng / ml leukemia inhibitory factor . the media were either the mpanstra maintenance medium , mescs medium , or another mescs medium in which the heat - inactivated serum was replaced with 15% knockout serum replacement ( gibco ) . for each of these reprogramming conditions , one series was maintained without xn and another with 1 mm xn . for the initiation of reprogramming , cells were plated at a density of 1.5 10 cells per 10 cm diameter dish . two weeks later , cells from one plate were frozen , another plate was used for alkaline phosphatase staining , and isolated colonies were clonally expanded from the remaining plate . cells were fixed for 20 minutes at room temperature with 4% formaldehyde in phosphate - buffered saline ( pbs ) . they were permeabilized by incubating them with 2% bovine serum albumin ( bsa ) , 0.2% dried milk , and 0.4% triton x-100 in pbs for 10 minutes at room temperature . cells were blocked for one hour at 4c with a 3% dilution in pbs of the serum from the source - animal species of the secondary antibody . the rabbit polyclonal anti - oct4 and mouse monoclonal anti - klf4 antibodies ( stemgent , cambridge , mass , usa ) were diluted at 1 : 200 in blocking solution and incubated overnight with the cells at 4c . incubation with 1 : 200 dilutions of secondary antibodies ( goat polyclonal anti - rabbit - fitc , santa cruz biotechnology inc . , santa cruz , calif , usa ; polyclonal rabbit anti - mouse - fitc , dako , glostrup , denmark ) was also performed overnight at 4c . dapi staining and mounting were performed using dapi - containing vectashield mounting medium ( vector laboratories , burlingame , calif , usa ) . cells were fixed in 4% formaldehyde for two minutes at room temperature and rinsed with tbst ( 20 mm tris - hcl , ph 7.4 , 0.15 m nacl , 0.05% tween-20 ) . they were then incubated , according to the manufacturer 's protocol , in a mixture of fast red violet , naphthol as - bi phosphate solution , and water from the alkaline phosphatase detection kit ( millipore , billerica , mass , usa ) . before observation under the microscope , the cells were rinsed with tbst and covered with pbs . to estimate the alkaline phosphatase - positive ( ap+ ) cell fraction , cells in colonies from representative 40x - magnification microscope fields the ap+ fraction was calculated by dividing the ap+ cell number by the total number of cells evaluated . live cells , alkaline phosphatase - tested colonies , and histological sections were viewed using an observer . 100 g of cell lysates from each cell strain was separated by sds - page ( 10% polyacrylamide ) . proteins were blotted to a hybond ecl membrane in tris - glycine - methanol buffer ( 25 mm tris - hcl , ph 7.5 , 190 mm glycine , 20% methanol ) for 2 hours at 120 volts on ice . the membrane was blocked with tris - buffered saline ( tbs ) containing 10% dried milk and 0.1% tween-20 at room temperature for one hour . polyclonal rabbit anti - pdx1 antibody ( millipore , billerica , mass , usa ) was diluted 1 : 5000 in blocking solution and incubated overnight at 4c with the membrane . following four 15-minute washes in tbst , the membrane was incubated with ecl anti - rabbit - hrp ( ge healthcare , waukesha , wis , usa ) diluted at 1 : 2000 in blocking solution . hrp - conjugated antibodies were detected using amersham ecl plus western blotting detection system ( ge healthcare ) , followed by auto - photography . mpanstra-12 and mpanstra-25 cells , mescs , and reprogrammed 12-kosr - xn-83 , 12-kosr - xn-89 , 25-kosr - xn-85 , and 25-kosr - xn-86 cells were collected by trypsinization followed by three washes with pbs . viable cell number was determined using a vi - cell xr cell viability analyzer ( beckman coulter , brea , calif , usa ) . for each cell type , two immunodeficient mice ( 3-month - old nude - foxn1nu females ; harlan laboratories ) were injected subcutaneously in each hind limb ( two injections per mouse , four injections per cell type ) with 5 10 cells using a 26 g needle . they were transferred to 70% ethanol and shipped to mass histology service ( worcester , mass , usa ) for processing , embedding , sectioning ( 5 m ) , and hematoxylin - eosin staining . asymmetric self - renewal is a unique feature of tissue stem cells . in adult tissues , tissue stem cells divide asymmetrically , resulting in the production of a new tissue stem cell and a lineage - committed progenitor , the latter having a limited proliferative potential . while this mechanism provides an equilibrium by maintaining a constant number of tissue stem cells in tissues , it limits the ex vivo expansion of tissue stem cells ( figure 1(a ) ) . asymmetric self - renewal by tissue stem cells in vitro leads to their dilution and eventual loss during serial passage . to overcome this asymmetric cell kinetics barrier sack promotes tissue stem cells ' expansion by reversibly converting asymmetric self - renewal kinetics to symmetric self - renewal kinetics . symmetric cell kinetics promotes exponential expansion of tissue stem cells to the exclusion of production of lineage - committed progeny cells . asymmetric cell kinetics requires a p53-dependent decrease in guanine ribonucleotide biosynthesis , specifically resulting from the repression of inosine-5-monophosphate dehydrogenase ii ( impdh ii ) ( figure 1(b ) ) [ 2325 ] . the sack method uses specific exogenous guanine ribonucleotide salvage precursors and/or genes that circumvent the biochemical consequences of impdh ii downregulation by acting either upstream or downstream of the reaction catalyzed by impdh ii , which is rate limiting for guanine ribonucleotide biosynthesis . in the presented study , we used sack - expanded adult mouse pancreatic stem cell strains derived from transgenic mice expressing the xanthine phosphoribosyl transferase ( xprt ) gene . it catalyzes the conversion of xn to xanthosine-5-monophosphate ( xmp ) , the product of the impdh ii reaction with its substrate inosine-5-monophosphate ( imp ) . xn supplementation allowed expansion of pancreatic stem cells strains from adult xprt - transgenic mice by promoting guanine ribonucleotide production downstream of the p53-regulation point . properties of these cells will be reported in detail elsewhere . in brief , they were derived from isolated islets ; their proliferation is optimal under xn - supplementation conditions ( i.e. , more tissue stem cells are produced ) , and they have pancreatic endocrine cell differentiation potential . although the cell strains are clonal , their cultures are composed of a mixture of tissue stem cells , lineage - committed pancreatic progenitor cells , and nonproliferating cells . we refer to these sack - expanded , clonal , adult ( i.e. , eight - month old mice ) mouse pancreatic stem cells as although all mpanstra strains required xn for their derivation , some are highly dependent on xn to achieve their maximum proliferative rate ( i.e. , xn - dependent , sack - d[ependent ] strains ) , whereas others maintain high rates of proliferation without xn supplementation ( i.e. , xn - independent , sack - i[ndependent ] strains ) . the sack - d clones are nontumorigenic pancreatic stem cell strains ( manuscript in preparation ) . tissue stem cells share more gene expression networks with pluripotent stem cells than do other somatic cells [ 27 , 28 ] . we considered that these gene expression similarities , in combination with sack - induced symmetric self - renewal , might predispose tissue stem cells for greater efficiency in conversion to a pluripotent phenotype . here , we report evidence that adult mouse pancreatic stem cells derived by the sack method can be converted to increased tissue potency without the use of exogenous transcription factors . based on the criterion of formation of teratomas with tissues that originate from all three embryonic germ layers ( i.e. , endoderm , mesoderm , and ectoderm ) , the cells attain full pluripotency . we also show that the sack agent xn significantly increases the efficiency of sack - d strains to convert to increased tissue potency . we propose that this ability reflects the common involvement of p53 gene regulation in induced pluripotent stem cell production [ 2932 ] and tissue stem cell self - renewal kinetics [ 19 , 20 , 24 , 25 ] . consistent with the concept of some shared gene expression networks between pluripotent stem cells and tissue stem cells , transcription factors used to induce formation of pluripotent stem cells are detected in mpanstra strains . under xn - supplementation conditions , two evaluated sack - d strains , mpanstra-12 and mpanstra-25 , show expression of nuclear klf4 in a subset of cells ( figure 2(a ) ) , while oct4 expression can only be detected in the mpanstra-12 strain ( figure 2(b ) ) . sto fibroblast feeder cells in comparison to mesc cultures were negative for klf4 fluorescence ( figure 2(a ) ) . given their basal expression of genes associated with pluripotency , we investigated whether maintaining mpanstra cells under culture conditions for pluripotent stem cells would promote their conversion to a pluripotent state . all six conditions used gelatin - coated dishes covered with a monolayer of sto fibroblast feeder cells . in the first two conditions , evaluated mpanstra strains were cultured in their own maintenance medium , which is supplemented with dialyzed fetal bovine serum ( dfbs ) , either without or with xn supplementation ( dfbs - ctrl and dfbs - xn , resp . ) . the third and fourth conditions employed culture medium commonly used for the growth of mouse embryonic stem cells ( mescs ) that was supplemented with a high concentration of heat - inactivated , nondialyzed fbs and other factors required for the maintenance of pluripotency . this condition was also evaluated either without or with xn supplementation ( hifbs - ctrl and hifbs - xn , resp . ) . the final two conditions were based on a more recently available defined condition used to maintain and grow pluripotent stem cells . basically , heat - inactivated serum is substituted with knockout serum replacement ( kosr ) . again , this medium was evaluated without or with xn supplementation ( kosr - ctrl and kosr - xn , resp . ) . the most prominent effect noted upon transfer of the mpanstra strains to the six trial reprogramming conditions was a general inhibition of the cell proliferation of sack - d strains . during the first week of growth , mpanstra cells plated in parallel at the same starting cell number in their routine maintenance conditions attained confluency . in contrast , only the sack - i strains ( mpanstra-11 and mpanstra-56 ) reached confluency in all reprogramming conditions . the sack - d strains ( mpanstra-12 and mpanstra-25 ) showed reduced cell proliferation in the reprogramming conditions , and even after two weeks , only cell colonies had formed . two weeks after the transfer , two types of colonies were observed for the sack - d strains in the reprogramming conditions : striated colonies and epithelial - like colonies ( figure 3(b ) ) . some colonies developed subsets of cells with alkaline phosphatase activity ( red cells in figure 3(c ) ) , a property of pluripotent stem cells . for each sack - d strain reprogramming condition , we picked 24 well - isolated colonies for clonal expansion . the proportion of striated versus epithelial colonies picked reflected their initial reprogramming fractions . following expansion , all the subclones originating from the mpanstra-25 strain and derived in dfbs reprogramming conditions uniformly exhibited the striated morphology , and none of them showed detectable alkaline phosphatase activity ( table 1a ) . there was a significant shift toward the epithelial morphology in the subclones from the hifbs conditions , with less than a third of the clones having the striated morphology . in the subclones showing alkaline phosphatase activity , the proportion of positive cells was estimated to vary from 5% to 50% ( see example in figure 4 , top right panel ) . more dramatic was the change in the kosr reprogramming conditions . only the subclones with the epithelial morphology could be expanded , and the xn - supplementation condition had more than twice the proportion of subclones showing alkaline phosphatase activity . moreover , the proportion of alkaline phosphatase - positive cells was estimated to range from 5% to 60% with xn supplementation , while never being higher than 20% in its absence . in the case of the mpanstra-12 subclones , about a third of these that were derived in the dfbs reprogramming conditions had the epithelial morphology . however , none of these epithelial subclones had detectable alkaline phosphatase activity , even though some of the striated subclones showed activity ( table 1b ) . in the hifbs reprogramming conditions , there was no obvious shift from striated to epithelial morphology , but three of the eight epithelial subclones obtained showed alkaline phosphatase activity under conditions for xn supplementation . for the kosr reprogramming conditions , we observed a significant shift toward the epithelial morphology with xn ( 15 out of 17 expanded clones ; table 1b ) , with two - thirds of them showing alkaline phosphatase activity . overall , in kosr medium , xn increased the rate of epithelial colony formation by mpanstra-12 subclones 1.8-fold ( p = 0.044 ; two - tailed fisher 's exact test ) . the finding that colonies with an epithelial morphology readily developed in dfbs reprogramming conditions only from the mpanstra-12 strain suggested that mpanstra-12 might be a developmentally less committed stem cell strain than the mpanstra-25 strain . in earlier studies , we have found that the sack method could expand distinct stem cell types from the same tissue . consistent with this hypothesis , mpanstra-12 cells do not express the early pancreatic marker pdx1 at detectable protein levels ( figure 5 ) , suggesting that mpanstra-12 is less committed toward a specific differentiation pathway ( e.g. , pancreatic ) . thereby , it could be more susceptible to conversion to increased tissue potency . to evaluate the developmental tissue potency of the subclones with epithelial morphology , we assayed them for their capacity to form teratomas following subcutaneous injections into immunodeficient mice . mouse escs and ipscs are characterized by their ability to form teratomas , that is , tumors that contain differentiated cells originating from the three embryonic germ layers . for each sack - d strain , we selected two subclones derived in the kosr - xn reprogramming conditions . for both sack - d mpanstra strains , this was the condition leading to the highest fraction of subclones showing both the epithelial morphology and alkaline phosphatase activity . for each strain , one selected subclone had no detectable alkaline phosphatase activity ( ap ; 12-kosr - xn-83 and 25-kosr - xn-86 ) , whereas the other had the highest proportion of alkaline phosphatase - positive cells ( ap+ ; 12-kosr - xn-89 ( see figure 4 ) and 25-kosr - xn-85 ; 90% and 60% , resp . ) . two mice were injected subcutaneously in their hind limbs with each of the four subclones , respectively ( i.e. , four injection sites for each subclone ) . in parallel , two mice were injected similarly with either mescs or the parental sack - d mpanstra cell strains . four weeks after the injections , the mice receiving the mescs showed tumors at all injection sites . the mesc - derived tumors had histological features of all three embryonic germ layers ( figure 6 ) . among the other cells injected , only the subclones derived from the mpanstra-12 strain formed small masses ( at all injection sites , both ap and ap+ ) four months after injection . the growth of these masses was slow , and two additional months were allowed before euthanizing the mice and analyzing the tumors ( figure 7 ) , at which point the other injected mice were also euthanized and their tumor - free status confirmed . histological analysis of the tumors derived from the reprogrammed mpanstra-12 cells ( figure 6 ) revealed the presence of tissues of mesodermal ( e.g. , cartilage ( ca ) and muscle ( mu ) ) , endodermal ( e.g. , digestive tract epithelium ( de ) ) , and ectodermal ( e.g. , nerves ( ne ) ) origin , confirming acquisition of pluripotency . ex vivo - expanded adult mouse pancreatic stem cell strains were evaluated for their ability to acquire phenotypic properties of pluripotent stem cells when transferred into culture conditions permissive for the derivation and maintenance of pluripotent stem cells . because of the requirement that tissue stem cells maintain an undifferentiated phenotype with respect to the tissue lineages that they replenish , we considered that they might have gene expression networks that were more susceptible to pluripotent reprogramming by environmental factors found in pluripotent cell culture media . subsequently , we found that the sack - d mpanstra strains show expression of a subset of transcription factors ( oct4 , klf4 ) implicated in pluripotency state control mechanisms . this finding advanced further the possibility that these cells were , in fact , predisposed to pluripotency reprogramming . supporting this tissue stem cell target hypothesis , both of the evaluated sack - d mpanstra stem cell strains showed evidence of reprogramming after exposure to pluripotent cell culture conditions . however , only strain mpanstra-12 achieved properties indicative of complete pluripotency conversion . since the parental sack - d strains were clonal , did not form tumors themselves , and were of endodermal origin , pluripotency was acquired after the transfer into reprogramming media . the particular differences noted between reprogrammed mpanstra-12 cells and reprogrammed mpanstra-25 cells may account for their difference in teratoma formation . the mpanstra-12 cells showed expression of two pluripotency factors ( klf4 and oct4 ) in some cells , whereas only klf4 was detected in mpanstra-25 cells . also , when compared with mpanstra-25 cells , expression of the pancreatic determination factor pdx1 was not detected in mpanstra-12 cells . these properties suggest that the mpanstra-12 cells were already less committed to the pancreatic differentiation pathway and that the gene expression networks necessary for reprogramming were more active , which could explain their increased potency . recent studies suggest that partial reprogramming of fibroblasts may provide attractive alternatives for producing cells that can replenish specific tissues [ 35 , 36 ] . such strategies highlight the well - recognized , but often understated , flaw in proposed pluripotency - based cell therapies , teratoma formation . the possibility of altering the tissue potency of tissue stem cells simply by transferring them into a defined culture environment underlies another approach to avoiding the inherent risks of pluripotency . in the future , culture environment might be used to instruct plentiful tissue stem cells ( e.g. , sack - expanded ) to switch their potency to a single tissue type of greater clinical need . the presence of xn in this reprogramming condition increased the frequency of subclones showing epithelial morphology and alkaline phosphatase activity . xn is known to act on the parental stem cell strains by increasing the rate of symmetric self - renewal over asymmetric self - renewal , their default pattern . making the target cells adopt a symmetric self - renewal cell division pattern could advance them closer to a pluripotent stem cell phenotype , characterized by symmetric self - renewal ( in the cases of mescs and ipscs ) . however , clearly more is at play than just acquisition of a symmetric self - renewal state . sack - i strains which undergo constitutive symmetric self - renewal were unable to produce reprogrammed cells . the molecular mechanisms responsible for the emergence of sack - i strains are not fully understood , but it seems most likely that they are pancreatic stem cells that acquired transforming cell mutations , prior to or after their culture . because no stably propagated adult pancreatic cell strains emerged in the absence of xprt transgenesis and xn supplementation , it seems likely that the proposed mutations occur during or after sack expansion . although transformed clones like the sack - i strains develop , the sack method greatly limits their frequency , so that sack - d stem cell strains can be obtained with a high efficiency . previous studies have established that supplemented purines like xn regulate the self - renewing pattern of tissue - specific stem cells by increasing guanine ribonucleotide biosynthesis . the upstream step in this pathway is catalyzed by impdh ii , whose expression is controlled by the p53 tumor suppressor protein . xn acts downstream of impdh ii regulation by p53 and , thereby , overcomes the action of p53 on guanine ribonucleotide biosynthesis without altering the action of p53 on other vital cellular functions . recently , several studies have demonstrated that loss of p53 function was a major factor responsible for more efficient derivation of ipscs [ 2932 ] . a large part of the effect of p53 loss on ipsc efficiency may be the increased symmetric self - renewal by tissue stem cells in treated cell preparations as a result of increased guanine ribonucleotide biosynthesis . as noted earlier , among the many challenges to using ipsc - derived cells in transplantation medicine , carcinogenic transformation , beyond the gnomonic teratoma formation of pluripotent stem cells injected into mature tissues , is a major one . thus , the recent major goal in ipsc research has been achieving complete reprogramming at high efficiency without resorting to gene transduction approaches . many studies have shown that gene transduction by a variety of strategies can result in insertional mutagenesis that can lead to cellular transformation and neoplasia [ 3 , 37 ] . gene transduction was recently circumvented by transducing cells directly with mrnas encoding reprogramming proteins or reprogramming proteins themselves [ 911 ] . also , transduction with only one pluripotency factor in combination with cell culture supplementation with specific small molecules has led to successful reprogramming [ 1214 ] . adding xn and/or other exogenous guanine ribonucleotide precursors ( e.g. , xanthosine , hypoxanthine ) to the reprogramming environment could provide an important element to increase ipsc production efficiency . moreover , the use of starting cell populations containing tissue - specific stem cells undergoing symmetric self - renewal could provide even more efficient production of ipscs . even with gene - free reprogramming , the long assumed inherent cellular transformation risk of pluripotency would still be present . previously , this risk had been attributed to poor regulation of pluripotent cells by heterologous tissue environments [ 39 , 40 ] . however , this view may change soon , because , recently , both human escs and human ipscs were discovered to contain many mutations in well - known oncogenes and tumor - suppressor genes , indicating that their production is inherently carcinogenic [ 41 , 42 ] . the reprogramming process of ipscs is associated with deletions of tumor suppressor genes , followed by duplications of oncogenes during expansion in culture . the slow tumor development that we observe after culture environment reprogramming of pancreatic tissue stem cells could lead to a safer alternative . specific tissue stem cell types could prove more sensitive for tissue potency reprogramming and/or less prone to cellular transformation . of course , recognizing such success will require assays that do not require tumor formation for evaluation . looking for the ability of reprogrammed cells to replace injured tissues or expand normal tissues of a different embryonic germ layer than their origin is the ideal assay concept . also , the development of better - defined culture conditions for directing reprogramming could increase the efficiency and decrease the time necessary for reprogramming . we demonstrated that sack - derived adult pancreatic tissue stem cells could acquire properties of pluripotent stem cells simply by culture under conditions used for production and routine maintenance of the latter . strain mpanstra-12 , the least developmentally committed sack - d stem cell strain , showed the greatest potency . it was the only one of the two evaluated sack - d strains that achieved teratoma formation . the combination of pancreatic tissue stem cells and xn induction of symmetric self - renewal may reflect the same cellular effect as the loss of p53 function for increasing reprogramming efficiency . we predict that starting the reprogramming process with tissue stem - cell - enriched populations , and xn supplementation ( or other exogenous guanine ribonucleotide precursors ) will independently and synergistically enhance pluripotent reprogramming into bona fide induced pluripotent stem cells while reducing the rate of emergence of clones with transforming cell mutations .

previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells ( ipscs ) have focused mainly on transcription factors and small molecule combinations . here , we report the results of our focus instead on the phenotype of the cells targeted for reprogramming . we find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics ( sack ) acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells . moreover , supplementation with the sack agent xanthine , which promotes symmetric self - renewal , significantly increases the efficiency and degree of acquisition of pluripotency properties . in transplantation analyses , clonal reprogrammed pancreatic stem cells produce slow - growing tumors with tissue derivative of all three embryonic germ layers . this acquisition of pluripotency , without transduction with exogenous transcription factors , supports the concept that tissue stem cells are predisposed to cellular reprogramming , particularly when symmetrically self - renewing .

1. Introduction 2. Materials and Methods 3. Background to the Study 4. Results 5. Discussion 6. Conclusion

direct reprogramming of somatic cells into pluripotent stem cells ( termed induced pluripotent stem cells ( ipscs ) ) has been achieved by inducing the expression of transcription factors associated with pluripotent cell states and cell transformation . the first successful attempt used over - expression of four transcription factors , oct4 , sox2 , klf4 , and c - myc , to convert somatic fibroblasts to ipscs [ 24 ] . in subsequent studies , ipscs were generated by overexpressing only the first three of these factors , and combinations using other transcription factors ( e.g. other strategies have been the use of one or two of the transcription factors in combination with small molecules to achieve reprogramming [ 1214 ] . on the other hand , the low efficiency of reprogramming continues to present a major challenge , especially with gene - free strategies . with the stochastic model for reprogramming now generally accepted , one approach to increasing reprogramming efficiency might be the use of cell populations whose active genetic networks inherently share similarities with those of pluripotent stem cells . previous ipsc studies have focused mainly on transcription factor combinations necessary for the induction and maintenance of a pluripotent state . to the authors ' knowledge , the possibility of tissue stem cells being involved as a predisposed population , though considered , previously has not been investigated specifically . in the closest example on the topic , cultures enriched for neural stem cells were shown to be more efficiently reprogrammed than routinely used fibroblast populations [ 1618 ] . however , this property was attributed to the expression of complementing factors in these cells , instead of their tissue stem cell phenotype per se . other specific investigations of the role of tissue stem cells may have been discouraged by the well - known difficulty of obtaining quantities of these cells sufficient for informative experimental analyses . here , we report the investigation of readily available adult mouse pancreatic stem cell strains for predisposition for pluripotency reprogramming . these clonal tissue stem cells were expanded by the method of suppression of asymmetric cell kinetics ( sack ) . the sack method is based on the concept that tissue stem cells are difficult to expand ex vivo because of their inherent asymmetric self - renewal kinetics [ 19 , 20 ] . in vivo , asymmetric self - renewal allows tissue stem cells to maintain their own number while continuously replenishing differentiated tissue cells . however , in vitro , the same process leads to the dilution and loss of tissue stem cells among their accumulating lineage - committed progeny cells . the sack method uses specific guanine ribonucleotide precursors to shift tissue stem cells reversibly from asymmetric self - renewal kinetics to symmetric self - renewal kinetics , which promotes their exponential expansion in culture . we found that , when simply transferred to culture conditions used for pluripotent stem cells , sack - expanded pancreatic stem cells acquired properties of pluripotent stem cells . moreover , inclusion of xanthine ( xn ) , the sack agent used for their derivation , significantly increased their acquisition of properties characteristic of pluripotency . these findings suggest that targeting symmetrically self - renewing tissue stem cells could significantly increase the efficiency of pluripotency reprogramming when combined with other reprogramming strategies . mouse pancreatic cell strain ( mpanstra ) cells ; ( see section 3 for detailed description ) were maintained in high - glucose dulbecco 's modified eagle 's medium ( dmem ) supplemented with 10% dialyzed fetal bovine serum ( dfbs ; gibco , invitrogen , carlsbad , calif , usa ) , 100 u / ml penicillin , 100 g / ml streptomycin , and 1 mm xanthine ( xn , sigma chemical co. , st . mouse embryonic stem cells ( mescs ) were cultured , on sto feeder layers inactivated with mitomycin c ( roche applied science , indianapolis , ind , usa ) , in high - glucose dmem supplemented with 20% heat - inactivated fbs ( sigma ) , 100 u / ml penicillin , 100 g / ml streptomycin , 2 mm l - glutamine , nonessential amino acids ( gibco ) , 50 m -mercaptoethanol , and 10 ng / ml leukemia inhibitory factor . for the initiation of reprogramming , cells were plated at a density of 1.5 10 cells per 10 cm diameter dish . the rabbit polyclonal anti - oct4 and mouse monoclonal anti - klf4 antibodies ( stemgent , cambridge , mass , usa ) were diluted at 1 : 200 in blocking solution and incubated overnight with the cells at 4c . before observation under the microscope , the cells were rinsed with tbst and covered with pbs . to estimate the alkaline phosphatase - positive ( ap+ ) cell fraction , cells in colonies from representative 40x - magnification microscope fields the ap+ fraction was calculated by dividing the ap+ cell number by the total number of cells evaluated . polyclonal rabbit anti - pdx1 antibody ( millipore , billerica , mass , usa ) was diluted 1 : 5000 in blocking solution and incubated overnight at 4c with the membrane . asymmetric self - renewal is a unique feature of tissue stem cells . in adult tissues , tissue stem cells divide asymmetrically , resulting in the production of a new tissue stem cell and a lineage - committed progenitor , the latter having a limited proliferative potential . while this mechanism provides an equilibrium by maintaining a constant number of tissue stem cells in tissues , it limits the ex vivo expansion of tissue stem cells ( figure 1(a ) ) . asymmetric self - renewal by tissue stem cells in vitro leads to their dilution and eventual loss during serial passage . to overcome this asymmetric cell kinetics barrier sack promotes tissue stem cells ' expansion by reversibly converting asymmetric self - renewal kinetics to symmetric self - renewal kinetics . symmetric cell kinetics promotes exponential expansion of tissue stem cells to the exclusion of production of lineage - committed progeny cells . asymmetric cell kinetics requires a p53-dependent decrease in guanine ribonucleotide biosynthesis , specifically resulting from the repression of inosine-5-monophosphate dehydrogenase ii ( impdh ii ) ( figure 1(b ) ) [ 2325 ] . the sack method uses specific exogenous guanine ribonucleotide salvage precursors and/or genes that circumvent the biochemical consequences of impdh ii downregulation by acting either upstream or downstream of the reaction catalyzed by impdh ii , which is rate limiting for guanine ribonucleotide biosynthesis . in the presented study , we used sack - expanded adult mouse pancreatic stem cell strains derived from transgenic mice expressing the xanthine phosphoribosyl transferase ( xprt ) gene . it catalyzes the conversion of xn to xanthosine-5-monophosphate ( xmp ) , the product of the impdh ii reaction with its substrate inosine-5-monophosphate ( imp ) . xn supplementation allowed expansion of pancreatic stem cells strains from adult xprt - transgenic mice by promoting guanine ribonucleotide production downstream of the p53-regulation point . , more tissue stem cells are produced ) , and they have pancreatic endocrine cell differentiation potential . although the cell strains are clonal , their cultures are composed of a mixture of tissue stem cells , lineage - committed pancreatic progenitor cells , and nonproliferating cells . we refer to these sack - expanded , clonal , adult ( i.e. , eight - month old mice ) mouse pancreatic stem cells as although all mpanstra strains required xn for their derivation , some are highly dependent on xn to achieve their maximum proliferative rate ( i.e. the sack - d clones are nontumorigenic pancreatic stem cell strains ( manuscript in preparation ) . tissue stem cells share more gene expression networks with pluripotent stem cells than do other somatic cells [ 27 , 28 ] . we considered that these gene expression similarities , in combination with sack - induced symmetric self - renewal , might predispose tissue stem cells for greater efficiency in conversion to a pluripotent phenotype . here , we report evidence that adult mouse pancreatic stem cells derived by the sack method can be converted to increased tissue potency without the use of exogenous transcription factors . based on the criterion of formation of teratomas with tissues that originate from all three embryonic germ layers ( i.e. , endoderm , mesoderm , and ectoderm ) , the cells attain full pluripotency . we also show that the sack agent xn significantly increases the efficiency of sack - d strains to convert to increased tissue potency . we propose that this ability reflects the common involvement of p53 gene regulation in induced pluripotent stem cell production [ 2932 ] and tissue stem cell self - renewal kinetics [ 19 , 20 , 24 , 25 ] . consistent with the concept of some shared gene expression networks between pluripotent stem cells and tissue stem cells , transcription factors used to induce formation of pluripotent stem cells are detected in mpanstra strains . under xn - supplementation conditions , two evaluated sack - d strains , mpanstra-12 and mpanstra-25 , show expression of nuclear klf4 in a subset of cells ( figure 2(a ) ) , while oct4 expression can only be detected in the mpanstra-12 strain ( figure 2(b ) ) . given their basal expression of genes associated with pluripotency , we investigated whether maintaining mpanstra cells under culture conditions for pluripotent stem cells would promote their conversion to a pluripotent state . in the first two conditions , evaluated mpanstra strains were cultured in their own maintenance medium , which is supplemented with dialyzed fetal bovine serum ( dfbs ) , either without or with xn supplementation ( dfbs - ctrl and dfbs - xn , resp . ) the third and fourth conditions employed culture medium commonly used for the growth of mouse embryonic stem cells ( mescs ) that was supplemented with a high concentration of heat - inactivated , nondialyzed fbs and other factors required for the maintenance of pluripotency . the final two conditions were based on a more recently available defined condition used to maintain and grow pluripotent stem cells . the most prominent effect noted upon transfer of the mpanstra strains to the six trial reprogramming conditions was a general inhibition of the cell proliferation of sack - d strains . in contrast , only the sack - i strains ( mpanstra-11 and mpanstra-56 ) reached confluency in all reprogramming conditions . the sack - d strains ( mpanstra-12 and mpanstra-25 ) showed reduced cell proliferation in the reprogramming conditions , and even after two weeks , only cell colonies had formed . two weeks after the transfer , two types of colonies were observed for the sack - d strains in the reprogramming conditions : striated colonies and epithelial - like colonies ( figure 3(b ) ) . some colonies developed subsets of cells with alkaline phosphatase activity ( red cells in figure 3(c ) ) , a property of pluripotent stem cells . for each sack - d strain reprogramming condition , we picked 24 well - isolated colonies for clonal expansion . there was a significant shift toward the epithelial morphology in the subclones from the hifbs conditions , with less than a third of the clones having the striated morphology . moreover , the proportion of alkaline phosphatase - positive cells was estimated to range from 5% to 60% with xn supplementation , while never being higher than 20% in its absence . in the case of the mpanstra-12 subclones , about a third of these that were derived in the dfbs reprogramming conditions had the epithelial morphology . however , none of these epithelial subclones had detectable alkaline phosphatase activity , even though some of the striated subclones showed activity ( table 1b ) . in the hifbs reprogramming conditions , there was no obvious shift from striated to epithelial morphology , but three of the eight epithelial subclones obtained showed alkaline phosphatase activity under conditions for xn supplementation . for the kosr reprogramming conditions , we observed a significant shift toward the epithelial morphology with xn ( 15 out of 17 expanded clones ; table 1b ) , with two - thirds of them showing alkaline phosphatase activity . in earlier studies , we have found that the sack method could expand distinct stem cell types from the same tissue . to evaluate the developmental tissue potency of the subclones with epithelial morphology , we assayed them for their capacity to form teratomas following subcutaneous injections into immunodeficient mice . mouse escs and ipscs are characterized by their ability to form teratomas , that is , tumors that contain differentiated cells originating from the three embryonic germ layers . for each sack - d strain , we selected two subclones derived in the kosr - xn reprogramming conditions . for each strain , one selected subclone had no detectable alkaline phosphatase activity ( ap ; 12-kosr - xn-83 and 25-kosr - xn-86 ) , whereas the other had the highest proportion of alkaline phosphatase - positive cells ( ap+ ; 12-kosr - xn-89 ( see figure 4 ) and 25-kosr - xn-85 ; 90% and 60% , resp . ) two mice were injected subcutaneously in their hind limbs with each of the four subclones , respectively ( i.e. the mesc - derived tumors had histological features of all three embryonic germ layers ( figure 6 ) . histological analysis of the tumors derived from the reprogrammed mpanstra-12 cells ( figure 6 ) revealed the presence of tissues of mesodermal ( e.g. , nerves ( ne ) ) origin , confirming acquisition of pluripotency . ex vivo - expanded adult mouse pancreatic stem cell strains were evaluated for their ability to acquire phenotypic properties of pluripotent stem cells when transferred into culture conditions permissive for the derivation and maintenance of pluripotent stem cells . because of the requirement that tissue stem cells maintain an undifferentiated phenotype with respect to the tissue lineages that they replenish , we considered that they might have gene expression networks that were more susceptible to pluripotent reprogramming by environmental factors found in pluripotent cell culture media . subsequently , we found that the sack - d mpanstra strains show expression of a subset of transcription factors ( oct4 , klf4 ) implicated in pluripotency state control mechanisms . this finding advanced further the possibility that these cells were , in fact , predisposed to pluripotency reprogramming . supporting this tissue stem cell target hypothesis , both of the evaluated sack - d mpanstra stem cell strains showed evidence of reprogramming after exposure to pluripotent cell culture conditions . also , when compared with mpanstra-25 cells , expression of the pancreatic determination factor pdx1 was not detected in mpanstra-12 cells . these properties suggest that the mpanstra-12 cells were already less committed to the pancreatic differentiation pathway and that the gene expression networks necessary for reprogramming were more active , which could explain their increased potency . the possibility of altering the tissue potency of tissue stem cells simply by transferring them into a defined culture environment underlies another approach to avoiding the inherent risks of pluripotency . in the future , culture environment might be used to instruct plentiful tissue stem cells ( e.g. xn is known to act on the parental stem cell strains by increasing the rate of symmetric self - renewal over asymmetric self - renewal , their default pattern . making the target cells adopt a symmetric self - renewal cell division pattern could advance them closer to a pluripotent stem cell phenotype , characterized by symmetric self - renewal ( in the cases of mescs and ipscs ) . however , clearly more is at play than just acquisition of a symmetric self - renewal state . sack - i strains which undergo constitutive symmetric self - renewal were unable to produce reprogrammed cells . the molecular mechanisms responsible for the emergence of sack - i strains are not fully understood , but it seems most likely that they are pancreatic stem cells that acquired transforming cell mutations , prior to or after their culture . although transformed clones like the sack - i strains develop , the sack method greatly limits their frequency , so that sack - d stem cell strains can be obtained with a high efficiency . previous studies have established that supplemented purines like xn regulate the self - renewing pattern of tissue - specific stem cells by increasing guanine ribonucleotide biosynthesis . the upstream step in this pathway is catalyzed by impdh ii , whose expression is controlled by the p53 tumor suppressor protein . a large part of the effect of p53 loss on ipsc efficiency may be the increased symmetric self - renewal by tissue stem cells in treated cell preparations as a result of increased guanine ribonucleotide biosynthesis . as noted earlier , among the many challenges to using ipsc - derived cells in transplantation medicine , carcinogenic transformation , beyond the gnomonic teratoma formation of pluripotent stem cells injected into mature tissues , is a major one . many studies have shown that gene transduction by a variety of strategies can result in insertional mutagenesis that can lead to cellular transformation and neoplasia [ 3 , 37 ] . also , transduction with only one pluripotency factor in combination with cell culture supplementation with specific small molecules has led to successful reprogramming [ 1214 ] . moreover , the use of starting cell populations containing tissue - specific stem cells undergoing symmetric self - renewal could provide even more efficient production of ipscs . even with gene - free reprogramming , the long assumed inherent cellular transformation risk of pluripotency would still be present . previously , this risk had been attributed to poor regulation of pluripotent cells by heterologous tissue environments [ 39 , 40 ] . the slow tumor development that we observe after culture environment reprogramming of pancreatic tissue stem cells could lead to a safer alternative . specific tissue stem cell types could prove more sensitive for tissue potency reprogramming and/or less prone to cellular transformation . looking for the ability of reprogrammed cells to replace injured tissues or expand normal tissues of a different embryonic germ layer than their origin is the ideal assay concept . also , the development of better - defined culture conditions for directing reprogramming could increase the efficiency and decrease the time necessary for reprogramming . we demonstrated that sack - derived adult pancreatic tissue stem cells could acquire properties of pluripotent stem cells simply by culture under conditions used for production and routine maintenance of the latter . it was the only one of the two evaluated sack - d strains that achieved teratoma formation . the combination of pancreatic tissue stem cells and xn induction of symmetric self - renewal may reflect the same cellular effect as the loss of p53 function for increasing reprogramming efficiency . we predict that starting the reprogramming process with tissue stem - cell - enriched populations , and xn supplementation ( or other exogenous guanine ribonucleotide precursors ) will independently and synergistically enhance pluripotent reprogramming into bona fide induced pluripotent stem cells while reducing the rate of emergence of clones with transforming cell mutations .

allergic rhinitis is a major global health problem affecting about 1020% of the population in all countries , ethnic groups , and ages . using a conservative estimate , it occurs in over 500 million people and its prevalence is increasing in most countries . it is a major chronic respiratory disease that weakens quality of life , school / work productivity , and performance . additionally , it is a substantial economic burden to societies . allergic rhinitis is an inheritable systemic inflammatory condition which links , for example , with asthma . however , it is very commonly underdiagnosed and undertreated , because of which specific programs and guidelines on the problem have been released , for example , by world health organization and european union [ 13 ] . allergic rhinitis is diagnosed when specific antigens are detected in the blood and the patient has symptoms that correspond with exposure to the sensitizing allergen . it is characterized by one or more symptoms , including nasal obstruction , rhinorrhea , sneezing , nasal itching , and eye irritation . subjects are challenged with the suspected allergen and changes in the symptoms are recorded and possibly the amount of secretions and the respiratory function of the nose are measured . nasal provocation tests are the standard procedure to evaluate the clinical response of the nasal mucosa to allergens with high specificity and sensitivity . it is of special relevance in the detection of patient with local allergic rhinitis and has been used for the clinical monitoring of antiallergic drugs and allergen - specific immunotherapy and also provides information on the etiology of occupational respiratory diseases of allergic origin . to rule out nonspecific nasal hyperreactivity , nasal provocation tests nasal breathing function is difficult to quantify directly by the patient 's own comprehension , history , and clinical examination , which calls for objective measurement methods . examples of these include acoustic rhinometry , peak nasal inspiratory flow measurement ( pnif ) , and rhinomanometry [ 6 , 7 ] . acoustic rhinometry assesses nasal geometry by measuring cross - sectional area of the nose as a function of the distance from the nostril . rhinomanometer measures the simultaneous nasal pressure and airflow from the values of which nasal airflow resistance is determined . the resistance is characteristically described as a number that derives from one or more breathing cycles of data . additionally , nasal cavities are measured one at a time and the total nasal resistance is calculated based on unilateral resistances . thus , it is not possible to get accurate instantaneous values . in practice , there are several minutes between the consecutive resistance values that a rhinomanometer can provide . in nasal provocation tests , the major response to measure is the rise in nasal airflow resistance , which can be rapid ( seconds or minutes ) , and the timing differs in different individuals . one possibility is to determine the resistance with the rhinomanometer in certain time intervals , but this can not be done very rapidly and it has been indicated to give inconsistent and variable results with low reproducibility [ 911 ] . thus , there is a need for a measurement giving accurate and continuous measurement data about the nasal breathing function . the nose is armed with a complex nervous system that includes parasympathetic , sympathetic , and sensory nerves . through the nervous system , the nose communicates with the cardiovascular apparatus , the lungs , and the gastrointestinal tract . through neural interactions , events that are initiated in the nose the allergic response comprises changes at all levels of the neural arc : central nervous system integration , sensory nerve function , and autonomic / enteric neuroeffector cell function . central sensitization can lead to the parasympathetic dominance which originated from the central nervous system . it would appear that the nervous system itself is altered and is rendered hyperactive in many allergic patients [ 12 , 13 ] . heart rate variability ( hrv ) analysis is an indirect noninvasive way to assess autonomic nervous system ( ans ) modulation . it quantifies the degree of fluctuation of the beat - to - beat differences in cardiac rhythm . the interactions between respiration , heart rate , and blood pressure fluctuations , which reflect the cardiovascular and cardiorespiratory couplings , are considered to be of paramount importance for the study of the ans . relatively few studies have examined the association between the allergic rhinitis and autonomic nervous system , especially using hrv analysis . lan et al . evaluated the effect of position on the autonomic nervous system of allergic and control volunteers by hrv analysis . they concluded that patients with allergic rhinitis may have poor sympathetic modulation in the sitting position . tacilar et al . measured 24-hour ecg recordings from pediatric allergic rhinitis patients with allergic rhinitis and healthy controls . their hrv analysis ' results implied sympathetic withdrawal and parasympathetic predominance in pediatric allergic rhinitis patients with allergic rhinitis . they found out that hrv indices predicting the parasympathetic predominance were increased in patients with allergic rhinitis . recently , we presented a novel method to assess nasal airflow resistance continuously and accurately . the pressure signal is measured with a thin nasopharyngeal catheter inserted into the nasopharynx and the airflow signal with the rib cage and abdominal effort belts calibrated with our new method . this makes it possible to discover rapid changes in nasal airflow resistance , which is essential , for example , during provocation tests . in this study , the continuous nasal airflow resistance is produced using the above - mentioned methods and dynamically changing autonomic nervous system parameters are computed to study their simultaneous changes during an allergen provocation test . to our knowledge , this kind of study has not been done before , apparently because it has not been possible to produce nasal airflow resistance curves in this precision . our objectives are to examine , firstly , whether there are associations between the dynamic reactions of nasal airflow resistance and autonomic nervous system parameters during the allergic reaction and , secondly , whether there are differences between the birch pollen allergic and the nonallergic groups . the study protocol was approved by the regional ethics committee of the northern ostrobothnia hospital district . in finland , the birch pollen is one of the most common causes of the intermittent seasonal allergic symptoms ; for this very reason , it was chosen as a provocation substance . for this preliminary study , ten ( three females ) birch pollen allergic and ten ( three females ) nonbirch pollen allergic adult volunteers were recruited . the diagnostic criteria for birch pollen allergic rhinitis were evidence of sensitization to birch pollen measured by the presence of allergen - specific ige in the serum and a history of nasal symptoms during the birch pollen season . the mean ( sd ) age of the allergic and nonallergic subjects was 24 ( 1 ) and 24 ( 3 ) years , respectively . the subjects had to be nonpregnant and free of surgical operations of nose , brain circulatory disorders , and heart diseases . medication that affects the nose was not allowed to be used during a specific time period before the measurement . the subjects had to be free of any acute respiratory symptoms during the prior two weeks to the measurement . before measurement , subjects refrained from having a smoke for at least four hours and heavy meal , caffeine , or other stimulants for at least two hours . the subjects were examined by an ear , nose , and throat specialist . before measurements , the specific ige for birch pollen was determined from blood for all of them . based on the blood samples , antibody levels of allergic subjects varied from moderate to very high . ecg , pressure , and respiratory effort belt signals were recorded with the commercial polygraphic recorder ( embletta gold , denver , colorado , usa ) . it had inductive respiratory effort belts for rib cage and abdomen with the sampling rate of 50 hz . the sampling rates of pressure and ecg were 50 hz and 200 hz , respectively . for calibrating the rib cage and abdominal effort belt signals , simultaneous respiratory airflow signal was recorded with a spirometer ( spirostar usb m9460 , medikro oy , kuopio , finland ) with the sampling rate of 100 hz . a water - based immunologically standardized commercial 1 : 10000 squ / ml birch extract ( allergologisk laboratorium a / s , copenhagen , denmark ) was used in the provocation . the diluent solution ( allergologisk laboratorium a / s , copenhagen , denmark ) of the allergen extract was used as a control solution . both solutions were administered bilaterally into the nasal cavities by pump spray . at the beginning of measurement , the rib cage effort belt was placed on the xiphoid process and the abdominal effort belt was placed near the umbilicus . next , the subjects sat peacefully for a period of 30 min prior to the measurement to adapt themselves to the environment and to allow heart rate and blood pressure to stabilize . during the actual measurements , they were instructed to sit in back upright position avoiding speaking and movements . first , respiratory effort belt signals were recorded along with the spirometer signal for one minute . the data was used for calibrating the rib cage and abdominal effort belt signals to flow signals , as described in section 2.3 . after calibration , the spirometer was removed from the subject and a thin catheter ( diameter : 1 mm ) was inserted 8 cm deep along the floor of nasal cavity into the nasopharynx , with the tip of the catheter lying 1 cm anterior from the back wall of nasopharynx . to inhibit the nasal secrete blocking , the nasal catheter air was blown with the syringe through the catheter before each measurement protocol phase and every time that the catheter blocking was detected . the measurement protocol consisted of the three phases . at the first protocol phase , the ecg , pressure , and respiratory effort belt signals were recorded for 10 min to get the baseline situation . at the second phase , to rule out the nonspecific nasal hyperreactivity , the nasal mucosa was challenged with a control solution . it was sprayed carefully on the anterior mucosa of both nasal cavities , after which the ecg , pressure , and respiratory effort belt signals were recorded for 5 min . at the third phase , the birch pollen solution was sprayed carefully on the anterior nasal mucosa of both nasal cavities . after that , the ecg , pressure , and respiratory effort belt signals were recorded for 20 min . after spraying the solution , the recording was initiated as soon as possible but first waiting for the immediate reactions such as sneezing and snuffling to settle . before analysis , all the detected disturbances , which originated , for example , from moving , snuffling , sneezing , and blowing of air with the syringe through the catheter , were deleted from signals before analysis . additionally , special care was taken to maintain the correct synchrony between the signals . in this study , we use our previously published novel method to estimate continuous nasal airflow resistance using pressure signal from the nasopharyngeal catheter inserted transnasally into the nasopharynx and calibrated rib cage and abdominal effort belt signals . recently , we published an improved respiratory effort belt calibration method , which reduces waveform errors considerably and is robust to breathing style changes . it is based on an optimally trained fir ( finite impulse response ) filter bank constructed as a miso ( multiple - input single - output ) system between respiratory effort belt signals and the spirometer and a delay element ( z ) ; see figure 2 . the method extends the traditional multiple linear regression method by using a number n of consecutive signal samples and linear filtering for each prediction . with the respiratory effort belts used in this study , the following realization of the filter bank was used:(1)yj=1tx1j+2tx2j+j , where 1 and 2 denote the n tap coefficients of filters fir1 and fir2 , respectively . superscript t indicates the matrix transpose in the formula , and j denotes the time index of the signals . vectors x1 and x2 include n consecutive signal samples from the rib cage signal and abdomen signal , respectively : xk[j ] = [ xk[j ] , xk[j 1], ,xk[j n + 1 ] ] , where k = 1,2 and j = n , , n. with this measurement data , the variable n was 3000 , which was the number of observations used in the calibration . during the calibration , tap coefficients 1 and 2 are estimated with the method of least squares from the available data . the least - squares estimator of the parameter vector = [ 1 , 2 ] is given by(2)^=xtx1xty , where x is ( n n + 1)(2 n ) matrix formed from the vectors x1 and x2:(3)x = x1tnx2tnx1tn+1x2tn+1x1tnx2tn.the length of the vector ^ is 2 n. thus , the flow signal predicted from the rib cage and abdominal effort belt signals through the fir filter bank is(4)y^=x^.in our previous study , the 0.3 sec length of fir filters was found to produce the best airflow prediction with the same respiratory effort belts . offers parametric means to describe the nonlinear pressure / flow relationship ; see figure 3 . in the model , the pressure / flow relationship is established as follows:(5)vr = v0+cr , where vr is the angle with radius r , v0 is the angle in the origin , and c is the curvature parameter . the resistance in radius r , denoted by rr , is given by(6)rr = xtanvr.the constant x is a normalization factor defined in . however , the broms model expects the data to be stationary , while the nasal system is nonstationary . our method to estimate continuous nasal airflow resistance is a least - mean - square ( lms ) extension to the broms model so that it can be used for calculating a continuous nasal airflow resistance through model adaptation to time - varying characteristics of the nasal functioning . in the extended model , an instantaneous nasal airflow resistance can be calculated after estimating the model parameters at each time instant from the input signals . the normalized pressure signal , p , and the estimate of flow signal , f , are the lms filter inputs . the update formulas of parameters v0 and c are(7)v0i+1=v0i+ivriv0i+ciri , ci+1=ci+irivriv0i+ciri , where(8)vri = tan1pifi , ri = p2i+f2i.the learning rate parameter and the initial values for v0 and c are defined in . hence , the instantaneous resistance values are calculated over the whole measurement data at any sampling frequency allowing for analysis of dynamic changes in the continuous nasal airflow resistance . at first , the baseline fluctuation of the measured ecg signals was removed with a savitzky - golay filter ( polynomial order 2 ) . r - peaks were automatically detected from the filtered ecg signals based on the expected time between adjacent r - waves and amplitude threshold . tachogram was derived from the r - r intervals and converted to equidistantly sampled series by interpolation , in this case to the frequency of 4 hz . the tachogram was filtered with the bandpass finite impulse response ( fir ) filters with the cutoff frequencies corresponding to lf and hf bands . the lf band was set between 0.04 hz and 0.15 hz and hf band was set between 0.15 hz and 0.4 hz , as recommended by the task force . the variance , which is the correlate of the signal power , was then computed in 3 min windows from the filtered signals . thereafter , the window was shifted ahead by 18 sec ( overlap of 90% ) and the variance was computed again , which produced the lf and hf power signals . typically , lf component is considered to reflect mostly the sympathetic modulation , hf component is considered to reflect mostly the parasympathetic modulation , and lf / hf ratio is considered to reflect mostly the sympathovagal balance , although this interpretation is not fully agreed among researches . statistical significance of the changes in nasal airflow resistance , lf , hf , and lf / hf ratio and their trends in the subjects was assessed by wilcoxon signed - rank test . statistical significance between the subject groups in its turn , was assessed by mann - whitney test ( wilcoxon rank - sum test ) . the null hypothesis was that there are no differences in the medians of given data sets . the results were reported as statistically significant if a two - sided p value was less than 0.05 . additionally , logistic regression was used to determine the best factors for predicting whether the subject is birch pollen allergic or not . ecg , respiratory effort signals , and pressure were recorded according to the measurement protocol described in section 2.2 . at first , the rib cage and abdominal effort belt signals were calibrated to flow of spirometer from the calibration recording . then , continuous nasal airflow resistance signals were computed from the nasal pressure and calibrated respiratory effort belt signals . lf , hf , and lf / hf ratio signals were computed from the ecg signal . in this paper , we studied only the third protocol phase , where the allergic reactions for the birch pollen challenge were expected to appear . interestingly , it was observed that there is a linear trend in the resistance signals and the hrv signals in all allergic subjects . thus , linear line fitting was performed to the trends to determine the slopes and their differences . for the allergic subjects , those parts of the nasal airflow resistance curves were selected to the trend analysis , where the resistance increased from the start of the protocol phase until some saturation point . however , individual variation of the time span of the resistance increase was large and for such reason analysis window varied between the subjects . importantly , it was observed that the trends in ans parameters turned out not to be the same in length as trends in resistance . to our knowledge , this is the first time this kind of differences in timing between continuous nasal airflow resistance and ans function is found out . we therefore selected different time windows for trend analysis for hrv signals in order to guarantee that only linear segments are considered . the starting time for the window was always the same as with the resistance signal for each subject , but the ending time depended on the actual end of the trend . for the nonallergic subjects , no obvious trends were observed . thus , the whole nasal airflow resistance , lf , hf , and lf / hf ratio signals from the third protocol phase were chosen to the trend analysis , excluding first the artefacts and the possible nonspecific nasal hyperreactivity at the beginning of the phase . an example case of concomitant dynamic changes in the nasal airflow resistance , lf , hf , and lf / hf ratio curves of birch pollen allergic subject after allergen provocation is shown in figure 4 . a clear upward trend can be observed in the nasal airflow resistance curve and downward trends can be seen in the lf and lf / hf ratio curves . a robust line fitting algorithm was applied to the curves to quantify the extent of slopes . a representative case of concomitant nasal airflow resistance , lf , hf , and lf / hf ratio curves of nonallergic subject after allergen provocation is shown in figure 5 . a gap at the beginning of resistance curve can be seen due to removing of artifacts during manual validation . table 1 presents the slopes and changes of nasal airflow resistance , lf , hf , and lf / hf ratio curves for each birch pollen allergic subject , together with their average values and standard deviations ( sd ) over the subject group . as can be seen from table 1 , lf and lf / hf ratio decreased consistently during the increase of nasal airflow resistance for all the birch pollen allergic subjects ( p = 0.000 ) , while the hf power changes had both positive and negative slopes . there was a statistically significant change in the resistance ( p = 0.002 ) , lf ( p = 0.002 ) , and lf / hf ratio ( p = 0.002 ) . table 2 presents the slopes and changes of resistance , lf , hf , and lf / hf ratio curves for each nonallergic subject , together with their average values and standard deviations over the subject group . in this group , lf and lf / hf ratio increased for 9/10 subjects . there was only one subject with decreasing lf and one subject with decreasing lf / hf ratio . a noticeable issue is that these two were different subjects and there was no subject with the simultaneous decreasing lf and decreasing lf / hf ratio . there was a statistically significant change in the lf ( p = 0.037 ) and lf / hf ratio ( p = 0.006 ) and no statistically significant change in resistance ( p = 0.865 ) and hf ( p = 0.432 ) . between the groups , a statistically significant difference was found in several parameters , the slope of resistance ( p < 0.001 ) , resistance at the end ( p = 0.029 ) , change of resistance ( p < 0.001 ) , slope of lf ( p < 0.001 ) , change of lf ( p < 0.001 ) , slope of lf / hf ratio ( p < 0.001 ) , and change of lf / hf ratio ( p < 0.001 ) , between the birch pollen allergic and nonallergic groups . however , there was no statistically significant difference in the resistance at the beginning ( p = 0.579 ) , slope of hf ( p = 0.529 ) , and change of hf ( p = 0.912 ) between the two groups . logistic regression was applied to determine parameters , which predict the allergy status ( allergic or nonallergic ) of the subjects most reliably . when the used parameter was the slope of lf / hf ratio , the logistic regression model predicted correctly 9/10 birch pollen allergic and 9/10 nonallergic subjects . when the parameter used was the slope of lf , the model predicted correctly 10/10 birch pollen allergic and 9/10 nonallergic subjects . the best result was achieved when both parameters , the slope of lf and the slope of lf / hf ratio , were used , in which case the model predicted correctly 10/10 birch pollen allergic and 10/10 nonallergic subjects . here , we conducted an experimental trial , where birch pollen allergic subjects and nonallergic control subjects had topical nasal challenge with birch pollen extract . we monitored both the immediate nasal reaction and simultaneous autonomic nervous system function by recording continuous nasal airflow resistance and by analyzing heart rate variability , respectively . we found that in allergic subjects the nasal airflow resistance increased and concomitantly the lf energy and lf / hf ratio decreased after allergen challenge indicating sympathetic withdrawal . in addition , the change in hf energy or the heart rate signal itself did not show similar dynamics . the sympathetic stimulus in the nose is known to cause vasoconstriction and thus increased nasal patency . thus , our findings indicate that in allergic subjects the nasal obstruction following topical nasal exposure to allergen is at least partially due to the dysfunction of the autonomic nervous system . the lf energy and lf / hf ratio were strong parameters that differentiated the allergy group from the nonallergy group . additionally , we found out that there are differences in timing between the length of increase in nasal airflow resistance and the length of trend in lf energy and lf / hf ratio signals . this finding was only possible with our proposed technique to estimate continuous nasal airflow resistance and hrv parameters . to our knowledge , this is the first time this kind of differences is found out . our findings are in accordance with those of pichon et al . who followed subjects with lower respiratory symptoms after diagnostic methacholine bronchial challenge using heart rate variability analysis and found that the hyperresponsive subjects had altered autonomic balance . however , pichon et al . found that the hyperresponsive subjects had higher parasympathetic tone ( hf component ) than those without airway responsiveness , both at baseline and after the challenge , whereas we found the decrease of sympathetic tone ( lf component ) to be responsible for the altered autonomic reaction in the allergic subjects after topical nasal challenge . , tacilar et al . , and yokusoglu et al . , who concluded that autonomic dysfunction may play a role in allergic rhinitis . measuring nasal reactions after topical nasal challenge is difficult in clinical work . for example , the weighing of nasal secretion after the challenge is done by a suctioning device which irritates the nose and can itself cause nasal obstruction and secretion . similarly , the measurement of nasal breathing with conventional rhinomanometry or acoustic rhinometry involves occlusion of one nostril that interferes with the normal nasal breathing . these disadvantages can be totally avoided if nasal allergy can be measured from the heart rate without touching the nose at all , like our present results indicate . still , the sample enabled us to find significant associations between the hrv results and the resistance values and allergy status . the study should be repeated with a larger data set in order to draw more general conclusions . secondly , it was observed that the timing of ans response to provocation was not the same as with nasal airflow resistance . automating the segment selection would be highly beneficial for practical applications and is left for future work . thirdly , the window size for hrv analysis was chosen as 3 min for the following reasons . traditional 5 min window was too long for our purposes because the rise of nasal airflow resistance was shorter than 5 min for some allergic subjects . however , due to the dynamics , 1 min window produced very unstable data . however , robustness to dynamic variation and , indeed , analysis of the variation itself still remains as future work . we presented a method to assess simultaneous dynamic changes in nasal airflow resistance and autonomic nervous system function during a provocation test . the continuous nasal airflow resistance was estimated with a method that applies lms filtering technique to the nasal pressure signals and calibrated rib cage and abdominal effort belt signals to update adaptively an extended broms model . lf and hf components of heart rate were quantified with bandpass fir filter in 3 min 90% overlapping windows . quantitative results of nasal airflow resistance , lf , hf , and lf / hf ratio changes were presented for birch pollen allergic and nonallergic subjects . the main finding was that when the nasal airflow resistance of birch pollen allergic subjects increases after the provocation , the lf energy and lf / hf ratio decrease with a clear trend . our findings further imply that the allergic and nonallergic subjects could be differentiated by following hrv after the provocation . we also found out that there are differences in timing between the length of increase in nasal airflow resistance and the length of trend in lf energy and lf / hf ratio signals . this finding was only possible with our proposed technique to estimate continuous nasal airflow resistance and hrv parameters . the proposed method opens entirely new opportunities to research accurately concomitant changes in nonstationary nasal breathing function and autonomic nervous system in provocation tests .

allergic rhinitis is a major chronic respiratory disease and an immunoneuronal disorder . we aimed at providing further knowledge on the function of the neural system in nasal allergic reaction . here , a method to assess simultaneously the nasal airflow resistance and the underlying function of autonomic nervous system ( ans ) is presented and used during the nasal provocation of allergic and nonallergic subjects . continuous nasal airflow resistance and spectral heart rate variability parameters show in detail the timing and intensity differences in subjects ' reactions . after the provocation , the nasal airflow resistance of allergic subjects showed a positive trend , whereas lf / hf ( low frequency / high frequency ) ratio and lf power showed a negative trend . this could imply a gradual sympathetic withdrawal in allergic subjects after the allergen provocation . the groups differed significantly by these physiological descriptors . the proposed method opens entirely new opportunities to research accurately concomitant changes in nasal breathing function and ans .

1. Introduction 2. Methods 3. Results 4. Discussion and Future Work 5. Conclusion

allergic rhinitis is a major global health problem affecting about 1020% of the population in all countries , ethnic groups , and ages . it is a major chronic respiratory disease that weakens quality of life , school / work productivity , and performance . allergic rhinitis is an inheritable systemic inflammatory condition which links , for example , with asthma . allergic rhinitis is diagnosed when specific antigens are detected in the blood and the patient has symptoms that correspond with exposure to the sensitizing allergen . subjects are challenged with the suspected allergen and changes in the symptoms are recorded and possibly the amount of secretions and the respiratory function of the nose are measured . nasal provocation tests are the standard procedure to evaluate the clinical response of the nasal mucosa to allergens with high specificity and sensitivity . it is of special relevance in the detection of patient with local allergic rhinitis and has been used for the clinical monitoring of antiallergic drugs and allergen - specific immunotherapy and also provides information on the etiology of occupational respiratory diseases of allergic origin . to rule out nonspecific nasal hyperreactivity , nasal provocation tests nasal breathing function is difficult to quantify directly by the patient 's own comprehension , history , and clinical examination , which calls for objective measurement methods . acoustic rhinometry assesses nasal geometry by measuring cross - sectional area of the nose as a function of the distance from the nostril . rhinomanometer measures the simultaneous nasal pressure and airflow from the values of which nasal airflow resistance is determined . additionally , nasal cavities are measured one at a time and the total nasal resistance is calculated based on unilateral resistances . in nasal provocation tests , the major response to measure is the rise in nasal airflow resistance , which can be rapid ( seconds or minutes ) , and the timing differs in different individuals . thus , there is a need for a measurement giving accurate and continuous measurement data about the nasal breathing function . through the nervous system , the nose communicates with the cardiovascular apparatus , the lungs , and the gastrointestinal tract . through neural interactions , events that are initiated in the nose the allergic response comprises changes at all levels of the neural arc : central nervous system integration , sensory nerve function , and autonomic / enteric neuroeffector cell function . central sensitization can lead to the parasympathetic dominance which originated from the central nervous system . it would appear that the nervous system itself is altered and is rendered hyperactive in many allergic patients [ 12 , 13 ] . heart rate variability ( hrv ) analysis is an indirect noninvasive way to assess autonomic nervous system ( ans ) modulation . it quantifies the degree of fluctuation of the beat - to - beat differences in cardiac rhythm . the interactions between respiration , heart rate , and blood pressure fluctuations , which reflect the cardiovascular and cardiorespiratory couplings , are considered to be of paramount importance for the study of the ans . relatively few studies have examined the association between the allergic rhinitis and autonomic nervous system , especially using hrv analysis . evaluated the effect of position on the autonomic nervous system of allergic and control volunteers by hrv analysis . they concluded that patients with allergic rhinitis may have poor sympathetic modulation in the sitting position . measured 24-hour ecg recordings from pediatric allergic rhinitis patients with allergic rhinitis and healthy controls . their hrv analysis ' results implied sympathetic withdrawal and parasympathetic predominance in pediatric allergic rhinitis patients with allergic rhinitis . they found out that hrv indices predicting the parasympathetic predominance were increased in patients with allergic rhinitis . recently , we presented a novel method to assess nasal airflow resistance continuously and accurately . the pressure signal is measured with a thin nasopharyngeal catheter inserted into the nasopharynx and the airflow signal with the rib cage and abdominal effort belts calibrated with our new method . this makes it possible to discover rapid changes in nasal airflow resistance , which is essential , for example , during provocation tests . in this study , the continuous nasal airflow resistance is produced using the above - mentioned methods and dynamically changing autonomic nervous system parameters are computed to study their simultaneous changes during an allergen provocation test . to our knowledge , this kind of study has not been done before , apparently because it has not been possible to produce nasal airflow resistance curves in this precision . our objectives are to examine , firstly , whether there are associations between the dynamic reactions of nasal airflow resistance and autonomic nervous system parameters during the allergic reaction and , secondly , whether there are differences between the birch pollen allergic and the nonallergic groups . the study protocol was approved by the regional ethics committee of the northern ostrobothnia hospital district . in finland , the birch pollen is one of the most common causes of the intermittent seasonal allergic symptoms ; for this very reason , it was chosen as a provocation substance . for this preliminary study , ten ( three females ) birch pollen allergic and ten ( three females ) nonbirch pollen allergic adult volunteers were recruited . the diagnostic criteria for birch pollen allergic rhinitis were evidence of sensitization to birch pollen measured by the presence of allergen - specific ige in the serum and a history of nasal symptoms during the birch pollen season . the mean ( sd ) age of the allergic and nonallergic subjects was 24 ( 1 ) and 24 ( 3 ) years , respectively . before measurements , the specific ige for birch pollen was determined from blood for all of them . based on the blood samples , antibody levels of allergic subjects varied from moderate to very high . the diluent solution ( allergologisk laboratorium a / s , copenhagen , denmark ) of the allergen extract was used as a control solution . at the beginning of measurement , the rib cage effort belt was placed on the xiphoid process and the abdominal effort belt was placed near the umbilicus . next , the subjects sat peacefully for a period of 30 min prior to the measurement to adapt themselves to the environment and to allow heart rate and blood pressure to stabilize . after calibration , the spirometer was removed from the subject and a thin catheter ( diameter : 1 mm ) was inserted 8 cm deep along the floor of nasal cavity into the nasopharynx , with the tip of the catheter lying 1 cm anterior from the back wall of nasopharynx . to inhibit the nasal secrete blocking , the nasal catheter air was blown with the syringe through the catheter before each measurement protocol phase and every time that the catheter blocking was detected . at the first protocol phase , the ecg , pressure , and respiratory effort belt signals were recorded for 10 min to get the baseline situation . at the second phase , to rule out the nonspecific nasal hyperreactivity , the nasal mucosa was challenged with a control solution . at the third phase , the birch pollen solution was sprayed carefully on the anterior nasal mucosa of both nasal cavities . after that , the ecg , pressure , and respiratory effort belt signals were recorded for 20 min . in this study , we use our previously published novel method to estimate continuous nasal airflow resistance using pressure signal from the nasopharyngeal catheter inserted transnasally into the nasopharynx and calibrated rib cage and abdominal effort belt signals . with the respiratory effort belts used in this study , the following realization of the filter bank was used:(1)yj=1tx1j+2tx2j+j , where 1 and 2 denote the n tap coefficients of filters fir1 and fir2 , respectively . superscript t indicates the matrix transpose in the formula , and j denotes the time index of the signals . vectors x1 and x2 include n consecutive signal samples from the rib cage signal and abdomen signal , respectively : xk[j ] = [ xk[j ] , xk[j 1], ,xk[j n + 1 ] ] , where k = 1,2 and j = n , , n. with this measurement data , the variable n was 3000 , which was the number of observations used in the calibration . the least - squares estimator of the parameter vector = [ 1 , 2 ] is given by(2)^=xtx1xty , where x is ( n n + 1)(2 n ) matrix formed from the vectors x1 and x2:(3)x = x1tnx2tnx1tn+1x2tn+1x1tnx2tn.the length of the vector ^ is 2 n. thus , the flow signal predicted from the rib cage and abdominal effort belt signals through the fir filter bank is(4)y^=x^.in our previous study , the 0.3 sec length of fir filters was found to produce the best airflow prediction with the same respiratory effort belts . the resistance in radius r , denoted by rr , is given by(6)rr = xtanvr.the constant x is a normalization factor defined in . however , the broms model expects the data to be stationary , while the nasal system is nonstationary . our method to estimate continuous nasal airflow resistance is a least - mean - square ( lms ) extension to the broms model so that it can be used for calculating a continuous nasal airflow resistance through model adaptation to time - varying characteristics of the nasal functioning . in the extended model , an instantaneous nasal airflow resistance can be calculated after estimating the model parameters at each time instant from the input signals . the normalized pressure signal , p , and the estimate of flow signal , f , are the lms filter inputs . the update formulas of parameters v0 and c are(7)v0i+1=v0i+ivriv0i+ciri , ci+1=ci+irivriv0i+ciri , where(8)vri = tan1pifi , ri = p2i+f2i.the learning rate parameter and the initial values for v0 and c are defined in . hence , the instantaneous resistance values are calculated over the whole measurement data at any sampling frequency allowing for analysis of dynamic changes in the continuous nasal airflow resistance . at first , the baseline fluctuation of the measured ecg signals was removed with a savitzky - golay filter ( polynomial order 2 ) . r - peaks were automatically detected from the filtered ecg signals based on the expected time between adjacent r - waves and amplitude threshold . thereafter , the window was shifted ahead by 18 sec ( overlap of 90% ) and the variance was computed again , which produced the lf and hf power signals . typically , lf component is considered to reflect mostly the sympathetic modulation , hf component is considered to reflect mostly the parasympathetic modulation , and lf / hf ratio is considered to reflect mostly the sympathovagal balance , although this interpretation is not fully agreed among researches . statistical significance of the changes in nasal airflow resistance , lf , hf , and lf / hf ratio and their trends in the subjects was assessed by wilcoxon signed - rank test . then , continuous nasal airflow resistance signals were computed from the nasal pressure and calibrated respiratory effort belt signals . lf , hf , and lf / hf ratio signals were computed from the ecg signal . interestingly , it was observed that there is a linear trend in the resistance signals and the hrv signals in all allergic subjects . for the allergic subjects , those parts of the nasal airflow resistance curves were selected to the trend analysis , where the resistance increased from the start of the protocol phase until some saturation point . however , individual variation of the time span of the resistance increase was large and for such reason analysis window varied between the subjects . to our knowledge , this is the first time this kind of differences in timing between continuous nasal airflow resistance and ans function is found out . the starting time for the window was always the same as with the resistance signal for each subject , but the ending time depended on the actual end of the trend . for the nonallergic subjects , no obvious trends were observed . thus , the whole nasal airflow resistance , lf , hf , and lf / hf ratio signals from the third protocol phase were chosen to the trend analysis , excluding first the artefacts and the possible nonspecific nasal hyperreactivity at the beginning of the phase . an example case of concomitant dynamic changes in the nasal airflow resistance , lf , hf , and lf / hf ratio curves of birch pollen allergic subject after allergen provocation is shown in figure 4 . a clear upward trend can be observed in the nasal airflow resistance curve and downward trends can be seen in the lf and lf / hf ratio curves . a representative case of concomitant nasal airflow resistance , lf , hf , and lf / hf ratio curves of nonallergic subject after allergen provocation is shown in figure 5 . table 1 presents the slopes and changes of nasal airflow resistance , lf , hf , and lf / hf ratio curves for each birch pollen allergic subject , together with their average values and standard deviations ( sd ) over the subject group . as can be seen from table 1 , lf and lf / hf ratio decreased consistently during the increase of nasal airflow resistance for all the birch pollen allergic subjects ( p = 0.000 ) , while the hf power changes had both positive and negative slopes . there was a statistically significant change in the resistance ( p = 0.002 ) , lf ( p = 0.002 ) , and lf / hf ratio ( p = 0.002 ) . table 2 presents the slopes and changes of resistance , lf , hf , and lf / hf ratio curves for each nonallergic subject , together with their average values and standard deviations over the subject group . in this group , lf and lf / hf ratio increased for 9/10 subjects . there was only one subject with decreasing lf and one subject with decreasing lf / hf ratio . a noticeable issue is that these two were different subjects and there was no subject with the simultaneous decreasing lf and decreasing lf / hf ratio . there was a statistically significant change in the lf ( p = 0.037 ) and lf / hf ratio ( p = 0.006 ) and no statistically significant change in resistance ( p = 0.865 ) and hf ( p = 0.432 ) . between the groups , a statistically significant difference was found in several parameters , the slope of resistance ( p < 0.001 ) , resistance at the end ( p = 0.029 ) , change of resistance ( p < 0.001 ) , slope of lf ( p < 0.001 ) , change of lf ( p < 0.001 ) , slope of lf / hf ratio ( p < 0.001 ) , and change of lf / hf ratio ( p < 0.001 ) , between the birch pollen allergic and nonallergic groups . logistic regression was applied to determine parameters , which predict the allergy status ( allergic or nonallergic ) of the subjects most reliably . when the used parameter was the slope of lf / hf ratio , the logistic regression model predicted correctly 9/10 birch pollen allergic and 9/10 nonallergic subjects . when the parameter used was the slope of lf , the model predicted correctly 10/10 birch pollen allergic and 9/10 nonallergic subjects . the best result was achieved when both parameters , the slope of lf and the slope of lf / hf ratio , were used , in which case the model predicted correctly 10/10 birch pollen allergic and 10/10 nonallergic subjects . here , we conducted an experimental trial , where birch pollen allergic subjects and nonallergic control subjects had topical nasal challenge with birch pollen extract . we monitored both the immediate nasal reaction and simultaneous autonomic nervous system function by recording continuous nasal airflow resistance and by analyzing heart rate variability , respectively . we found that in allergic subjects the nasal airflow resistance increased and concomitantly the lf energy and lf / hf ratio decreased after allergen challenge indicating sympathetic withdrawal . in addition , the change in hf energy or the heart rate signal itself did not show similar dynamics . thus , our findings indicate that in allergic subjects the nasal obstruction following topical nasal exposure to allergen is at least partially due to the dysfunction of the autonomic nervous system . the lf energy and lf / hf ratio were strong parameters that differentiated the allergy group from the nonallergy group . additionally , we found out that there are differences in timing between the length of increase in nasal airflow resistance and the length of trend in lf energy and lf / hf ratio signals . this finding was only possible with our proposed technique to estimate continuous nasal airflow resistance and hrv parameters . who followed subjects with lower respiratory symptoms after diagnostic methacholine bronchial challenge using heart rate variability analysis and found that the hyperresponsive subjects had altered autonomic balance . found that the hyperresponsive subjects had higher parasympathetic tone ( hf component ) than those without airway responsiveness , both at baseline and after the challenge , whereas we found the decrease of sympathetic tone ( lf component ) to be responsible for the altered autonomic reaction in the allergic subjects after topical nasal challenge . , who concluded that autonomic dysfunction may play a role in allergic rhinitis . measuring nasal reactions after topical nasal challenge is difficult in clinical work . for example , the weighing of nasal secretion after the challenge is done by a suctioning device which irritates the nose and can itself cause nasal obstruction and secretion . similarly , the measurement of nasal breathing with conventional rhinomanometry or acoustic rhinometry involves occlusion of one nostril that interferes with the normal nasal breathing . still , the sample enabled us to find significant associations between the hrv results and the resistance values and allergy status . secondly , it was observed that the timing of ans response to provocation was not the same as with nasal airflow resistance . thirdly , the window size for hrv analysis was chosen as 3 min for the following reasons . traditional 5 min window was too long for our purposes because the rise of nasal airflow resistance was shorter than 5 min for some allergic subjects . however , robustness to dynamic variation and , indeed , analysis of the variation itself still remains as future work . we presented a method to assess simultaneous dynamic changes in nasal airflow resistance and autonomic nervous system function during a provocation test . the continuous nasal airflow resistance was estimated with a method that applies lms filtering technique to the nasal pressure signals and calibrated rib cage and abdominal effort belt signals to update adaptively an extended broms model . lf and hf components of heart rate were quantified with bandpass fir filter in 3 min 90% overlapping windows . quantitative results of nasal airflow resistance , lf , hf , and lf / hf ratio changes were presented for birch pollen allergic and nonallergic subjects . the main finding was that when the nasal airflow resistance of birch pollen allergic subjects increases after the provocation , the lf energy and lf / hf ratio decrease with a clear trend . our findings further imply that the allergic and nonallergic subjects could be differentiated by following hrv after the provocation . we also found out that there are differences in timing between the length of increase in nasal airflow resistance and the length of trend in lf energy and lf / hf ratio signals . this finding was only possible with our proposed technique to estimate continuous nasal airflow resistance and hrv parameters . the proposed method opens entirely new opportunities to research accurately concomitant changes in nonstationary nasal breathing function and autonomic nervous system in provocation tests .

mid october 2009 general practitioner sandra bijl published an article in the dutch doctors journal medisch contact . she described one of the many dilemmas she faced since the introduction of marketized health care in the netherlands . doctor bijl s practice consists of many immigrant patients , with a low education and a low income . many of them are single mothers , struggling to keep their families afloat and former refugees who suffered personal losses in their country of origin that they still do not know how to deal with . bijl : of course i can just pay attention to their physical pains . quick service and they will get back very soon . as an entrepreneur i might say that this would be profitable : little effort and lots of benefits for me . but as a human being devoted to her profession one of my patients has been suffering from headaches for the past 12 years . not because of a tumor , or high blood pressure or bad eyesight , but because her grown up children use their mother as their personal slave . it took me 30 min , and i will not get paid for all of them . she used to visit me once every fortnight , but since the talk i havent seen her for months . as an entrepreneur i fail if i deliver this type of care , but as a professional , a conversation like that is very rewarding . abbreviated and translated by the authors ) . during the 1990s and the first decade of the twenty - first century numerous politicians and policy - makers argued that health care should be consumer - driven and consumer - oriented. care providers should become entrepreneurial ; they should cater according to consumer preferences with regard to prices and quality of care . several authors have observed that these changes may lead to changes in medical professional ethics . eliot freidson points out that the logic of professionalism differs from the logic of market consumerism and from the logic of bureaucratic managerialism . professionals consider their work a secular calling , not a mere job or a means to maximize their income , as the example of dr bijl shows . professionals ( and medical doctors are archetypal professionals ) are devoted to a transcendent value , such as the health of the patient ( which is not the same as fulfilling the patient s wishes ) . introducing consumerism or managerialism ( or both ) may threaten the soul of professionalism . referring to freidson duyvendak et al . eve and hodgkin argue that rising consumerism and the creation of an internal market within the british national health service will challenge doctors professional ethics . krizova observes that marketization and other changes in health care systems may lead to a decline of professional autonomy which might cause a decrease in altruistic or service - oriented attitudes towards patients similar concerns raised by a number of other authors , mostly medical ethicists , are discussed by randall and williams . although there are also some dissenting opinions ( e.g. garrett argues that market principles need not destroy professional ethics because sticking to one s professional ethics might be the most profitable strategy for market actors in the long run and applbaum questions the use and legitimacy of professional ethics in the first place , thus would not deplore its demise ) , the general feeling seems to be pessimistic . we will try to add some empirics to the debate on the relationship between market principles and medical professional ethics . in our study we set out to ascertain how market reforms have changed medical professional ethics in the netherlands . within western europe the netherlands has gone farthest in introducing markets and managed competition in health care ( cf . [ 3 , 13 , 19 ] ) , which makes the country very suitable for an empirical study of the relationship between marketization and medical professional ethics . the details of the new dutch system as well as its economic effects have been discussed in several other publications [ 2 , 12 , 14 , 17 , 22 , 23 ] . for this study it is important to know that : private insurers have to contract hospitals to deliver medical services . some of these services still have fixed prices , others ( mostly elective surgery : inguinal ruptures , varicose veins , knee operations etc . ) these packages may include treatment guarantees ( e.g. if you are diagnosed with breast cancer , your health insurer may guarantee that you will get surgery within 2 weeks ) . these treatment guarantees may diminish physicians room to manoeuvre with their operating schedules;the payment system for general practitioners has been changed , in a much more fee - for - service direction . gps who perform minor surgeries or who use new diagnostic tools may charge much more for this service than they could before 2006 . policy makers hoped to accomplish a substitution effect as minor surgeries in hospitals are more expensive than similar surgeries in gp practices . private insurers have to contract hospitals to deliver medical services . some of these services still have fixed prices , others ( mostly elective surgery : inguinal ruptures , varicose veins , knee operations etc . ) these packages may include treatment guarantees ( e.g. if you are diagnosed with breast cancer , your health insurer may guarantee that you will get surgery within 2 weeks ) . these treatment guarantees may diminish physicians room to manoeuvre with their operating schedules ; the payment system for general practitioners has been changed , in a much more fee - for - service direction . gps who perform minor surgeries or who use new diagnostic tools may charge much more for this service than they could before 2006 . policy makers hoped to accomplish a substitution effect as minor surgeries in hospitals are more expensive than similar surgeries in gp practices . although the complete overhaul of the insurance system took place in 2006 , elements of market competition were introduced before that date . medical professionals in the netherlands have a few years of experience with market elements by now . one year after the new insurance system was adopted the council for public health and health care ( an advisory council to the government and one of the more fervent proponents of the new system ) observed that the introduction of market elements in the dutch health care system had enlarged the influence of insurers and hospital managers on the attribution and the distribution of health care services . the council foresaw that further marketization might challenge medical professional autonomy as well as medical ethical principles and admonished policymakers and professionals to be vigilant in this respect . in our research we wanted to find out what kind of market induced phenomena professionals encounter in their daily work , how they feel about them and whether these phenomena would change medical professional ethics . we chose to study two specialties within the medical profession that differ with regard to their working conditions : general practitioners ( gps ) and surgeons . we assumed that both groups would have been confronted with the effects of marketization by now ; gps because their payment system has been changed and surgeons because several operations have become the object of market negotiations and may have been included in treatment guarantee packages . most dutch citizens have their own gp whom they can consult for health - related problems . patients are referred to surgeons by their gp ( who functions as a gatekeeper to hospital care ) . we assumed that working conditions might influence the way in which doctors are confronted with elements of marketization . for example : gps might be confronted most directly with demanding patient - consumers whereas surgeons might have to deal with eager hospital managers , looking for quick wins . this would allow us to ask respondents for all sort examples of marketization they encountered in their work which we could not construe beforehand . moreover we could ask them to discuss these examples and their ideas about them at length , thus allowing for more insights and more nuance than would have been possible in a written questionnaire . we performed 27 interviews with surgeons and 28 with general practitioners in 2008 and 2009 . we strove to interview both male and female doctors , doctors of different generations , doctors working in different regions of the country ( more and less urbanized ) and surgeons working in different types of hospitals ( academic hospitals where surgeons have a fixed salary as well as smaller hospitals in the periphery where surgeons get paid on a fee for service basis ) we did not include surgeons working in newly founded private clinics ( zelfstandige behandelcentra ) who specialize in certain types of surgery ( e.g. cosmetic surgery , eye surgery or varicose vein surgery ) . although these newly founded clinics may be considered a direct result of marketization in health care we assumed that medical staff in these clinics would be a special segment of the profession . we wanted to find out how market changes play out for rank and file medical professionals in obviously the number of respondents is too small to establish correlations ( such as : surgeons in the periphery are more affected by the marketization of health care than surgeons operating in academic hospitals ) , but our attempt to achieve variation will at least prevent us from drawing conclusions based on the experiences of one particular type of doctors in one particular hospital or one specific municipality . table 1 provides an overview of the respondents and their background.table 1overview of respondents28 general practitionersmale15female13between 25 and 409between 40 and 55855 and older11urbanized17non urbanized1127 surgeonsmale16female11between 25 and 409between 40 and 55955 and older9academic hospital ( fixed salary)6non academic hospital ( fee for service)21 overview of respondents the interviews were semi - structured and part of a broader research project , relating to two other topics besides marketization ( the growing percentage of well educated , well informed patients and the growing percentage of female doctors).1 with regard to marketization we asked our respondents whether they had noticed any changes in their work as an effect of marketization . most respondents had an understanding of what marketization meant in the context of dutch health care in general and their own work in particular,2 but for those respondents who asked for a clarification we provided some examples , such as : more competition between care providers , more marketing and public relations , fear of losing customers , shifting priorities . if they had noticed change as a result of marketization we asked them to give examples and to describe their thoughts and feelings about the changes they saw . if they had not noticed any change we would ask them if they had witnessed changes outside their direct environment , that is : in other hospitals , other gp practices , other parts of the country or other specialties . we did not specifically ask our respondents if they had noticed any bending or breaching of medical professional principles , since such questions would be leading in nature . however , we had considered three specific medical - ethical principles that might change as a consequence of marketization . brothers , a moral norm which might change as a consequence of more and harsher competition among doctors;the principle that patients ought to be treated according to urgency and medical need , which might change if certain patients come to be more profitable than others andthe primum non nocere , first of all do no harm principle , which might change because a more market oriented ethic could make physicians more inclined to sell unnecessary and thus potentially harmful treatments or diagnostic interventions . brothers , a moral norm which might change as a consequence of more and harsher competition among doctors ; the principle that patients ought to be treated according to urgency and medical need , which might change if certain patients come to be more profitable than others and the primum non nocere , first of all do no harm principle , which might change because a more market oriented ethic could make physicians more inclined to sell unnecessary and thus potentially harmful treatments or diagnostic interventions . the interviews were transcribed verbatim and were then analysed in a two phase model . during the first phase the first author coded the interview material using the computer programme atlas - ti . atlas - ti allows the researcher to first establish broad categories and then subdivide these into smaller categories . for our research project we used three broad categories , related to the three topics we wanted to investigate . for this article we selected all interview fragments related to market developments in the health care system ( one of the three broader categories ) . the first researcher coded these fragments in smaller categories . during the second phase the two other researchers read all interview transcriptions and checked the codes attributed to the interview fragments by the first researcher , so as to enhance the validity of our analysis . before we discuss the findings related to the three different principles it is important to establish whether respondents saw any changes at all in their daily work , related to marketization . despite the fact that we selected our two professional groups because we assumed that they would have been confronted with marketization directly , a minority of respondents ( 1 surgeon and 7 gps ) reported that they did not notice any difference caused by marketization in their day to day work . the surgeon and two gps explained that in certain environments it was possible to ignore the changes.many people seem to think , let them talk all they want , we will just do our work ( su jv4).in [ my village ] everything stays the same . , 36).3 many people seem to think , let them talk all they want , we will just do our work ( su jv4 ) . in , 36).3 two gps found that the market elements in the new system were too small to cause any real change , and the remaining three gps explained that market elements ( such as marketing and competition ) were also prominent when they first started their professional career.when i first started [ other doctors ] were very hesitant . there was certainly no shortage of gps , so it may be a bit much to say that you had to fight your way in , but for sure there was rivalry in the beginning ( gp om7 , p. 38).female doctors were competitors back then . when i applied for this practice , there was much resistance among other doctors , because in fact they did not want to have a female colleague ( gp ov3 , p. 39 ) . there was certainly no shortage of gps , so it may be a bit much to say that you had to fight your way in , but for sure there was rivalry in the beginning ( gp om7 , p. 38 ) . when i applied for this practice , there was much resistance among other doctors , because in fact they did not want to have a female colleague ( gp ov3 , p. 39 ) . the most important change for surgeons , mentioned by eleven of them , pertained to the fact that they now had to sell themselves , they had to advertise or market their performance . before the marketization took place the doctors association in the netherlands ( the knmg ) the introduction of market elements in dutch health care not only made the anti - advertisement principle obsolete , it actually made it illegal for the doctors association to uphold this traditional rule of medical professional ethics . hence the fact that marketing was often mentioned as a major change is not surprising . some of them did a visiting tour among gps in the neighborhood , so as to encourage these gps to send their patients to their hospital.4 one surgeon reported that his hospital had managed ( with quite some effort ) to become the first google hit for certain types of operations . other public relations activities involved publishing advertorials in local newspapers , distributing leaflets , inviting a pop group to sing in the hospital to generate more publicity and buying advertising space on the back of a local bus.two surgeons were quite positive about this change of practices : so yes , well , you may be proud if you perform so well ( su jm1).i always felt it was a pity that we could do absolutely nothing in terms of advertising . you could not say on the radio : come over here , because we re so good at this or that . so yes , i rather like it that you can promote yourself now . because you do invest a lot in your work . and then , if you go home nobody knows what good things you ve been doing . so it feels good to make that public ( su mv1 , p. 12 , 13 two surgeons were quite positive about this change of practices : so yes , well , you may be proud if you perform so well ( su jm1 ) . i always felt it was a pity that we could do absolutely nothing in terms of advertising . you could not say on the radio : come over here , because we re so good at this or that . so yes , i rather like it that you can promote yourself now . because you do invest a lot in your work . and then , if you go home nobody knows what good things you ve been doing . so it feels good to make that public ( su mv1 , p. 12 , 13 ) . four others did not like the self promotion at all.so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . the remaining surgeons regarded public relations and marketing as developments that were thrust upon them , to which they would have to adjust sooner or later.i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . i do nt particularly look forward to having my picture on a bus , but hey , if you have to , you have to ( su mv5 , p. 21 ) . i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . i do nt particularly look forward to having my picture on a bus , but hey , if you have to , you have to ( su mv5 , p. 21 ) . among gps the issue of marketing and commercials was less prominent , although six of them mentioned this effect of marketization . she studied websites hosted by real estate companies to find out about new inhabitants in her neighborhood , she placed advertisements in a students magazine and she distributed leaflets throughout her neighborhood . most gps reported matter of factly about this new development . two gps morally disapproved of the changes in professional attitude and behavior.i just want to be a kind and good doctor to people . i would not want an evening clinic or a drivers license medical examination as attention grabbers for my practice ( gp jm2 , p. 49 ) . i would not want an evening clinic or a drivers license medical examination as attention grabbers for my practice ( gp jm2 , p. 49 ) . traditional medical professional ethics held that health care should be distributed according to medical need . the declaration of the rights of the patient of the world medical association clearly states : in circumstances where a choice must be made between potential patients for a particular treatment which is in limited supply , all such patients are entitled to a fair selection procedure for that treatment . that choice must be based on medical criteria and made without discrimination . in the netherlands politicians orchestrated a public debate on choices in health care during the 1980s and early 1990s which led to a parliamentary confirmation of the need principle : if choices were necessary , medical care should go to those most in need of it . is this medical professional principle endangered by the market elements in the new health care system ? ten of the surgeons we interviewed mentioned that the new system made them pay more attention to minor afflictions than they did in the past . their hospitals had invested in clinical paths and speedy treatment for patients suffering from varicose veins and inguinal ruptures . the standardization of these simple treatments became a number one priority.we are organizing a clinical path for varicose veins . we want to see to it that if a patient comes in he gets an examination straight away and then arrange the next appointment right after that ( su mv3 , p. 8).young colleagues of mine just announce : we re gon na make a clinical path for varicose veins and we re gon na make big money there ( su dm 3 , p. 16 ) . we are organizing a clinical path for varicose veins . together with the dermatologist we are making a protocol . we want to see to it that if a patient comes in he gets an examination straight away and then arrange the next appointment right after that ( su mv3 , p. 8) . young colleagues of mine just announce : we re gon na make a clinical path for varicose veins and we re gon na make big money there ( su dm 3 , p. 16 ) . less popular but also mentioned occasionally were special clinics for people who had taken a fall , for people with urinary problems , and for obese patients hoping for a stomach reduction . six surgeons observed that the extra attention for routine surgery was taken away from other patients or other medical duties.of course i am not operating more because of maketization . it s a shift of attention , at best ( su mv2 , p. 12).right , and because one patient gets a speedy treatment , somebody else who suffers from something else , will be helped later . but i do nt hear anybody about that ( su om3 , p. 17).we have indicated that bariatric surgery takes up a lot of time , and that we have less time to spend on oncology . it s a shift of attention , at best ( su mv2 , p. 12 ) . right , and because one patient gets a speedy treatment , somebody else who suffers from something else , will be helped later . but i do nt hear anybody about that ( su om3 , p. 17 ) . we have indicated that bariatric surgery takes up a lot of time , and that we have less time to spend on oncology . we re working on that ( su jv4 , p. 3 ) . among gps investing extra resources gps do a lot of things that they did not do before the change of the health care system . to be able to do so they invest in new equipment ( tympanometers to be able to measure fluid behind the ear , ecg machines to be able to make cardiograms , sophisticated 24/7 blood pressure meters ) and they employ personnel ( administrative staff , a physician assistant or another gp ) . twelve of our gp respondents mentioned that they had bought new equipment or hired new staff to be able to perform extra medical activities . whereas surgeons testified about a shift in priorities in favour of patients with minor afflictions , several gps ( n = 5 ) witnessed a change for the better for patients with chronic conditions.marketization has led to an improvement in the care for the chronically ill . diabetes care , but even more clearly for copd patients ( gp mv3 , p. 8).in diabetes care , but even more clearly for copd patients ( gp mv3 , p. 8) . in our practice the improvement is probably due to the growing competition in the care for the chronically ill . when that happens , gps miss them as well as the income they generated.i think it s a pity that diabetics no longer visit a gp . and that they do nt see their gp for their regular check up . because if you come to your gp with some ordinary condition i can ask you how you re doing otherwise . to me those were the nicest elements of gp practice and i know patients feel the same way ( gp ov4 , p. 47 ) . i think it s a pity that diabetics no longer visit a gp . and that they do nt see their gp for their regular check up . because if you come to your gp with some ordinary condition i can ask you how you re doing otherwise . to me those were the nicest elements of gp practice and i know patients feel the same way ( gp ov4 , p. 47 ) . within gp practice the shift of priorities is much more in accordance with the traditional distribution - according - medical - need principle . spending extra money on equipment and personnel probably leads to a net enlargement of the total amount of medical care,5 whereas the extra attention to people with chronic conditions might conceivably be construed as offering extra care to those most in need ( although this remains debatable ; if the extra services would be spent on diabetics rather than on patients with less common but much more debilitating diseases , this would not hold ) . one of the first principles of medical professional ethics , featuring prominently in the hippocratic oath is primum non nocere : first of all , do no harm . it is a principle that might be challenged by the introduction of market principles in health care . if a patient wants a certain operation which the doctor thinks is unnecessary or futile , should the doctor then go along with the patient s wishes ( following the rhetoric of demand driven care ) , despite the fact that an operation might harm the patient ( after all , every surgical procedure involves certain risks ) , or should the doctor refuse ? and what if operating although useless or unnecessary would bring money , to the doctor and to the hospital ? six of our surgeon respondents pointed out that this might lead to tensions.they perform gallbladder surgery here on people with relatively minor complaints . many people have gallstones and if they are not in much pain , i doubt if an operation will do them much good ( su jm1 , p. 4).look if people suffer from terminal cancer , and you know that an operation wo nt do any good , many patients will say : all the same , even if there s only one percent chance of success , will you please operate ? and as the influence of the market grows , the doctor may say , well , i can sit here and talk for an hour or so , to explain this patient that an operation is pointless . but i may also think : he wants an operation , everybody tells me we ought to deliver patient centred care , so i am politically correct if i operate , i get more money if i operate , and i prefer operating to talking anyway . so what incentive do i have left to explain my patient that he should not undergo surgery ? ( su om1 , p. 15 ) . they perform gallbladder surgery here on people with relatively minor complaints . many people have gallstones and if they are not in much pain , i doubt if an operation will do them much good ( su jm1 , p. 4 ) . if people suffer from terminal cancer , and you know that an operation wo nt do any good , many patients will say : all the same , even if there s only one percent chance of success , will you please operate ? and as the influence of the market grows , the doctor may say , well , i can sit here and talk for an hour or so , to explain this patient that an operation is pointless . but i may also think : he wants an operation , everybody tells me we ought to deliver patient centred care , so i am politically correct if i operate , i get more money if i operate , and i prefer operating to talking anyway . so what incentive do i have left to explain my patient that he should not undergo surgery ? ( su om1 , p. 15 ) . another surgeon reported that she would hold firm in this respect.sometimes i explain to my patients that surgery would not benefit them . i tell them : i like to operate , that s why i chose this profession , i earn money if i operate on you , so sure , i could operate , but it would not be good for you ( su mv3 , p. 9 ) . i like to operate , that s why i chose this profession , i earn money if i operate on you , so sure , i could operate , but it would not be good for you ( su mv3 , p. 9 ) . another surgeon pointed out that surgery , unlike other specialties , would not allow for this type of manipulation with indications.you ca nt fool around in surgery . you either have an inguinal rupture or you do nt . [ ] you can not fake in this profession . there was a doctor here who specialised in throats and ears . if you do that type of thing you can look in all sorts of body holes and say something about them . quite easy to cheat . you admit someone ; have a look in one of his holes , you say : no lump there and bob s your uncle ( su mv1 , p. 14 ) . you either have an inguinal rupture or you do nt . [ ] you can not fake in this profession . there was a doctor here who specialised in throats and ears . if you do that type of thing you can look in all sorts of body holes and say something about them . quite easy to cheat . you admit someone ; have a look in one of his holes , you say : no lump there and bob s your uncle ( su mv1 , p. 14 ) . in dutch gp practice the do no harm principle used to be interpreted as follows : any medical performance by any doctor is a form of medicalization and thereby potentially harmful.if a patient can recover without therapy or medication it is far better to forego treatment.if a patient really needs medication or therapy , he should get it , but preferably as little as possible.thus , if a patient can be treated at home by his gp this is to be preferred over hospital treatment . hospital doctors tend to over treat , a hospital is a sickening environment and hospital treatment takes the patient out of his private surrounding which is unsettling and potentially unhealthy . any medical performance by any doctor is a form of medicalization and thereby potentially harmful . if a patient can recover without therapy or medication it is far better to forego treatment . if a patient really needs medication or therapy , he should get it , but preferably as little as possible . thus , if a patient can be treated at home by his gp this is to be preferred over hospital treatment . hospital doctors tend to over treat , a hospital is a sickening environment and hospital treatment takes the patient out of his private surrounding which is unsettling and potentially unhealthy . this gp interpretation of primum non nocere suited the dutch government in the 1980s and 1990s . hospital care is expensive and gps were strict gatekeepers to hospital care , thus contributing to the government s intention to cut back on medical care . several gp respondents informed us about the traditional gp ideology.that really put a mark on dutch gps . and i think , this whole idea of let s keep it all affordable , that s also part of what it means to be a gp ( gp om3 , p. 53).it also because of the costs ( gp om6 , p. 27 ) . that really put a mark on dutch gps . and i think , this whole idea of let s keep it all affordable , that s also part of what it means to be a gp ( gp om3 , p. 53 ) . many gp respondents ( n = 22 ) felt that this medical professional principle , their traditional gp ideology , was endangered by the marketization of health care . they were treating conditions that did not really need treatment according to their former ideology . they were performing examinations which they would have condemned as unnecessary in the past.for instance , say you ve got a metal splinter in your eye . if i take it out with a cotton swab i get 9 euros . if i use a tiny drill to get it out , that is classified as a pseudo specialist intervention , and then i get 51 euros . so you see , it s kind of tempting to pick up the drill and get it out like that . while actually it s not necessary , and a drill always creates a little wound so that s a threshold for a doctor but i am sure there is more drilling going on than necessary ( gp mm1 , p. 14).colleagues of mine want to invite all patients over 50 in their practice to make an ecg . [ ] the same thing happens with a lung function meter ( gp mv2 , p. 13).a i would wonder : what do you want with it ? [ ] and now i say : okay , patient wants it , fine . i am not the one who s going to pay for it ( gp mm3 , p. 24 ) . if i take it out with a cotton swab i get 9 euros . if i use a tiny drill to get it out , that is classified as a pseudo specialist intervention , and then i get 51 euros . so you see , it s kind of tempting to pick up the drill and get it out like that . while actually it s not necessary , and a drill always creates a little wound so that s a threshold for a doctor . but i am sure there is more drilling going on than necessary ( gp mm1 , p. 14 ) . colleagues of mine want to invite all patients over 50 in their practice to make an ecg . [ ] the same thing happens with a lung function meter ( gp mv2 , p. 13 ) . a full blood examination . in the past i would wonder : what do you want with it ? i am not the one who s going to pay for it ( gp mm3 , p. 24 ) . some gp respondents reported that they still want to cling to their traditional reticence and that they do nt want to give in.gp medicine is like rowing upstream . against the patient s demand even . that may sound strange , because you have to think along with your patient , but i think that patients do ask a lot of unhealthy things ( gp om2 , p. i sometimes say to a patient , look you re not in a supermarket here where you can come in and just take whatever you want gp medicine is like rowing upstream . against the patient s demand even . that may sound strange , because you have to think along with your patient , but i think that patients do ask a lot of unhealthy things ( gp om2 , p. 31 ) . i sometimes say to a patient , look you re not in a supermarket here where you can come in and just take whatever you want however , the same gp also told that he felt he had no choice but to give in , because he feared that patients would leave him and find another gp . the majority of gps seem to be reinterpreting the do no harm principle . they have discovered that patients are not harmed by examinations and blood tests . quite the contrary , patients seem to like it ( gp mm1 , p. 12 ) . gps have bought equipment which enables them to examine patients in their own office , rather than referring them to a hospital , which contributes to the idea that patients will not be harmed , as patients do nt have to spend half a day waiting in a hospital , they can be examined on the spot ( gp mv2 , p. 10 ) . although screenings and examinations do not harm patients and many patients rather like to be examined thoroughly every now and then ( gp mm4 , p. 13 ) , screenings and examinations do cost a lot of money , while it is unclear whether they will improve public health . in the past the costs of health care were an integral part of the gps ideology , of their particular interpretation of the do no harm principle . this seems to have changed , due to marketization.[politicians ] hoped to lower the costs of health care . there s a rising demand for total body scans and the like . because everybody wants to have everything and everything is being refunded by health insurers ( gp mv 3 , p. 55).you should not make gp care commercial . the minister is completely wrong doing that . [ ] because gps will then try to please their patients and that is not always what s best for the patient ( gp mm4 , p. 33).i think the argument that you should not harm your patient will remain prominent . but cooperating to keep down the costs of health care ; that s not appealing any more ( gp mm1 , p. 29 ) . there s a rising demand for total body scans and the like . because everybody wants to have everything and everything is being refunded by health insurers ( gp mv 3 , p. 55 ) . the minister is completely wrong doing that . [ ] because gps will then try to please their patients and that is not always what s best for the patient ( gp mm4 , p. 33 ) . i think the argument that you should not harm your patient will remain prominent . but cooperating to keep down the costs of health care ; that s not appealing any more ( gp mm1 , p. 29 ) . before we discuss the findings related to the three different principles it is important to establish whether respondents saw any changes at all in their daily work , related to marketization . despite the fact that we selected our two professional groups because we assumed that they would have been confronted with marketization directly , a minority of respondents ( 1 surgeon and 7 gps ) reported that they did not notice any difference caused by marketization in their day to day work . the surgeon and two gps explained that in certain environments it was possible to ignore the changes.many people seem to think , let them talk all they want , we will just do our work ( su jv4).in [ my village ] everything stays the same . , 36).3 many people seem to think , let them talk all they want , we will just do our work ( su jv4 ) . in , 36).3 two gps found that the market elements in the new system were too small to cause any real change , and the remaining three gps explained that market elements ( such as marketing and competition ) were also prominent when they first started their professional career.when i first started [ other doctors ] were very hesitant . there was certainly no shortage of gps , so it may be a bit much to say that you had to fight your way in , but for sure there was rivalry in the beginning ( gp om7 , p. 38).female doctors were competitors back then . when i applied for this practice , there was much resistance among other doctors , because in fact they did not want to have a female colleague ( gp ov3 , p. 39 ) . there was certainly no shortage of gps , so it may be a bit much to say that you had to fight your way in , but for sure there was rivalry in the beginning ( gp om7 , p. 38 ) . when i applied for this practice , there was much resistance among other doctors , because in fact they did not want to have a female colleague ( gp ov3 , p. 39 ) . the most important change for surgeons , mentioned by eleven of them , pertained to the fact that they now had to sell themselves , they had to advertise or market their performance . before the marketization took place the doctors association in the netherlands ( the knmg ) the introduction of market elements in dutch health care not only made the anti - advertisement principle obsolete , it actually made it illegal for the doctors association to uphold this traditional rule of medical professional ethics . hence the fact that marketing was often mentioned as a major change is not surprising . some of them did a visiting tour among gps in the neighborhood , so as to encourage these gps to send their patients to their hospital.4 one surgeon reported that his hospital had managed ( with quite some effort ) to become the first google hit for certain types of operations . other public relations activities involved publishing advertorials in local newspapers , distributing leaflets , inviting a pop group to sing in the hospital to generate more publicity and buying advertising space on the back of a local bus.two surgeons were quite positive about this change of practices : so yes , well , you may be proud if you perform so well ( su jm1).i always felt it was a pity that we could do absolutely nothing in terms of advertising . you could not say on the radio : come over here , because we re so good at this or that . so yes , i rather like it that you can promote yourself now . because you do invest a lot in your work . and then , if you go home nobody knows what good things you ve been doing . two surgeons were quite positive about this change of practices : so yes , well , you may be proud if you perform so well ( su jm1 ) . i always felt it was a pity that we could do absolutely nothing in terms of advertising . you could not say on the radio : come over here , because we re so good at this or that . so yes , i rather like it that you can promote yourself now . because you do invest a lot in your work . and then , if you go home nobody knows what good things you ve been doing . so it feels good to make that public ( su mv1 , p. 12 , 13 ) . four others did not like the self promotion at all.so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . the remaining surgeons regarded public relations and marketing as developments that were thrust upon them , to which they would have to adjust sooner or later.i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . i do nt particularly look forward to having my picture on a bus , but hey , if you have to , you have to ( su mv5 , p. 21 ) . i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . i do nt particularly look forward to having my picture on a bus , but hey , if you have to , you have to ( su mv5 , p. 21 ) . among gps the issue of marketing and commercials was less prominent , although six of them mentioned this effect of marketization . she studied websites hosted by real estate companies to find out about new inhabitants in her neighborhood , she placed advertisements in a students magazine and she distributed leaflets throughout her neighborhood . most gps reported matter of factly about this new development . two gps morally disapproved of the changes in professional attitude and behavior.i just want to be a kind and good doctor to people . i would not want an evening clinic or a drivers license medical examination as attention grabbers for my practice ( gp jm2 , p. 49 ) . i would not want an evening clinic or a drivers license medical examination as attention grabbers for my practice ( gp jm2 , p. 49 ) . traditional medical professional ethics held that health care should be distributed according to medical need . the declaration of the rights of the patient of the world medical association clearly states : in circumstances where a choice must be made between potential patients for a particular treatment which is in limited supply , all such patients are entitled to a fair selection procedure for that treatment . that choice must be based on medical criteria and made without discrimination . in the netherlands politicians orchestrated a public debate on choices in health care during the 1980s and early 1990s which led to a parliamentary confirmation of the need principle : if choices were necessary , medical care should go to those most in need of it . is this medical professional principle endangered by the market elements in the new health care system ? ten of the surgeons we interviewed mentioned that the new system made them pay more attention to minor afflictions than they did in the past . their hospitals had invested in clinical paths and speedy treatment for patients suffering from varicose veins and inguinal ruptures . the standardization of these simple treatments became a number one priority.we are organizing a clinical path for varicose veins . we want to see to it that if a patient comes in he gets an examination straight away and then arrange the next appointment right after that ( su mv3 , p. 8).young colleagues of mine just announce : we re gon na make a clinical path for varicose veins and we re gon na make big money there ( su dm 3 , p. 16 ) . we are organizing a clinical path for varicose veins . together with the dermatologist we are making a protocol . we want to see to it that if a patient comes in he gets an examination straight away and then arrange the next appointment right after that ( su mv3 , p. 8) . young colleagues of mine just announce : we re gon na make a clinical path for varicose veins and we re gon na make big money there ( su dm 3 , p. 16 ) . less popular but also mentioned occasionally were special clinics for people who had taken a fall , for people with urinary problems , and for obese patients hoping for a stomach reduction . six surgeons observed that the extra attention for routine surgery was taken away from other patients or other medical duties.of course i am not operating more because of maketization . it s a shift of attention , at best ( su mv2 , p. 12).right , and because one patient gets a speedy treatment , somebody else who suffers from something else , will be helped later . but i do nt hear anybody about that ( su om3 , p. 17).we have indicated that bariatric surgery takes up a lot of time , and that we have less time to spend on oncology . it s a shift of attention , at best ( su mv2 , p. 12 ) . right , and because one patient gets a speedy treatment , somebody else who suffers from something else , will be helped later . but i do nt hear anybody about that ( su om3 , p. 17 ) . we have indicated that bariatric surgery takes up a lot of time , and that we have less time to spend on oncology . we re working on that ( su jv4 , p. 3 ) . among gps investing extra resources gps do a lot of things that they did not do before the change of the health care system . to be able to do so they invest in new equipment ( tympanometers to be able to measure fluid behind the ear , ecg machines to be able to make cardiograms , sophisticated 24/7 blood pressure meters ) and they employ personnel ( administrative staff , a physician assistant or another gp ) . twelve of our gp respondents mentioned that they had bought new equipment or hired new staff to be able to perform extra medical activities . whereas surgeons testified about a shift in priorities in favour of patients with minor afflictions , several gps ( n = 5 ) witnessed a change for the better for patients with chronic conditions.marketization has led to an improvement in the care for the chronically ill . diabetes care , but even more clearly for copd patients ( gp mv3 , p. 8).in our practice we see it with diabetics . diabetes care , but even more clearly for copd patients ( gp mv3 , p. 8) . in our practice the improvement is probably due to the growing competition in the care for the chronically ill . when that happens , gps miss them as well as the income they generated.i think it s a pity that diabetics no longer visit a gp . and that they do nt see their gp for their regular check up . because if you come to your gp with some ordinary condition i can ask you how you re doing otherwise . to me those were the nicest elements of gp practice and i know patients feel the same way ( gp ov4 , p. 47 ) . i think it s a pity that diabetics no longer visit a gp . and that they do nt see their gp for their regular check up . because if you come to your gp with some ordinary condition i can ask you how you re doing otherwise . to me those were the nicest elements of gp practice and i know patients feel the same way ( gp ov4 , p. 47 ) . within gp practice the shift of priorities is much more in accordance with the traditional distribution - according - medical - need principle . spending extra money on equipment and personnel probably leads to a net enlargement of the total amount of medical care,5 whereas the extra attention to people with chronic conditions might conceivably be construed as offering extra care to those most in need ( although this remains debatable ; if the extra services would be spent on diabetics rather than on patients with less common but much more debilitating diseases , this would not hold ) . one of the first principles of medical professional ethics , featuring prominently in the hippocratic oath is primum non nocere : first of all , do no harm . it is a principle that might be challenged by the introduction of market principles in health care . if a patient wants a certain operation which the doctor thinks is unnecessary or futile , should the doctor then go along with the patient s wishes ( following the rhetoric of demand driven care ) , despite the fact that an operation might harm the patient ( after all , every surgical procedure involves certain risks ) , or should the doctor refuse ? and what if operating although useless or unnecessary would bring money , to the doctor and to the hospital ? six of our surgeon respondents pointed out that this might lead to tensions.they perform gallbladder surgery here on people with relatively minor complaints . many people have gallstones and if they are not in much pain , i doubt if an operation will do them much good ( su jm1 , p. 4).look if people suffer from terminal cancer , and you know that an operation wo nt do any good , many patients will say : all the same , even if there s only one percent chance of success , will you please operate ? and as the influence of the market grows , the doctor may say , well , i can sit here and talk for an hour or so , to explain this patient that an operation is pointless . but i may also think : he wants an operation , everybody tells me we ought to deliver patient centred care , so i am politically correct if i operate , i get more money if i operate , and i prefer operating to talking anyway . so what incentive do i have left to explain my patient that he should not undergo surgery ? ( su om1 , p. 15 ) . they perform gallbladder surgery here on people with relatively minor complaints . many people have gallstones and if they are not in much pain , i doubt if an operation will do them much good ( su jm1 , p. 4 ) . if people suffer from terminal cancer , and you know that an operation wo nt do any good , many patients will say : all the same , even if there s only one percent chance of success , will you please operate ? and as the influence of the market grows , the doctor may say , well , i can sit here and talk for an hour or so , to explain this patient that an operation is pointless . but i may also think : he wants an operation , everybody tells me we ought to deliver patient centred care , so i am politically correct if i operate , i get more money if i operate , and i prefer operating to talking anyway . so what incentive do i have left to explain my patient that he should not undergo surgery ? ( su om1 , p. 15 ) . another surgeon reported that she would hold firm in this respect.sometimes i explain to my patients that surgery would not benefit them . i like to operate , that s why i chose this profession , i earn money if i operate on you , so sure , i could operate , but it would not be good for you ( su mv3 , p. 9 ) . i tell them : i like to operate , that s why i chose this profession , i earn money if i operate on you , so sure , i could operate , but it would not be good for you ( su mv3 , p. 9 ) . another surgeon pointed out that surgery , unlike other specialties , would not allow for this type of manipulation with indications.you ca nt fool around in surgery . there was a doctor here who specialised in throats and ears . if you do that type of thing you can look in all sorts of body holes and say something about them . quite easy to cheat . you admit someone ; have a look in one of his holes , you say : no lump there and bob s your uncle ( su mv1 , p. 14 ) . you either have an inguinal rupture or you do nt . [ ] you can not fake in this profession . there was a doctor here who specialised in throats and ears . if you do that type of thing you can look in all sorts of body holes and say something about them . quite easy to cheat . you admit someone ; have a look in one of his holes , you say : no lump there and bob s your uncle ( su mv1 , p. 14 ) . in dutch gp practice the do no harm principle used to be interpreted as follows : any medical performance by any doctor is a form of medicalization and thereby potentially harmful.if a patient can recover without therapy or medication it is far better to forego treatment.if a patient really needs medication or therapy , he should get it , but preferably as little as possible.thus , if a patient can be treated at home by his gp this is to be preferred over hospital treatment . hospital doctors tend to over treat , a hospital is a sickening environment and hospital treatment takes the patient out of his private surrounding which is unsettling and potentially unhealthy . any medical performance by any doctor is a form of medicalization and thereby potentially harmful . if a patient can recover without therapy or medication it is far better to forego treatment . if a patient really needs medication or therapy , he should get it , but preferably as little as possible . thus , if a patient can be treated at home by his gp this is to be preferred over hospital treatment . hospital doctors tend to over treat , a hospital is a sickening environment and hospital treatment takes the patient out of his private surrounding which is unsettling and potentially unhealthy . this gp interpretation of primum non nocere suited the dutch government in the 1980s and 1990s . hospital care is expensive and gps were strict gatekeepers to hospital care , thus contributing to the government s intention to cut back on medical care . several gp respondents informed us about the traditional gp ideology.that really put a mark on dutch gps . and i think , this whole idea of let s keep it all affordable , that s also part of what it means to be a gp ( gp om3 , p. 53).it also because of the costs ( gp om6 , p. 27 ) . that really put a mark on dutch gps . and i think , this whole idea of let s keep it all affordable , that s also part of what it means to be a gp ( gp om3 , p. 53 ) . . many gp respondents ( n = 22 ) felt that this medical professional principle , their traditional gp ideology , was endangered by the marketization of health care . they were treating conditions that did not really need treatment according to their former ideology . they were performing examinations which they would have condemned as unnecessary in the past.for instance , say you ve got a metal splinter in your eye . if i take it out with a cotton swab i get 9 euros . if i use a tiny drill to get it out , that is classified as a pseudo specialist intervention , and then i get 51 euros . so you see , it s kind of tempting to pick up the drill and get it out like that . while actually it s not necessary , and a drill always creates a little wound so that s a threshold for a doctor . but i am sure there is more drilling going on than necessary ( gp mm1 , p. 14).colleagues of mine want to invite all patients over 50 in their practice to make an ecg . [ ] the same thing happens with a lung function meter ( gp mv2 , p. 13).a i would wonder : what do you want with it ? [ ] and now i say : okay , patient wants it , fine . i am not the one who s going to pay for it ( gp mm3 , p. 24 ) . if i use a tiny drill to get it out , that is classified as a pseudo specialist intervention , and then i get 51 euros . so you see , it s kind of tempting to pick up the drill and get it out like that . while actually it s not necessary , and a drill always creates a little wound so that s a threshold for a doctor . but i am sure there is more drilling going on than necessary ( gp mm1 , p. 14 ) . colleagues of mine want to invite all patients over 50 in their practice to make an ecg . [ ] the same thing happens with a lung function meter ( gp mv2 , p. 13 ) . a full blood examination . in the past i would wonder : what do you want with it ? [ ] and now i say : okay , patient wants it , fine . i am not the one who s going to pay for it ( gp mm3 , p. 24 ) . some gp respondents reported that they still want to cling to their traditional reticence and that they do nt want to give in.gp medicine is like rowing upstream . against the patient s demand even . that may sound strange , because you have to think along with your patient , but i think that patients do ask a lot of unhealthy things ( gp om2 , p. i sometimes say to a patient , look you re not in a supermarket here where you can come in and just take whatever you want . that may sound strange , because you have to think along with your patient , but i think that patients do ask a lot of unhealthy things ( gp om2 , p. 31 ) . i sometimes say to a patient , look you re not in a supermarket here where you can come in and just take whatever you want however , the same gp also told that he felt he had no choice but to give in , because he feared that patients would leave him and find another gp . the majority of gps seem to be reinterpreting the do no harm principle . they have discovered that patients are not harmed by examinations and blood tests . quite the contrary , patients seem to like it ( gp mm1 , p. 12 ) . gps have bought equipment which enables them to examine patients in their own office , rather than referring them to a hospital , which contributes to the idea that patients will not be harmed , as patients do nt have to spend half a day waiting in a hospital , they can be examined on the spot ( gp mv2 , p. 10 ) . although screenings and examinations do not harm patients and many patients rather like to be examined thoroughly every now and then ( gp mm4 , p. 13 ) , screenings and examinations do cost a lot of money , while it is unclear whether they will improve public health . in the past the costs of health care were an integral part of the gps ideology , of their particular interpretation of the do no harm principle . this seems to have changed , due to marketization.[politicians ] hoped to lower the costs of health care . there s a rising demand for total body scans and the like . because everybody wants to have everything and everything is being refunded by health insurers ( gp mv 3 , p. 55).you should not make gp care commercial . the minister is completely wrong doing that . [ ] because gps will then try to please their patients and that is not always what s best for the patient ( gp mm4 , p. 33).i think the argument that you should not harm your patient will remain prominent . but cooperating to keep down the costs of health care ; that s not appealing any more ( gp mm1 , p. 29 ) . there s a rising demand for total body scans and the like . because everybody wants to have everything and everything is being refunded by health insurers ( gp mv 3 , p. 55 ) . the minister is completely wrong doing that . [ ] because gps will then try to please their patients and that is not always what s best for the patient ( gp mm4 , p. 33 ) . i think the argument that you should not harm your patient will remain prominent . but cooperating to keep down the costs of health care ; that s not appealing any more ( gp mm1 , p. 29 ) . during the 1990s and the first decade of the 21st century a lot of things changed in dutch health care . guidelines and standards for medical treatment became more important due to a growing emphasis on evidence based care . the economy went up and down , which had repercussions in the health care system . and it is difficult , perhaps even impossible to prove that certain developments in daily practice brought up by our respondents ( e.g. the changes in diabetic care ) were caused by marketization rather than , for example , standardization . moreover , we have to be careful drawing conclusions based on a limited number of interviews among just two medical specialties . what holds for surgeons in dutch hospitals need not necessarily hold for other specialists in dutch hospitals , let alone hospitals in other countries contemplating market reforms . still we strove for diversity among our respondents , and we explicitly asked them to relate their own as well as other doctors experiences with and feelings about the market reforms . also it seems significant that changes in medical professional ethics have occurred just a few years after the introduction of market reforms . our research shows that the dutch council for public health and health care and the authors discussed in the introduction were right when they pointed out that the introduction of market elements in health care would challenge traditional medical professional ethics . marketing activities seem to be widespread in hospital surroundings and some marketing also goes on in gp practices . some physicians abhor the self - promotion involved , others welcome the new opportunities , whereas yet others consider this a fact of life , with which they will learn to live eventually . a lot of medical time and energy is spent on minor , routine operations with which hospitals can make a lot of money . this sometimes goes to the detriment of patients in need of major , risky surgical procedures . in gp practices more attention goes to patients with chronic conditions who may arguably qualify as those most in need of medical care . lastly , the primum non nocere principle is still being upheld in surgery . among gps whereas it used to mean : do not examine or treat a patient unless this is really necessary , it now comes to mean : you may examine and treat a patient as he or she prefers as long as you do not actually harm your patient . the traditional gp ideology , which may be conceived as a very broad interpretation of the do no harm principle , is challenged most by the market elements in the new system . our findings are summarized in table 2.table 2summary of findingsprinciple of medical ethicssurgeonsgpsyou shall not advertisesubstantial change : marketing has become normal although some surgeons dislike itsmall change : marketing is not very widespread , but gps feel ( and some fear ) this may changedistribute according to medical needsubstantial change : much more attention to minor afflictionssmall change in favour of patients with chronic conditions , but this may concur with the moral principleprimum non noceresmall change : surgeons feel tempted to operate more , but do not give insubstantial change : the do no harm principle is reinterpreted . medical attention is no longer considered harmful per se obviously it is too early to render a final verdict on the effects of market reforms in the dutch health care system . the advent of advertising due to competition among doctors and hospitals could diminish patients trust in doctors , but it could also be appreciated as a source of information . as to the other changes , neither of these would have to create immediate problems if the costs of health care were allowed to expand . one could then just pay for the extra time spent on minor surgery and on the growing number of preventive examinations in gp practice , making sure that other patients with other conditions would not have to suffer the consequences . however , recent economic reports have pointed out that marketization has driven up costs and that most of these costs are still paid with public money despite all the market rhetoric [ 5 , 16 , 24 ] . if health care policy makers can not accept ever rising costs and do not want to defer costs to citizens private expenses , they should realize that marketization within the boundaries of a public budget causes changes in professional medical ethics . these changes should be weighed and taken into account whenever further policy changes are considered .

to explore whether market reforms in a health care system affect medical professional ethics of hospital - based specialists on the one hand and physicians in independent practices on the other . qualitative interviews with 27 surgeons and 28 general practitioners in the netherlands , held 23 years after a major overhaul of the dutch health care system involving several market reforms . surgeons now regularly advertise their work ( while this was forbidden in the past ) and pay more attention to patients with relatively minor afflictions , thus deviating from codes of ethics that oblige physicians to treat each other as brothers and to treat patients according to medical need . dutch gps have abandoned their traditional reticence and their fear of medicalization . they now seem to treat more in accordance with patients preferences and less in accordance with medical need . market reforms do affect medical professional principles , and it is doubtful whether these changes were intended when dutch policy makers decided to introduce market elements in the health care system . policy makers in other countries considering similar reforms should pay attention to these results .

Introduction Method Results Change or No Change? Marketing Yourself (Advertising) Shifting Priorities Primum Non Nocere Conclusion

mid october 2009 general practitioner sandra bijl published an article in the dutch doctors journal medisch contact . she described one of the many dilemmas she faced since the introduction of marketized health care in the netherlands . during the 1990s and the first decade of the twenty - first century numerous politicians and policy - makers argued that health care should be consumer - driven and consumer - oriented. several authors have observed that these changes may lead to changes in medical professional ethics . garrett argues that market principles need not destroy professional ethics because sticking to one s professional ethics might be the most profitable strategy for market actors in the long run and applbaum questions the use and legitimacy of professional ethics in the first place , thus would not deplore its demise ) , the general feeling seems to be pessimistic . we will try to add some empirics to the debate on the relationship between market principles and medical professional ethics . in our study we set out to ascertain how market reforms have changed medical professional ethics in the netherlands . within western europe the netherlands has gone farthest in introducing markets and managed competition in health care ( cf . [ 3 , 13 , 19 ] ) , which makes the country very suitable for an empirical study of the relationship between marketization and medical professional ethics . these treatment guarantees may diminish physicians room to manoeuvre with their operating schedules ; the payment system for general practitioners has been changed , in a much more fee - for - service direction . although the complete overhaul of the insurance system took place in 2006 , elements of market competition were introduced before that date . medical professionals in the netherlands have a few years of experience with market elements by now . one year after the new insurance system was adopted the council for public health and health care ( an advisory council to the government and one of the more fervent proponents of the new system ) observed that the introduction of market elements in the dutch health care system had enlarged the influence of insurers and hospital managers on the attribution and the distribution of health care services . in our research we wanted to find out what kind of market induced phenomena professionals encounter in their daily work , how they feel about them and whether these phenomena would change medical professional ethics . we chose to study two specialties within the medical profession that differ with regard to their working conditions : general practitioners ( gps ) and surgeons . moreover we could ask them to discuss these examples and their ideas about them at length , thus allowing for more insights and more nuance than would have been possible in a written questionnaire . we performed 27 interviews with surgeons and 28 with general practitioners in 2008 and 2009 . we strove to interview both male and female doctors , doctors of different generations , doctors working in different regions of the country ( more and less urbanized ) and surgeons working in different types of hospitals ( academic hospitals where surgeons have a fixed salary as well as smaller hospitals in the periphery where surgeons get paid on a fee for service basis ) we did not include surgeons working in newly founded private clinics ( zelfstandige behandelcentra ) who specialize in certain types of surgery ( e.g. although these newly founded clinics may be considered a direct result of marketization in health care we assumed that medical staff in these clinics would be a special segment of the profession . we wanted to find out how market changes play out for rank and file medical professionals in obviously the number of respondents is too small to establish correlations ( such as : surgeons in the periphery are more affected by the marketization of health care than surgeons operating in academic hospitals ) , but our attempt to achieve variation will at least prevent us from drawing conclusions based on the experiences of one particular type of doctors in one particular hospital or one specific municipality . table 1 provides an overview of the respondents and their background.table 1overview of respondents28 general practitionersmale15female13between 25 and 409between 40 and 55855 and older11urbanized17non urbanized1127 surgeonsmale16female11between 25 and 409between 40 and 55955 and older9academic hospital ( fixed salary)6non academic hospital ( fee for service)21 overview of respondents the interviews were semi - structured and part of a broader research project , relating to two other topics besides marketization ( the growing percentage of well educated , well informed patients and the growing percentage of female doctors).1 with regard to marketization we asked our respondents whether they had noticed any changes in their work as an effect of marketization . most respondents had an understanding of what marketization meant in the context of dutch health care in general and their own work in particular,2 but for those respondents who asked for a clarification we provided some examples , such as : more competition between care providers , more marketing and public relations , fear of losing customers , shifting priorities . if they had not noticed any change we would ask them if they had witnessed changes outside their direct environment , that is : in other hospitals , other gp practices , other parts of the country or other specialties . we did not specifically ask our respondents if they had noticed any bending or breaching of medical professional principles , since such questions would be leading in nature . brothers , a moral norm which might change as a consequence of more and harsher competition among doctors;the principle that patients ought to be treated according to urgency and medical need , which might change if certain patients come to be more profitable than others andthe primum non nocere , first of all do no harm principle , which might change because a more market oriented ethic could make physicians more inclined to sell unnecessary and thus potentially harmful treatments or diagnostic interventions . brothers , a moral norm which might change as a consequence of more and harsher competition among doctors ; the principle that patients ought to be treated according to urgency and medical need , which might change if certain patients come to be more profitable than others and the primum non nocere , first of all do no harm principle , which might change because a more market oriented ethic could make physicians more inclined to sell unnecessary and thus potentially harmful treatments or diagnostic interventions . for this article we selected all interview fragments related to market developments in the health care system ( one of the three broader categories ) . the surgeon and two gps explained that in certain environments it was possible to ignore the changes.many people seem to think , let them talk all they want , we will just do our work ( su jv4).in [ my village ] everything stays the same . in , 36).3 two gps found that the market elements in the new system were too small to cause any real change , and the remaining three gps explained that market elements ( such as marketing and competition ) were also prominent when they first started their professional career.when i first started [ other doctors ] were very hesitant . before the marketization took place the doctors association in the netherlands ( the knmg ) the introduction of market elements in dutch health care not only made the anti - advertisement principle obsolete , it actually made it illegal for the doctors association to uphold this traditional rule of medical professional ethics . other public relations activities involved publishing advertorials in local newspapers , distributing leaflets , inviting a pop group to sing in the hospital to generate more publicity and buying advertising space on the back of a local bus.two surgeons were quite positive about this change of practices : so yes , well , you may be proud if you perform so well ( su jm1).i always felt it was a pity that we could do absolutely nothing in terms of advertising . four others did not like the self promotion at all.so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . the remaining surgeons regarded public relations and marketing as developments that were thrust upon them , to which they would have to adjust sooner or later.i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . traditional medical professional ethics held that health care should be distributed according to medical need . in the netherlands politicians orchestrated a public debate on choices in health care during the 1980s and early 1990s which led to a parliamentary confirmation of the need principle : if choices were necessary , medical care should go to those most in need of it . is this medical professional principle endangered by the market elements in the new health care system ? ten of the surgeons we interviewed mentioned that the new system made them pay more attention to minor afflictions than they did in the past . among gps investing extra resources gps do a lot of things that they did not do before the change of the health care system . whereas surgeons testified about a shift in priorities in favour of patients with minor afflictions , several gps ( n = 5 ) witnessed a change for the better for patients with chronic conditions.marketization has led to an improvement in the care for the chronically ill . within gp practice the shift of priorities is much more in accordance with the traditional distribution - according - medical - need principle . spending extra money on equipment and personnel probably leads to a net enlargement of the total amount of medical care,5 whereas the extra attention to people with chronic conditions might conceivably be construed as offering extra care to those most in need ( although this remains debatable ; if the extra services would be spent on diabetics rather than on patients with less common but much more debilitating diseases , this would not hold ) . one of the first principles of medical professional ethics , featuring prominently in the hippocratic oath is primum non nocere : first of all , do no harm . it is a principle that might be challenged by the introduction of market principles in health care . six of our surgeon respondents pointed out that this might lead to tensions.they perform gallbladder surgery here on people with relatively minor complaints . in dutch gp practice the do no harm principle used to be interpreted as follows : any medical performance by any doctor is a form of medicalization and thereby potentially harmful.if a patient can recover without therapy or medication it is far better to forego treatment.if a patient really needs medication or therapy , he should get it , but preferably as little as possible.thus , if a patient can be treated at home by his gp this is to be preferred over hospital treatment . this gp interpretation of primum non nocere suited the dutch government in the 1980s and 1990s . many gp respondents ( n = 22 ) felt that this medical professional principle , their traditional gp ideology , was endangered by the marketization of health care . some gp respondents reported that they still want to cling to their traditional reticence and that they do nt want to give in.gp medicine is like rowing upstream . gps have bought equipment which enables them to examine patients in their own office , rather than referring them to a hospital , which contributes to the idea that patients will not be harmed , as patients do nt have to spend half a day waiting in a hospital , they can be examined on the spot ( gp mv2 , p. 10 ) . in the past the costs of health care were an integral part of the gps ideology , of their particular interpretation of the do no harm principle . the surgeon and two gps explained that in certain environments it was possible to ignore the changes.many people seem to think , let them talk all they want , we will just do our work ( su jv4).in [ my village ] everything stays the same . , 36).3 many people seem to think , let them talk all they want , we will just do our work ( su jv4 ) . in , 36).3 two gps found that the market elements in the new system were too small to cause any real change , and the remaining three gps explained that market elements ( such as marketing and competition ) were also prominent when they first started their professional career.when i first started [ other doctors ] were very hesitant . before the marketization took place the doctors association in the netherlands ( the knmg ) the introduction of market elements in dutch health care not only made the anti - advertisement principle obsolete , it actually made it illegal for the doctors association to uphold this traditional rule of medical professional ethics . other public relations activities involved publishing advertorials in local newspapers , distributing leaflets , inviting a pop group to sing in the hospital to generate more publicity and buying advertising space on the back of a local bus.two surgeons were quite positive about this change of practices : so yes , well , you may be proud if you perform so well ( su jm1).i always felt it was a pity that we could do absolutely nothing in terms of advertising . four others did not like the self promotion at all.so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . so basically you have to show off with your product , and i do nt like that at all [ ] because i think health care is not something fancy or popular , it s just something that has to be decent all around ( su jv3 , p. 67 ) . the remaining surgeons regarded public relations and marketing as developments that were thrust upon them , to which they would have to adjust sooner or later.i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . i do nt really like this whole idea of competition in health care but if we do it all the same , then , perhaps you have to go along , and advertise . traditional medical professional ethics held that health care should be distributed according to medical need . in the netherlands politicians orchestrated a public debate on choices in health care during the 1980s and early 1990s which led to a parliamentary confirmation of the need principle : if choices were necessary , medical care should go to those most in need of it . is this medical professional principle endangered by the market elements in the new health care system ? ten of the surgeons we interviewed mentioned that the new system made them pay more attention to minor afflictions than they did in the past . among gps investing extra resources gps do a lot of things that they did not do before the change of the health care system . whereas surgeons testified about a shift in priorities in favour of patients with minor afflictions , several gps ( n = 5 ) witnessed a change for the better for patients with chronic conditions.marketization has led to an improvement in the care for the chronically ill . within gp practice the shift of priorities is much more in accordance with the traditional distribution - according - medical - need principle . spending extra money on equipment and personnel probably leads to a net enlargement of the total amount of medical care,5 whereas the extra attention to people with chronic conditions might conceivably be construed as offering extra care to those most in need ( although this remains debatable ; if the extra services would be spent on diabetics rather than on patients with less common but much more debilitating diseases , this would not hold ) . one of the first principles of medical professional ethics , featuring prominently in the hippocratic oath is primum non nocere : first of all , do no harm . it is a principle that might be challenged by the introduction of market principles in health care . six of our surgeon respondents pointed out that this might lead to tensions.they perform gallbladder surgery here on people with relatively minor complaints . they perform gallbladder surgery here on people with relatively minor complaints . in dutch gp practice the do no harm principle used to be interpreted as follows : any medical performance by any doctor is a form of medicalization and thereby potentially harmful.if a patient can recover without therapy or medication it is far better to forego treatment.if a patient really needs medication or therapy , he should get it , but preferably as little as possible.thus , if a patient can be treated at home by his gp this is to be preferred over hospital treatment . this gp interpretation of primum non nocere suited the dutch government in the 1980s and 1990s . many gp respondents ( n = 22 ) felt that this medical professional principle , their traditional gp ideology , was endangered by the marketization of health care . they were performing examinations which they would have condemned as unnecessary in the past.for instance , say you ve got a metal splinter in your eye . in the past i would wonder : what do you want with it ? some gp respondents reported that they still want to cling to their traditional reticence and that they do nt want to give in.gp medicine is like rowing upstream . gps have bought equipment which enables them to examine patients in their own office , rather than referring them to a hospital , which contributes to the idea that patients will not be harmed , as patients do nt have to spend half a day waiting in a hospital , they can be examined on the spot ( gp mv2 , p. 10 ) . in the past the costs of health care were an integral part of the gps ideology , of their particular interpretation of the do no harm principle . during the 1990s and the first decade of the 21st century a lot of things changed in dutch health care . the economy went up and down , which had repercussions in the health care system . what holds for surgeons in dutch hospitals need not necessarily hold for other specialists in dutch hospitals , let alone hospitals in other countries contemplating market reforms . still we strove for diversity among our respondents , and we explicitly asked them to relate their own as well as other doctors experiences with and feelings about the market reforms . also it seems significant that changes in medical professional ethics have occurred just a few years after the introduction of market reforms . our research shows that the dutch council for public health and health care and the authors discussed in the introduction were right when they pointed out that the introduction of market elements in health care would challenge traditional medical professional ethics . in gp practices more attention goes to patients with chronic conditions who may arguably qualify as those most in need of medical care . the traditional gp ideology , which may be conceived as a very broad interpretation of the do no harm principle , is challenged most by the market elements in the new system . our findings are summarized in table 2.table 2summary of findingsprinciple of medical ethicssurgeonsgpsyou shall not advertisesubstantial change : marketing has become normal although some surgeons dislike itsmall change : marketing is not very widespread , but gps feel ( and some fear ) this may changedistribute according to medical needsubstantial change : much more attention to minor afflictionssmall change in favour of patients with chronic conditions , but this may concur with the moral principleprimum non noceresmall change : surgeons feel tempted to operate more , but do not give insubstantial change : the do no harm principle is reinterpreted . medical attention is no longer considered harmful per se obviously it is too early to render a final verdict on the effects of market reforms in the dutch health care system . as to the other changes , neither of these would have to create immediate problems if the costs of health care were allowed to expand . one could then just pay for the extra time spent on minor surgery and on the growing number of preventive examinations in gp practice , making sure that other patients with other conditions would not have to suffer the consequences .

psychiatric disorders are disadvantageous behavioral phenotypes that are subject to therapeutic treatments in modern human society . nevertheless , psychiatric disorders have been present in humans with constant or , recently , increased prevalence , a phenomenon that persists even after discounting for changing diagnostic criteria [ 24 ] . this raises the question as to why psychiatric disorders that are disadvantageous and lead to decreased reproductive successes in humans have not vanished but have been maintained in the process of evolution . in regard to the biological mechanisms of psychiatric disorders , two major issues should be taken into consideration . first , although psychiatric disorders are genetic and inheritable , environmental factors are often associated with their pathogenesis . in such environmental factors , stress appears to be one of the most important factors . some psychiatric disorders , such as attention deficit / hyperactivity disorder ( adhd ) and autism spectrum disorder ( asd ) , are childhood onset disorders , and , therefore , the involvement of neurodevelopmental deficits is relatively clear in these disorders . in contrast , the onsets of other psychiatric disorders , such as schizophrenia and major depressive disorder ( mdd ) , occur during adulthood in most cases . nevertheless , neurodevelopmental deficits , which could occur even before birth , have been implicated even among adult onset psychiatric disorders . in this paper , we discuss the connections between these two major issues with evolutionary perspectives . this leads to the proposal of a hypothesis that the stress - induced neurodevelopmental changes that occur during the pre- and/or early neonatal periods may be understood to be adaptation against expected postnatal stressful environments , and , therefore , psychiatric disorders associated with such prenatal stress - induced neurodevelopmental changes may have evolved and remained in humans as adaptation strategies against adverse environmental conditions . stress is an adverse environmental factor that is likely strongly associated with psychiatric disorders . for instance , stress has been shown to be involved in the onset , exacerbation , and precipitation of symptoms in schizophrenia and mdd . epidemiological studies have reported that antenatal maternal exposure to stress during pregnancy also increases the risk of developing psychiatric disorders , such as schizophrenia , asd , and adhd in offspring [ 711 ] . consistent with the strong association between stress and psychiatric disorders , stress , especially in the form of a chronic , repeated exposure , has been shown to cause an assortment of brain dysfunctions , including cognitive deficits in the working memory , long - term memory , and behavioral flexibility , as well as affective impairments , such as anhedonia , heightened anxiety , and fear conditioning . a deficit in long - term memory is one of the most investigated brain dysfunctions which is caused by chronic stress , and this deficit is observed across different species , such as rodents , nonhuman primates , and humans . indeed , long - term memory is a brain function that plays a pivotal role in the survival of many organisms as it is required for the forging of foods and avoidance of predators . thus , a deficit in long - term memory significantly endangers organism survival and reproduction . when these disadvantageous stress effects on brain function are considered from evolutionary perspectives , several questions , such as those listed below , arise : research has shown that a similar , if not identical , pattern of stress - induced long - term memory deficit is observed in rodents , nonhuman primates , and humans . because the divergence between rodents and primates is estimated to have occurred approximately 60~100 million years ago , the biological process that causes stress - induced memory impairment ( i.e. , neuroplasticity deficit ) had already been present in organisms before the divergence of rodents and primates and has been maintained for the past 100 million years , even though it appear to be a disadvantageous phenotype that could affect reproductive success.recent studies have revealed that stress induces behavioral changes and associated gene expressions through epigenetic mechanisms . moreover , such stress - induced epigenetic changes are inheritable across generations through parental gametes [ 2224 ] . thus , organisms have become equipped with biological mechanisms that can deliberately transmit stress - induced changes to descendants , with quite atypical , lamarckian - like mechanism , although stress - induced changes are thought to be disadvantageous .epidemiological studies have reported that antenatal maternal stress exposure during pregnancy increases the risk of psychiatric disorders in the offspring . consistent with these epidemiological investigations , animal studies have shown that prenatal stress causes neurodevelopmental deficits , which in turn disrupt various cognitive and affective functions after birth [ 2630 ] . a fetal brain is more plastic than an adult brain , as evidenced by the greater recovery of fetal brains following insult compared to adult brains [ 31 , 32 ] . thus , although a fetal brain is highly plastic and is in an immature state , compensation against stress - induced neurodevelopmental deficits does not seem to take place . research has shown that a similar , if not identical , pattern of stress - induced long - term memory deficit is observed in rodents , nonhuman primates , and humans . because the divergence between rodents and primates is estimated to have occurred approximately 60~100 million years ago , the biological process that causes stress - induced memory impairment ( i.e. , neuroplasticity deficit ) had already been present in organisms before the divergence of rodents and primates and has been maintained for the past 100 million years , even though it appear to be a disadvantageous recent studies have revealed that stress induces behavioral changes and associated gene expressions through epigenetic mechanisms . moreover , such stress - induced epigenetic changes are inheritable across generations through parental gametes [ 2224 ] . thus , organisms have become equipped with biological mechanisms that can deliberately transmit stress - induced changes to descendants , with quite atypical , lamarckian - like mechanism , although stress - induced changes are thought to be disadvantageous . epidemiological studies have reported that antenatal maternal stress exposure during pregnancy increases the risk of psychiatric disorders in the offspring . consistent with these epidemiological investigations , animal studies have shown that prenatal stress causes neurodevelopmental deficits , which in turn disrupt various cognitive and affective functions after birth [ 2630 ] . a fetal brain is more plastic than an adult brain , as evidenced by the greater recovery of fetal brains following insult compared to adult brains [ 31 , 32 ] . thus , although a fetal brain is highly plastic and is in an immature state , compensation against stress - induced neurodevelopmental deficits does not seem to take place . in the following sections , we further discuss these issues , along with specific focuses on ( 1 ) the interaction of prenatal and postnatal stress effects on neurodevelopment and ( 2 ) the transgenerational inheritance of stress - induced neural system alterations . the following two studies investigated prenatal stress - induced neurodevelopmental alterations in rodents to illustrate how such alterations could be understood as environmental adaptations . first , an elegant series of studies by kaiser and colleagues of the effects of prenatal chronic stress associated with social crowdedness in guinea pigs has shown that offspring exposed to a prenatal social crowdedness stress , in which dames were repeatedly placed in a cage with an exceeding number of mates during pregnancy , exhibited gender - specific behavioral and molecular changes [ 3337 ] . thus , in normal male and female animals , the expressions of androgen / estrogen receptors in limbic areas were high and low , respectively ; however , these receptor expressions decreased in the male offspring , whereas the expressions increased in the female offspring among animals exposed to prenatal social crowdedness stress . prenatal social crowdedness stress also caused behavioral alterations , such as more frequent observations of adult male offspring resting with body contact with cage mates , as well as decreased sexual motivation despite an increased nonsexually motivated ( i.e. , play ) courtship behavior . because these behaviors are usually observed in the juvenile period and wane by adulthood , the persistence of these behaviors in adult animals exposed to prenatal stress indicates that delayed maturation processes extended even into adulthood . on the other hand , female animals exposed to prenatal social crowdedness stress exhibited play behavior , which is a male - specific behavior and , therefore , unusual to be observed in females , in addition to male - like courtship behavior , indicating that the behaviors of such female animals exposed to prenatal stress become more like those of males . these alterations caused by prenatal social crowdedness , which make male animals feminized and infantilized and female animals masculine , could be understood as adaption strategies against the specific environmental condition , that is , an overpopulated society , in which animals exposed to prenatal social crowdedness stress expect to spend their postnatal life . thus , in an overly populated social group with a hierarchy , the best strategy for a male is to avoid fighting while there are many competitors and wait until the number of competitors eventually decreases , which is expected to occur given resource limitation , to gain a higher social rank within the society . in this context , in contrast , regardless of the crowdedness of the social group , a female needs to secure resources ( housing / foods ) to care for her children , for which the best adaptive strategy is to be a stronger competitor with male characteristics . of particular importance are these behavioral changes caused by prenatal stress function as adaption strategies when the postnatal environment is consistent with the prenatal environment that caused the stress . however , these behavioral alterations caused by prenatal stress result in maladaptations or deficits if the postnatal environment is inconsistent with the prenatal environment , that is , a normal stress - free condition . in fact , the idea that prenatal and postnatal environmental inconsistencies cause disease has been already proposed and is known as the barker hypothesis , as well as a thrifty phenotype , although such an idea has not yet been applied to neurodevelopment processes and the pathogenesis of psychiatric disorders . the barker hypothesis , as its name indicates , is coined by the uk medical researcher , david barker , who found that there were associations between low birth weight and the increased risks of lifestyle diseases , such as obesity , diabetes , cancer , and cardiovascular diseases in adulthood . such associations could be explained as follows : a baby may be born with a lower birth weight partly due to the insufficient nutrition of the mother during pregnancy . consequently , such a baby adapts to a low nutrition condition by decreasing their metabolism to conserve energy . however , when such a baby with a low metabolic rate is raised with normal nutrition after birth , the child will continue to conserve energy , leading to lifestyle diseases in adulthood . thus , the cause of such lifestyle diseases is a mismatch between the actual and expected environments that prenatal environmental adaptation has developed for . another study illustrated the neurodevelopmental changes associated with the prenatal environment , which could be understood as adaptations to the effects of prenatal and postnatal nutrition restriction on cortical synaptic development . for instance , in the prefrontal cortex , synaptic development has been shown to continue until adulthood , with synaptogenesis continuing throughout the juvenile period and synaptic pruning taking place in adolescence . given that cortical synaptic development is a process involving both the prenatal and postnatal periods , this process is influenced by both the prenatal and the postnatal environments . thus , consistency between the prenatal and the postnatal environments should be an important determining factor of cortical development . leuba and rabinowicz have shown that the number of dendritic spines , where excitatory synaptic contacts are made , of pyramidal neurons in the visual cortex is lower in mice that are raised in undernutritional conditions compared to mice raised in normal nutrition conditions [ 41 , 42 ] . however , when offspring born from dames that had a nutritional intake that was restricted during pregnancy are raised in an undernutritional condition , the number of dendritic spines in such offspring does not differ from that of normal mice , suggesting that the synaptic development alterations caused by prenatal undernutrition counteracts those caused by postnatal undernutrition . thus , in this case , it appears that the alteration caused by a prenatal environment works as a preparation for the expected postnatal environment , through which the prenatal environment - associated alteration is normalized . importantly , however , if a postnatal environment is not matched with the condition expected from the prenatal environment , such a prenatal environment - associated alteration results in maladaptation or a deficit . we have recently investigated the effects of prenatal and postnatal restraint stress interactions on spatial memory in mice . there have already been many studies showing that prenatal restraint stress causes spatial memory impairments . in addition , adult rodents subjected to chronic restraint stress have similar , if not identical , spatial memory deficits . however , it is still unknown how the prenatal and postnatal stress interaction ( prenatal and postnatal environmental consistency ) affects spatial memory . a preliminary result in our study indicates that when offspring born from dames exposed to prenatal restraint stress are exposed to 2 weeks of chronic restraint stress again during the juvenile period , these mice exhibit spatial memory comparable to that of normal mice , suggesting that matching between the prenatal and postnatal environments results in the normalization of the spatial memory function . collectively , this evidence suggests that there are two different types of adaptation processes associated with prenatal environmental conditions . one is an adaptive plasticity change in which an alteration associated with a prenatal environment may work better in a postnatal environment if matched with the prenatal environment . the other is a preparatory plasticity change , in which an alteration associated with a prenatal environment may be normalized when the postnatal environment matches the prenatal environment . epidemiological studies have shown that adverse antenatal maternal environmental conditions , such as stress , virus infection , and malnutrition , during pregnancy increase the risks of the offspring developing psychiatric disorders , such as schizophrenia , mdd , adhd , and asd [ 711 ] . considering that neurodevelopmental changes induced by prenatal environmental factors , such as stress , may be adaptions to the expected postnatal stressful environment , neurodevelopmental changes that increase the risks of psychiatric disorders could also be understood as environmental adaptation strategies against specific environments that are the sources of stress to organisms . thus , some behavioral traits that are associated with psychiatric disorders could be understood to be either preparatory or adaptive changes to cope with expected postnatal environments . psychiatric symptoms that are already apparent in childhood , such as those of adhd and asd , may be understood to be adaptive changes , such that adhd and asd symptoms may be expressed regardless of the consistency between the prenatal and postnatal environmental conditions . expressions of behavioral phenotypes associated with these disorders at early ages are crucially important for survival and reproductive success in animals in a wild environment and a hunter - gather society in case of humans . adhd symptoms consist of hyperactivity , inattention , and impulsivity , all of which facilitate survival in a prey - predator interaction , with hyperactivity and inattention enabling exploration and scanning of predators in wider area and impulsivity leading to quick decision for escape from predators . in addition , individuals with asd have been suggested to have similar behavioral characteristics to those of animals , although whether cognitive processes are also similar between asd individuals and animals is still under debate . thus , subjects with asd symptoms are expected to have more successful survival and reproduction in wild environments than normal subjects . one example of such cases may be victor of aveyron , who was a boy found in the forests of southern france more than 200 years ago . a description by the medical doctor of behavioral characteristics of this boy who was living in the wild environment has been suggested to resemble asd symptoms [ 4749 ] . there are also psychiatric conditions with delayed onsets , such as schizophrenia and mdd , which typically emerge in adolescence and adulthood and may be understood to be preparatory changes , such that expressions of the disorders are due to mismatches between the prenatal and postnatal environments . expressions of symptoms of these disorders in a normal environment are mostly disadvantages for successful reproduction . in contrast , in an adverse environment , normal subjects have been shown to exhibit brain dysfunction . thus , any strategy that maintains brain function normal in such an adverse environment could yield a higher reproductive success . in this context , delayed expressions of symptoms of these psychiatric disorders in early adulthood are also advantageous for reproduction to determine whether these phenotypes should be maintained or eliminated , depending on whether an environment is adverse or normal at the time of reproduction . considering the evolutionary aspect of stress - induced behavioral and neuronal changes , the most important issue is that such stress - induced changes have to be inheritable . first , the inheritance of environmentally induced changes is not compatible with darwinian evolution ( natural selection ) [ 50 , 51 ] . in contrast , such inheritance is consistent with the evolutionary theory proposed by lamarck , which has been rejected by the majority of evolutionary biologists . second , the inheritance of deficits , which are disadvantageous for reproduction success , contradicts evolutionary theory , as evolution should be accompanied by successful reproduction . regarding the later issue , as we have discussed in the previous section , there is accumulating evidence that leads us to suggest that stress - induced changes may not necessarily be deficits but could be advantageous adaptive strategies depending on the environmental context . in fact , the finding that stress - induced behavioral changes could be inherited by descendants was previously reported in 1970 . in this study , wehmer and colleagues found that stress administered before and during pregnancy in dames caused behavioral alterations , such as heightened anxiety in the offspring . moreover , such alterations were transgenerationally inherited into grandoffspring that did not experience stress at all . even before this report thus , he demonstrated that crossveinless induced by heat in fruit flies was inherited by descendants through an unidentified mechanism that incorporated the phenotypic change into a genetic mutation , which is now known as genetic assimilation . more recently , instances of transgenerational inheritance of environmentally induced behavioral , physiological , and neuronal alterations in both animals and humans have been documented . a number of review articles of these findings are already available [ 22 , 5458 ] . accumulating evidence suggests that epigenetic regulation of gene expression through mechanisms such as histone acetylation and dna methylation is the molecular mechanisms that mediate the transgenerational inheritance of environmentally induced behavioral , physiological , and neuronal alterations [ 56 , 59 ] . these biochemical modifications of dna have been shown to be transmitted into descendants through paternal and/or maternal gametes . accordingly , the transgenerational inheritance of epigenetic - based alterations should involve gamete - specific gene expression , that is , genomic imprinting [ 6062 ] . it is interesting to note that the genomic imprinting of several candidate genes associated with psychiatric disorders has been reported . most studies that have investigated transgenerational epigenetic inheritance in mammals follow its inheritance for a few generations ; it remains unclear whether the biochemical processes , such as histone modification of chromatin structures and dna methylation , could be maintained across additional generations . nonetheless , transgenerational epigenetic inheritance has also been shown to play a significant role in the domestication of animals , such as the white leghorn , which is domesticated from a red jungle fowl , suggesting that transgenerational epigenetic inheritance could be a sustainable mechanism in many generations . psychiatric disorders have strong genetic backgrounds , including genetic mutations and variations ( e.g. , copy number variations and single nucleotide polymorphisms ) . thus , even if the epigenetic bases of environmentally induced changes are transgenerational , they require a mechanism for translating epigenetic changes into equivalent genetic changes to demonstrate that this mechanism is involved in the causes of psychiatric disorders . one speculative explanation is that epigenetic processes may somehow promote genetic recombination , which in turn leads to genetic mutations and variants associated with psychiatric disorders . such a link between epigenetic changes and genetic mutations may eventually prove that the mechanisms of the onset of psychiatric disorders often require gene x environment interactions [ 65 , 66 ] . in this regard , psychiatric disorders may also involve mechanisms similar to those of phenotypic plasticity . when considering the transgenerational inheritance of stress - induced behavioral and neuronal alterations , it is particularly important to note that stress - induced changes are not uniform but are variable and dependent on the environments that generate stress . this is supported by a number of findings , such as the fact that chronic restraint , but not unpredictable stress , causes heightened anxiety and remodeling of the neural network in the basolateral amygdala in a gender - specific manner [ 68 , 69 ] . moreover , prenatal restraint stress has been shown to cause a spatial memory impairment , whereas other prenatal stress procedures or prenatal administration of the synthetic stress hormone , dexamethasone , do not cause a spatial memory impairment in offspring . indeed , restraint stress is accompanied by the restriction of spatial information while animals are placed in restrainers . in animal studies , physical stress procedures , such as restraint , elevated platforms , cold , pain ( e.g. , foot - shock ) , and unpredictable ( a mixture of different stressors administered each day ) stress , have been frequently utilized . in addition , social stress procedures , such as social defeat , crowdedness , and isolation , have also been commonly employed . there has been no systematic investigation to compare the behavioral and neuronal alterations caused by different stressors , and , consequently , the studies that have investigated stress effects quite often assume that stress - induced alterations caused by different stressors are similar or identical . indeed however , such an increase of stress hormones could be a signal for adaptation , and how the system alters may depend on the specific stressful environment to which the system was exposed . collectively , it is plausible that stress - induced changes may consist of heritable and nonheritable components , of which inheritable stress - induced changes may be understood to be environmental adaptation strategies ( figure 1 ) . in this regard , unpredictable stress is remarkable in that the stressful environments change daily such that there is no particular environment for adaptation . this stress procedure has been frequently used in animal research , as habituation to stress environments can be minimized by this procedure . thus , a comparison of the transgenerational inheritance of stress - induced changes between unpredictable stress and other stress procedures would be a promising direction of investigation . based on the above discussions , psychiatric disorders may be understood to be an array of behavioral traits that have emerged as environmental adaptation against adverse stressful environments in the evolution of organisms . environment , they may enable higher survival and reproductive rates in adverse environments . thus , psychiatric disorders may have been present in humans and possibly other organisms as reservoirs to prevent the extinction of species against severe and stressful environments which endanger their survival and reproduction . this may be better intuitively understood by considering the model illustrated in figure 2 . when a specific behavioral trait or brain function is considered within a population , an exceeding or insufficient function distributed at the extreme ends of the distribution may correspond to psychiatric conditions in the normal environment ( figure 2(a ) ) . on the other hand , in a severe and stressful environment , some populations with over- or underfunctioning in the distribution in the normal environment may no longer be considered abnormal ( figure 2(b ) ) . these adaptive phenotypes associated with psychiatric disorders may not be selected over time and , therefore , may not become common traits among populations , since a severe , stressful event may not last for sufficiently long time or happen only in a microenvironment . for this model first , psychiatric disorders are continuous , but not discrete , deviants from normal conditions . empirical evidence supports the continuous relationships of psychiatric symptoms , such as psychosis , externalizing behaviors , and autistic traits , with normal conditions . second , if psychiatric disorders are the extreme ends of normality , there would always be one extreme end of a psychiatric condition that corresponds to the other extreme ends ( e.g. , hyper- versus hypofunction ) . thus , there should be pairs of psychiatric conditions that have oppositional relationships with each other ( figure 3 ) . in fact , there are several diagnostically distinct psychiatric disorders that appear to have such diametrical ( oppositional ) phenotypic relationships with each other . crespi and colleagues have shown that schizophrenia and asd are one such case of a diametrical relationship , as evidenced by the overlapping candidate genes suggested in both schizophrenia and asd , although the copy number variations in several of these genes are opposite , with copy number expansions in one disorder and deletions in the other . we have also recently suggested that mdd and adhd may have some diametrical relationships in several of their phenotypes ( psychomotor retardation versus hyperactivity , thought suppression versus impulsivity , and rumination versus inattention ) , which may be associated with hyper- and hypoactivity , respectively , of the habenula , one of the key brain regions regulating monoamine transmission in mdd and adhd , respectively . these arguments also inevitably predict that although , in the current clinical situation , each psychiatric disorder has been categorized , they may not be biologically discrete conditions but rather spectrums . in this paper , we have explained , using the supporting literature , that there are three emerging psychiatric disorder issues that can be viewed from an evolutionary perspective . these issues are as follows : ( 1 ) some behavioral traits associated with psychiatric disorders may work beneficially in specific stressful environments that are otherwise difficult for organisms to survive and maintain reproductive success in ; ( 2 ) behavioral and associated neuronal changes caused by prenatal stressful environments could be adaptive strategies against postnatal environments that are expected to be stressful ; and ( 3 ) some , but not all , behavioral and associated neuronal changes caused by prenatal and postnatal stressful environments could be inheritable . collectively , a hypothesis that psychiatric disorders may have emerged as adaptation strategies against adverse environmental conditions in the evolution of organisms is formulated . thus , psychiatric conditions may be a strategy for creating biodiversity to maintain the species in case unusual environmental changes occur . for instance , although psychiatric disorders involve genetic mutations , no empirical evidence to date has documented the mechanisms that translate epigenetic changes into equivalent genetic mutations . nevertheless , our hypothesis may also yield several novel insights into our understanding of psychiatric disorders , such as ( 1 ) why stress is often associated with the onset , relapse , and exacerbation of symptoms in several psychiatric disorders ; ( 2 ) why the onset of symptoms in some psychiatric disorders is apparent early in development , whereas others have delayed onset in adulthood ; ( 3 ) why psychiatric disorders may not be discrete conditions from the normal but continuous extremes ; and ( 4 ) why symptoms of different psychiatric disorders may have oppositional relationships , which in turn suggests that psychiatric disorders could exist on a spectrum and are not categorical . indeed , the concept proposed here is highly speculative at this moment , and investigations to validate , modify , or reject the concept have been awaited . for instance , higher incidence of schizophrenia has been shown to be associated with urbanicity [ 75 , 76 ] . although which aspects of environmental factors in the urbanicity are involved and whether either urban birth or urban residence or both of them are involved in such higher prevalence of the disorder have been still unclear , this observation appears to be somewhat controversial to the proposed concept . further epidemiological studies such as those investigating an incident rate of schizophrenia in subjects born in an urban city and upbringing in a rural area and vice versa would yield more insights on this issue . moreover , another major limitation of the concept also includes how these environmental adaptation processes could interact with genetic predispositions of psychiatric disorders which has also remained unclear at this moment . future investigations of the molecular and neural network changes associated with stress under evolutionary perspectives would be able to further our understanding of the biological mechanisms and therapeutic treatments of psychiatric disorders .

psychiatric disorders are disadvantageous behavioral phenotypes in humans . accordingly , a recent epidemiological study has reported decreased fecundity in patients with psychiatric disorders , such as schizophrenia and autism spectrum disorders . moreover , the fecundity of the relatives of these patients is not exceedingly higher compared to the fecundity of the relatives of normal subjects . collectively , the prevalence of psychiatric disorders among humans is expected to decrease over generations . nevertheless , in reality , the prevalence rates of psychiatric disorders in humans either have been constant over a long period of time or have even increased more recently . several attempts to explain this fact have been made using biological mechanisms , such as de novo gene mutations or variants , although none of these explanations is fully comprehensive . here , we propose a hypothesis towards understanding the biological mechanisms of psychiatric disorders from evolutionary perspectives . this hypothesis considers that behavioral phenotypes associated with psychiatric disorders might have emerged in the evolution of organisms as a neurodevelopmental adaptation against adverse environmental conditions associated with stress .

1. Introduction 2. Stress Effects on Brain Function from an Evolutionary Perspective 3. Prenatal Stress-Induced Neurodevelopmental Alterations as Environmental Adaptations 4. Inheritance of Stress-Induced Alterations 5. Understanding Psychiatric Disorders as Environmental Adaptation 6. Conclusions

psychiatric disorders are disadvantageous behavioral phenotypes that are subject to therapeutic treatments in modern human society . nevertheless , psychiatric disorders have been present in humans with constant or , recently , increased prevalence , a phenomenon that persists even after discounting for changing diagnostic criteria [ 24 ] . this raises the question as to why psychiatric disorders that are disadvantageous and lead to decreased reproductive successes in humans have not vanished but have been maintained in the process of evolution . in regard to the biological mechanisms of psychiatric disorders , two major issues should be taken into consideration . first , although psychiatric disorders are genetic and inheritable , environmental factors are often associated with their pathogenesis . in such environmental factors , stress appears to be one of the most important factors . some psychiatric disorders , such as attention deficit / hyperactivity disorder ( adhd ) and autism spectrum disorder ( asd ) , are childhood onset disorders , and , therefore , the involvement of neurodevelopmental deficits is relatively clear in these disorders . in contrast , the onsets of other psychiatric disorders , such as schizophrenia and major depressive disorder ( mdd ) , occur during adulthood in most cases . nevertheless , neurodevelopmental deficits , which could occur even before birth , have been implicated even among adult onset psychiatric disorders . in this paper , we discuss the connections between these two major issues with evolutionary perspectives . this leads to the proposal of a hypothesis that the stress - induced neurodevelopmental changes that occur during the pre- and/or early neonatal periods may be understood to be adaptation against expected postnatal stressful environments , and , therefore , psychiatric disorders associated with such prenatal stress - induced neurodevelopmental changes may have evolved and remained in humans as adaptation strategies against adverse environmental conditions . stress is an adverse environmental factor that is likely strongly associated with psychiatric disorders . for instance , stress has been shown to be involved in the onset , exacerbation , and precipitation of symptoms in schizophrenia and mdd . epidemiological studies have reported that antenatal maternal exposure to stress during pregnancy also increases the risk of developing psychiatric disorders , such as schizophrenia , asd , and adhd in offspring [ 711 ] . consistent with the strong association between stress and psychiatric disorders , stress , especially in the form of a chronic , repeated exposure , has been shown to cause an assortment of brain dysfunctions , including cognitive deficits in the working memory , long - term memory , and behavioral flexibility , as well as affective impairments , such as anhedonia , heightened anxiety , and fear conditioning . a deficit in long - term memory is one of the most investigated brain dysfunctions which is caused by chronic stress , and this deficit is observed across different species , such as rodents , nonhuman primates , and humans . indeed , long - term memory is a brain function that plays a pivotal role in the survival of many organisms as it is required for the forging of foods and avoidance of predators . when these disadvantageous stress effects on brain function are considered from evolutionary perspectives , several questions , such as those listed below , arise : research has shown that a similar , if not identical , pattern of stress - induced long - term memory deficit is observed in rodents , nonhuman primates , and humans . because the divergence between rodents and primates is estimated to have occurred approximately 60~100 million years ago , the biological process that causes stress - induced memory impairment ( i.e. moreover , such stress - induced epigenetic changes are inheritable across generations through parental gametes [ 2224 ] . thus , organisms have become equipped with biological mechanisms that can deliberately transmit stress - induced changes to descendants , with quite atypical , lamarckian - like mechanism , although stress - induced changes are thought to be disadvantageous .epidemiological studies have reported that antenatal maternal stress exposure during pregnancy increases the risk of psychiatric disorders in the offspring . thus , although a fetal brain is highly plastic and is in an immature state , compensation against stress - induced neurodevelopmental deficits does not seem to take place . because the divergence between rodents and primates is estimated to have occurred approximately 60~100 million years ago , the biological process that causes stress - induced memory impairment ( i.e. moreover , such stress - induced epigenetic changes are inheritable across generations through parental gametes [ 2224 ] . thus , organisms have become equipped with biological mechanisms that can deliberately transmit stress - induced changes to descendants , with quite atypical , lamarckian - like mechanism , although stress - induced changes are thought to be disadvantageous . epidemiological studies have reported that antenatal maternal stress exposure during pregnancy increases the risk of psychiatric disorders in the offspring . thus , although a fetal brain is highly plastic and is in an immature state , compensation against stress - induced neurodevelopmental deficits does not seem to take place . in the following sections , we further discuss these issues , along with specific focuses on ( 1 ) the interaction of prenatal and postnatal stress effects on neurodevelopment and ( 2 ) the transgenerational inheritance of stress - induced neural system alterations . first , an elegant series of studies by kaiser and colleagues of the effects of prenatal chronic stress associated with social crowdedness in guinea pigs has shown that offspring exposed to a prenatal social crowdedness stress , in which dames were repeatedly placed in a cage with an exceeding number of mates during pregnancy , exhibited gender - specific behavioral and molecular changes [ 3337 ] . thus , in normal male and female animals , the expressions of androgen / estrogen receptors in limbic areas were high and low , respectively ; however , these receptor expressions decreased in the male offspring , whereas the expressions increased in the female offspring among animals exposed to prenatal social crowdedness stress . prenatal social crowdedness stress also caused behavioral alterations , such as more frequent observations of adult male offspring resting with body contact with cage mates , as well as decreased sexual motivation despite an increased nonsexually motivated ( i.e. because these behaviors are usually observed in the juvenile period and wane by adulthood , the persistence of these behaviors in adult animals exposed to prenatal stress indicates that delayed maturation processes extended even into adulthood . thus , in an overly populated social group with a hierarchy , the best strategy for a male is to avoid fighting while there are many competitors and wait until the number of competitors eventually decreases , which is expected to occur given resource limitation , to gain a higher social rank within the society . in this context , in contrast , regardless of the crowdedness of the social group , a female needs to secure resources ( housing / foods ) to care for her children , for which the best adaptive strategy is to be a stronger competitor with male characteristics . in fact , the idea that prenatal and postnatal environmental inconsistencies cause disease has been already proposed and is known as the barker hypothesis , as well as a thrifty phenotype , although such an idea has not yet been applied to neurodevelopment processes and the pathogenesis of psychiatric disorders . the barker hypothesis , as its name indicates , is coined by the uk medical researcher , david barker , who found that there were associations between low birth weight and the increased risks of lifestyle diseases , such as obesity , diabetes , cancer , and cardiovascular diseases in adulthood . such associations could be explained as follows : a baby may be born with a lower birth weight partly due to the insufficient nutrition of the mother during pregnancy . consequently , such a baby adapts to a low nutrition condition by decreasing their metabolism to conserve energy . another study illustrated the neurodevelopmental changes associated with the prenatal environment , which could be understood as adaptations to the effects of prenatal and postnatal nutrition restriction on cortical synaptic development . for instance , in the prefrontal cortex , synaptic development has been shown to continue until adulthood , with synaptogenesis continuing throughout the juvenile period and synaptic pruning taking place in adolescence . leuba and rabinowicz have shown that the number of dendritic spines , where excitatory synaptic contacts are made , of pyramidal neurons in the visual cortex is lower in mice that are raised in undernutritional conditions compared to mice raised in normal nutrition conditions [ 41 , 42 ] . however , when offspring born from dames that had a nutritional intake that was restricted during pregnancy are raised in an undernutritional condition , the number of dendritic spines in such offspring does not differ from that of normal mice , suggesting that the synaptic development alterations caused by prenatal undernutrition counteracts those caused by postnatal undernutrition . thus , in this case , it appears that the alteration caused by a prenatal environment works as a preparation for the expected postnatal environment , through which the prenatal environment - associated alteration is normalized . importantly , however , if a postnatal environment is not matched with the condition expected from the prenatal environment , such a prenatal environment - associated alteration results in maladaptation or a deficit . a preliminary result in our study indicates that when offspring born from dames exposed to prenatal restraint stress are exposed to 2 weeks of chronic restraint stress again during the juvenile period , these mice exhibit spatial memory comparable to that of normal mice , suggesting that matching between the prenatal and postnatal environments results in the normalization of the spatial memory function . collectively , this evidence suggests that there are two different types of adaptation processes associated with prenatal environmental conditions . the other is a preparatory plasticity change , in which an alteration associated with a prenatal environment may be normalized when the postnatal environment matches the prenatal environment . epidemiological studies have shown that adverse antenatal maternal environmental conditions , such as stress , virus infection , and malnutrition , during pregnancy increase the risks of the offspring developing psychiatric disorders , such as schizophrenia , mdd , adhd , and asd [ 711 ] . considering that neurodevelopmental changes induced by prenatal environmental factors , such as stress , may be adaptions to the expected postnatal stressful environment , neurodevelopmental changes that increase the risks of psychiatric disorders could also be understood as environmental adaptation strategies against specific environments that are the sources of stress to organisms . thus , some behavioral traits that are associated with psychiatric disorders could be understood to be either preparatory or adaptive changes to cope with expected postnatal environments . psychiatric symptoms that are already apparent in childhood , such as those of adhd and asd , may be understood to be adaptive changes , such that adhd and asd symptoms may be expressed regardless of the consistency between the prenatal and postnatal environmental conditions . expressions of behavioral phenotypes associated with these disorders at early ages are crucially important for survival and reproductive success in animals in a wild environment and a hunter - gather society in case of humans . in addition , individuals with asd have been suggested to have similar behavioral characteristics to those of animals , although whether cognitive processes are also similar between asd individuals and animals is still under debate . thus , subjects with asd symptoms are expected to have more successful survival and reproduction in wild environments than normal subjects . one example of such cases may be victor of aveyron , who was a boy found in the forests of southern france more than 200 years ago . there are also psychiatric conditions with delayed onsets , such as schizophrenia and mdd , which typically emerge in adolescence and adulthood and may be understood to be preparatory changes , such that expressions of the disorders are due to mismatches between the prenatal and postnatal environments . expressions of symptoms of these disorders in a normal environment are mostly disadvantages for successful reproduction . in contrast , in an adverse environment , normal subjects have been shown to exhibit brain dysfunction . in this context , delayed expressions of symptoms of these psychiatric disorders in early adulthood are also advantageous for reproduction to determine whether these phenotypes should be maintained or eliminated , depending on whether an environment is adverse or normal at the time of reproduction . considering the evolutionary aspect of stress - induced behavioral and neuronal changes , the most important issue is that such stress - induced changes have to be inheritable . first , the inheritance of environmentally induced changes is not compatible with darwinian evolution ( natural selection ) [ 50 , 51 ] . second , the inheritance of deficits , which are disadvantageous for reproduction success , contradicts evolutionary theory , as evolution should be accompanied by successful reproduction . regarding the later issue , as we have discussed in the previous section , there is accumulating evidence that leads us to suggest that stress - induced changes may not necessarily be deficits but could be advantageous adaptive strategies depending on the environmental context . in fact , the finding that stress - induced behavioral changes could be inherited by descendants was previously reported in 1970 . in this study , wehmer and colleagues found that stress administered before and during pregnancy in dames caused behavioral alterations , such as heightened anxiety in the offspring . moreover , such alterations were transgenerationally inherited into grandoffspring that did not experience stress at all . more recently , instances of transgenerational inheritance of environmentally induced behavioral , physiological , and neuronal alterations in both animals and humans have been documented . a number of review articles of these findings are already available [ 22 , 5458 ] . accordingly , the transgenerational inheritance of epigenetic - based alterations should involve gamete - specific gene expression , that is , genomic imprinting [ 6062 ] . it is interesting to note that the genomic imprinting of several candidate genes associated with psychiatric disorders has been reported . most studies that have investigated transgenerational epigenetic inheritance in mammals follow its inheritance for a few generations ; it remains unclear whether the biochemical processes , such as histone modification of chromatin structures and dna methylation , could be maintained across additional generations . nonetheless , transgenerational epigenetic inheritance has also been shown to play a significant role in the domestication of animals , such as the white leghorn , which is domesticated from a red jungle fowl , suggesting that transgenerational epigenetic inheritance could be a sustainable mechanism in many generations . thus , even if the epigenetic bases of environmentally induced changes are transgenerational , they require a mechanism for translating epigenetic changes into equivalent genetic changes to demonstrate that this mechanism is involved in the causes of psychiatric disorders . one speculative explanation is that epigenetic processes may somehow promote genetic recombination , which in turn leads to genetic mutations and variants associated with psychiatric disorders . such a link between epigenetic changes and genetic mutations may eventually prove that the mechanisms of the onset of psychiatric disorders often require gene x environment interactions [ 65 , 66 ] . this is supported by a number of findings , such as the fact that chronic restraint , but not unpredictable stress , causes heightened anxiety and remodeling of the neural network in the basolateral amygdala in a gender - specific manner [ 68 , 69 ] . moreover , prenatal restraint stress has been shown to cause a spatial memory impairment , whereas other prenatal stress procedures or prenatal administration of the synthetic stress hormone , dexamethasone , do not cause a spatial memory impairment in offspring . in animal studies , physical stress procedures , such as restraint , elevated platforms , cold , pain ( e.g. in addition , social stress procedures , such as social defeat , crowdedness , and isolation , have also been commonly employed . there has been no systematic investigation to compare the behavioral and neuronal alterations caused by different stressors , and , consequently , the studies that have investigated stress effects quite often assume that stress - induced alterations caused by different stressors are similar or identical . thus , a comparison of the transgenerational inheritance of stress - induced changes between unpredictable stress and other stress procedures would be a promising direction of investigation . based on the above discussions , psychiatric disorders may be understood to be an array of behavioral traits that have emerged as environmental adaptation against adverse stressful environments in the evolution of organisms . thus , psychiatric disorders may have been present in humans and possibly other organisms as reservoirs to prevent the extinction of species against severe and stressful environments which endanger their survival and reproduction . when a specific behavioral trait or brain function is considered within a population , an exceeding or insufficient function distributed at the extreme ends of the distribution may correspond to psychiatric conditions in the normal environment ( figure 2(a ) ) . on the other hand , in a severe and stressful environment , some populations with over- or underfunctioning in the distribution in the normal environment may no longer be considered abnormal ( figure 2(b ) ) . these adaptive phenotypes associated with psychiatric disorders may not be selected over time and , therefore , may not become common traits among populations , since a severe , stressful event may not last for sufficiently long time or happen only in a microenvironment . for this model first , psychiatric disorders are continuous , but not discrete , deviants from normal conditions . empirical evidence supports the continuous relationships of psychiatric symptoms , such as psychosis , externalizing behaviors , and autistic traits , with normal conditions . second , if psychiatric disorders are the extreme ends of normality , there would always be one extreme end of a psychiatric condition that corresponds to the other extreme ends ( e.g. in fact , there are several diagnostically distinct psychiatric disorders that appear to have such diametrical ( oppositional ) phenotypic relationships with each other . crespi and colleagues have shown that schizophrenia and asd are one such case of a diametrical relationship , as evidenced by the overlapping candidate genes suggested in both schizophrenia and asd , although the copy number variations in several of these genes are opposite , with copy number expansions in one disorder and deletions in the other . we have also recently suggested that mdd and adhd may have some diametrical relationships in several of their phenotypes ( psychomotor retardation versus hyperactivity , thought suppression versus impulsivity , and rumination versus inattention ) , which may be associated with hyper- and hypoactivity , respectively , of the habenula , one of the key brain regions regulating monoamine transmission in mdd and adhd , respectively . these arguments also inevitably predict that although , in the current clinical situation , each psychiatric disorder has been categorized , they may not be biologically discrete conditions but rather spectrums . these issues are as follows : ( 1 ) some behavioral traits associated with psychiatric disorders may work beneficially in specific stressful environments that are otherwise difficult for organisms to survive and maintain reproductive success in ; ( 2 ) behavioral and associated neuronal changes caused by prenatal stressful environments could be adaptive strategies against postnatal environments that are expected to be stressful ; and ( 3 ) some , but not all , behavioral and associated neuronal changes caused by prenatal and postnatal stressful environments could be inheritable . collectively , a hypothesis that psychiatric disorders may have emerged as adaptation strategies against adverse environmental conditions in the evolution of organisms is formulated . for instance , although psychiatric disorders involve genetic mutations , no empirical evidence to date has documented the mechanisms that translate epigenetic changes into equivalent genetic mutations . nevertheless , our hypothesis may also yield several novel insights into our understanding of psychiatric disorders , such as ( 1 ) why stress is often associated with the onset , relapse , and exacerbation of symptoms in several psychiatric disorders ; ( 2 ) why the onset of symptoms in some psychiatric disorders is apparent early in development , whereas others have delayed onset in adulthood ; ( 3 ) why psychiatric disorders may not be discrete conditions from the normal but continuous extremes ; and ( 4 ) why symptoms of different psychiatric disorders may have oppositional relationships , which in turn suggests that psychiatric disorders could exist on a spectrum and are not categorical . indeed , the concept proposed here is highly speculative at this moment , and investigations to validate , modify , or reject the concept have been awaited . for instance , higher incidence of schizophrenia has been shown to be associated with urbanicity [ 75 , 76 ] . although which aspects of environmental factors in the urbanicity are involved and whether either urban birth or urban residence or both of them are involved in such higher prevalence of the disorder have been still unclear , this observation appears to be somewhat controversial to the proposed concept . further epidemiological studies such as those investigating an incident rate of schizophrenia in subjects born in an urban city and upbringing in a rural area and vice versa would yield more insights on this issue . moreover , another major limitation of the concept also includes how these environmental adaptation processes could interact with genetic predispositions of psychiatric disorders which has also remained unclear at this moment . future investigations of the molecular and neural network changes associated with stress under evolutionary perspectives would be able to further our understanding of the biological mechanisms and therapeutic treatments of psychiatric disorders .

this epidemic of overweight and obesity has major implications , since both overweight and obesity are independently associated with increased mortality [ 35 ] . adipose tissue is not metabolically inert but synthesizes and secretes proinflammatory mediators , including interleukin 6 ( il-6 ) [ 69 ] , tumor necrosis factor ( tnf ) [ 7 , 10 , 11 ] , il-8 , and monocyte chemoattractant [ protein 1 ] ( mcp-1 ) [ 12 , 13 ] . several studies reported that high sensitivity c - reactive protein levels are elevated in overweight and obese patients [ 1426 ] . compared with lean subjects , overweight and obese patients also have higher levels of il-6 [ 9 , 19 , 24 , 2628 ] , tnf [ 24 , 2630 ] , mcp-1 , as well as of other markers of inflammation , including il-8 [ 31 , 32 ] , il-18 , soluble tnf receptor-2 ( stnfr2 ) [ 27 , 34 , 35 ] , soluble e - selectin [ 26 , 36 , 37 ] , soluble intercellular adhesion molecule-1 ( sicam-1 ) [ 18 , 26 , 27 , 34 , 3638 ] , and soluble vascular cell adhesion molecule-1 ( svcam-1 ) [ 26 , 36 ] . in obese patients , inflammatory markers correlate with visceral adipose tissue mass [ 20 , 21 , 27 , 33 , 34 , 3941 ] and with waist circumference , an index of visceral adiposity [ 17 , 2123 , 27 , 32 , 35 , 39 ] . the activation of inflammation that is present in obesity might contribute to the increased vascular morbidity and mortality of these patients , since elevated levels of inflammatory markers are associated with increased vascular risk [ 4246 ] . in this review , we summarize the effects of lifestyle changes , antiobesity agents , and bariatric surgery on serological inflammatory markers in obese patients . lifestyle measures are the cornerstone of the management of obesity . in randomized controlled studies , diet and exercise reduced the risk of type 2 diabetes mellitus ( t2 dm ) in overweight patients with impaired glucose tolerance ( igt ) [ 48 , 49 ] . in observational studies , weight loss achieved with lifestyle changes was associated with reduced incidence of coronary heart disease ( chd ) and with lower all - cause mortality . several studies evaluated the effects of diet and exercise alone or in combination on inflammatory markers in obese patients . several small studies ( n = 11100 ) assessed the effects of low - calorie , very low - calorie , low - fat , or low - carbohydrate diets on inflammatory markers in obese patients ( table 1 ) [ 20 , 22 , 28 , 5275 ] . the duration of the intervention ranged from 3 weeks to 17 months [ 20 , 22 , 28 , 5275 ] . the majority of studies noted a significant decrease in crp after weight loss induced by dietary modification in otherwise healthy overweight or obese adults [ 20 , 22 , 52 , 54 , 5660 , 62 , 6468 , 70 , 7275 ] , in obese patients with the metabolic syndrome , in overweight patients with raised triglycerides ( tg ) , in severely obese patients , or in obese patients with t2 dm . in some studies , the magnitude of decrease in hscrp levels was linearly correlated to the amount of weight loss [ 20 , 22 , 67 ] . only few reports did not record a significant fall in hscrp concentration after weight loss [ 28 , 63 ] . furthermore , several studies compared the effects of different diets on inflammatory markers [ 5254 , 56 , 57 , 59 , 62 , 6568 , 7073 ] . reported a significant 24% decrease in serum hscrp levels after 3 months of replacement of high - fat foods with reduced - fat alternatives . in another study , a carbohydrate restricted diet ( crd ) with daily intake of eggs decreased hscrp levels compared with a crd without eggs , indicating that eggs significantly contribute to the anti - inflammatory effects of crd possibly due to the antioxidant lutein which is present in eggs and modulates certain inflammatory responses . a low - glycemic - load diet was also associated with a significantly greater improvement in serum hscrp concentration compared with a low - fat diet ( 48 versus 5% ) despite the similar weight loss in both diets . in contrast , a more recent study reported a greater fall in hscrp levels after a high - carbohydrate , low - saturated - fat diet compared with an isocaloric very - low - carbohydrate , high - saturated - fat diet , even though the latter group lost more weight and abdominal fat . however , most studies comparing diets of different composition reported a similar decrease in hscrp concentration among groups , suggesting that the weight loss per se rather than the dietary composition is the primary determinant of the fall in inflammatory markers during diet [ 5254 , 56 , 57 , 6567 , 7173 ] . some studies reported a reduction in serum tnf levels with diet [ 31 , 72 , 74 , 76 , 77 ] while others have not [ 28 , 70 , 75 ] . similarly , serum il-6 levels fell after diet - induced weight loss in some [ 28 , 31 , 72 , 76 ] , but not all studies [ 60 , 74 ] . regarding the effects of diet on il-8 , a significant reduction , increase , or no change has been reported . in addition , caloric restriction did not affect circulating levels of the anti - inflammatory cytokine il-10 . however , limited data suggest that diet - induced weight loss is associated with a proportional reduction in il-18 and il-20 levels . weight loss by caloric restriction also induces significant decreases in sicam-1 [ 36 , 52 , 55 , 61 , 62 , 72 ] and svcam-1 levels [ 36 , 61 , 80 ] . only one study reported no change in sicam-1 and svcam-1 levels and another study reported an increase in svcam-1 levels . the conflicting results mentioned above could be attributed to the differences in baseline characteristics of the studied population that might influence inflammatory markers ( i.e. , age , gender and coexisting diseases ) , the brief duration of the intervention , the insufficient sample size , or the degree of body weight reduction . overall , the greatest reductions in serological markers of inflammation were observed in studies where an at least 10% weight loss was achieved . interestingly , recent studies demonstrated that brief periods of reduced food intake can increase resistance to ischemia reperfusion injury in rodents and modify circulating levels of pro - inflammatory agents in humans [ 81 , 82 ] . the effects of short - term interventions on inflammation appear to be mostly due to the calorie restriction and negative energy balance whereas in long - term interventions weight loss and adipose mass reduction also contribute to the suppression of inflammation . however , existing studies do not show a major difference between the effects of short and long terms of calorie restriction on the levels of inflammatory markers . the 2 studies that did not report an effect of diet on hscrp levels lasted for 3 weeks and 3 months , respectively [ 28 , 63 ] . in addition , both short- and long - lasting dietary interventions ( 3 weeks to 17 months ) induced a reduction in hscrp levels in several studies [ 20 , 22 , 5260 , 62 , 6475 ] . a number of small studies ( n = 8199 ) evaluated the effects of exercise training , resistance training , or aerobic exercise training for 212 months on inflammatory markers ( table 1 ) [ 35 , 8393 ] . some [ 8387 ] but not all [ 8890 ] studies reported a significant reduction in serum hscrp levels . interestingly , body weight changed only slightly in some studies that reported a significant fall in hscrp levels [ 8385 ] . serum tf levels fell after aerobic exercise training in most [ 35 , 87 , 91 ] but not all studies . in contrast , serum il-6 levels did not change with exercise in most reports [ 83 , 85 , 89 ] and fell only in 1 study . the concentration of il-10 , an anti - inflammatory cytokine , increased after 6 months of aerobic exercise in overweight patients with t2 dm and serum il-18 concentration decreased in the same study , but remained unchanged in another . in addition , sicam-1 and svcam-1 levels were not affected after one year of resistance training . in a recent controlled exercise training intervention , 189 overweight and mildly obese patients were randomized to 6 months of inactivity or 1 of 3 types of aerobic exercise training regimens : low - amount / moderate intensity , low - amount / vigorous intensity , and high - amount / vigorous intensity . despite a mean reduction of adipose tissue mass by 9 11% ( or 2.5 3.0 kg ) , exercise training did not affect hscrp , il-6 , or tf levels to a significant degree . differences in the type , frequency , intensity , and duration of exercise , in the amount of achieved weight loss , and/or in the duration of the study may partly explain the observed discrepancies in the effects of exercise on markers of inflammation . moreover , the participants were mainly overweight or mildly obese , with variable baseline characteristics ( age , gender , previous physical activity , all of which might have influenced the results ) . finally , it has been reported that both strenuous exercise ( which can lead to muscle damage ) and overtraining ( which might induce oxidative stress ) might act as an inflammatory stimulus and raise inflammatory markers . more data are available on the effects of combining both diet and exercise on inflammatory markers ( table 1 ) [ 26 , 29 , 40 , 94114 ] . all studies ( n = 12190 , duration 4 weeks to 2 years ) reported a significant decrease in bmi in overweight [ 94 , 111 , 112 ] , obese [ 26 , 29 , 40 , 95107 , 109 , 111 , 112 ] and morbidly obese patients [ 110 , 113 , 114 ] . interestingly , hscrp levels significantly decreased in all but 1 of these studies [ 26 , 29 , 40 , 9496 , 98114 ] ; the remaining study reported a nonsignificant fall in hscrp concentration ( p = .71 ) . in accordance with diet - only studies , hscrp reductions after diet and exercise correlated positively with changes in body weight and fat mass . the majority of studies also showed a significant decrease in serum il-6 concentration after a multidisciplinary program including diet and exercise [ 26 , 94 , 97 , 98 , 100103 , 107 , 110113 ] and only few studies did not [ 95 , 96 , 109 ] . furthermore , all studies that evaluated the effects of caloric restriction and exercise on serum il-18 levels observed a significant decrease [ 100102 , 106 , 111 ] . limited data also suggest that diet and increased physical activity decrease il-7 , il-8 , sicam-1 [ 26 , 104 ] , and svcam-1 levels . in contrast , the effects of diet and exercise on serum tnf levels were less consistent with both significant decreases [ 26 , 40 , 101 , 105 ] and no change reported [ 94 , 95 , 97 , 109 , 112 ] . finally , 4 studies assessed the separate and combined effects of exercise and diet intervention on several cytokines . nicklas et al . reported that an 18-month dietary intervention resulted in significant reductions in hscrp and il-6 concentrations ( but not in tnf levels ) whereas exercise training had no effect ; however , weight loss was observed only in patients on diet . patients on both diet and exercise did not show greater reductions in hscrp and il-6 levels than those on diet alone . in another study , 6 months of diet plus exercise , but not exercise alone , decreased hscrp and il-6 levels ( but not tnf levels ) in obese postmenopausal women despite a similar weight and fat loss in both groups . randomly allocated 188 men with vascular risk factors to 4 groups : diet , exercise , combined diet and exercise , and control . serum tnf levels increased significantly in the 3 intervention groups but the increase was smaller in the combined diet and exercise group and probably represents a chance finding . in another study , 33 obese postmenopausal women with t2 dm were assigned to diet , exercise , or diet and exercise for 14 weeks . serum hscrp levels decreased by approximately 15% with all interventions even though weight loss was observed only in patients assigned to diet alone or with exercise . the conflicting effects of the combination of diet and exercise on inflammatory markers are probably due to the short duration of the majority of the studies , the small number of participants , or the varying weight loss achieved . the lack of a decrease in serum tnf concentration may be a result of its transient production and short half - life . similar to the studies that assessed dietary modifications alone , a weight loss of at least 10% resulted in the greatest improvements in serological markers of inflammation in studies where diet was combined with exercise . overall , diet and exercise alone or in combination have favourable effects on serum inflammatory markers . the inconsistent findings in some studies may be due to ( apart from the reasons already mentioned ) differences in the studied population ( i.e. , overweight , obese , patients with diabetes ) , in the baseline concentration of cytokines and inflammatory status of each patient , as well as in the laboratory methods used for the measurement of cytokines . orlistat induces weight loss by reducing intestinal fat absorption ( by up to 30% ) by binding to pancreatic lipases and partially inhibiting the hydrolysis of dietary fat ( triglycerides ) into absorbable free fatty acids and monoacylglycerols . compared with placebo , orlistat reduces body weight by approximately 2.9 kg ( 95% confidence interval ( ci ) 2.53.2 kg ) in studies lasting more than 1 year . in addition , orlistat lowers low density lipoprotein cholesterol ( ldl - c ) levels , reduces blood pressure ( bp ) , and improves glycemic control in diabetic patients [ 120 , 121 ] . more importantly , in xenical in the prevention of diabetes in obese subjects ( xendos ) study , orlistat reduced the risk of developing t2 dm in obese patients with impaired glucose tolerance . a limited number of studies evaluated the effects of orlistat on inflammatory markers in obese patients ( table 1 ) . in some reports , treatment with orlistat for up to 1 year reduced hscrp levels [ 123125 ] . however , in other studies the fall in crp levels during orlistat treatment did not reach statistical significance [ 126 , 127 ] . in addition , in a placebo - controlled study that evaluated the effects of long - term ( 3 years ) treatment with orlistat , hscrp levels decreased significantly only in patients who lost more than 11% of their body weight . it is of interest to point out that all 3 studies that reported a decrease in hscrp levels during orlistat treatment [ 123125 ] lasted for 6 months or more and hscrp levels were measured . in contrast , negative studies evaluated crp levels , which are less sensitive for evaluating inflammation than hscrp [ 126 , 127 ] . moreover , baseline crp levels were within the normal range in both negative studies and this might have limited their statistical power to show a significant decrease in crp levels [ 126 , 127 ] . finally , both negative studies had shorter follow - up than the studies that reported a decrease in hscrp levels during orlistat treatment ( 3 and 4 months , resp . ) and the potential antiinflammatory effect of orlistat might have not become apparent yet at study completion [ 123127 ] . a reduction in serum il-6 [ 123 , 124 , 129 ] and tnf levels was also reported after treatment with orlistat [ 123 , 127 , 129 ] . orlistat did not affect serum il-6 levels only in one study , which however had a short follow - up as already mentioned ( 4 months versus 6 months or more in other reports ) . interestingly , the decrease in hscrp and tnf in some studies was greater in the diet plus orlistat group compared with patients who were only on diet even after correcting for the greater weight loss observed in the orlistat group [ 123 , 125 , 129 ] . overall , it appears that 6 months or more of treatment with orlistat and a weight loss greater than 10% are required for a significant reduction in inflammatory markers with this agent . sibutramine lowers body weight by increasing satiety and by reducing appetite through the selective inhibition of the neuronal reuptake of serotonin and noradrenaline within the hypothalamus [ 130 , 131 ] . in studies lasting more than 1 year , sibutramine reduced body weight by approximately 4.2 kg ( 95% ci 3.64.7 kg ) compared with placebo [ 120 , 130 ] . sibutramine also lowers triglyceride ( tg ) levels but increases bp and pulse rate [ 120 , 130 ] . in some studies , sibutramine reduced serum levels of hscrp [ 132 , 133 ] , il-6 , and tnf and increased serum levels of il-10 when given for 36 months ( table 1 ) . a weight loss of 6.17.1 kg ( 5.4%6.9% ) was observed during the latter studies [ 133 , 134 ] and correlated with the reduction in il-6 levels . in another study , treatment with sibutramine for 6 months did not affect hscrp levels ; however , the weight loss achieved in this study was substantially smaller ( 2.4 kg or 2.9% ) . therefore , similarly with orlistat , weight loss appears to be the major factor that determines the anti - inflammatory effect of sibutramine . in a nonrandomized , nonblinded comparative study , only sibutramine reduced hscrp levels after 6 months whereas changes in hscrp levels were nonsignificant in patients given orlistat . however , weight loss was greater in the sibutramine group compared with the orlistat group ( 5.4% versus 2.5% , resp . ) , suggesting again that the degree of weight loss correlates with the anti - inflammatory action of these antiobesity agents . whether orlistat or sibutramine exerts an anti - inflammatory action independently of weight loss is unclear . it should emphasized that the committee for medicinal products for human use of the european medicines agency recently concluded that the risks of sibutramine are greater than its benefits and recommended the suspension of marketing authorization of this agent across the european union ( http://www.ema.europa.eu/pdfs/human/referral/sibutramine/3940810en.pdf ; accessed 25/1/2010 ) . the food and drug administration also stated that sibutramine is not to be used in patients with a history of coronary heart disease , stroke or transient ischemic attack , heart failure , heart arrhythmias , peripheral arterial disease , or uncontrolled hypertension ( http://www.fda.gov/safety/medwatch/safetyinformation/safetyalertsforhumanmedicalproducts/ucm198221.htm ; accessed 25/1/2010 ) . these recommendations were based on the results of the sibutramine cardiovascular outcome trial ( scout ) , which showed an increased risk of nonfatal cardiovascular events with sibutramine compared with placebo in patients with known or high risk for cardiovascular disease ( http://www.ema.europa.eu/pdfs/human/referral/sibutramine/3940810en.pdf and http://www.fda.gov/safety/medwatch/safetyinformation/safetyalertsforhumanmedicalproducts/ucm198221.htm ; accessed 25/1/2010 ) . metformin enhances insulin sensitivity , lowers hepatic glucose production , and is a first - line treatment for t2 dm [ 136 , 137 ] . metformin frequently induces a modest weight loss in diabetic patients . in 4 studies in patients with t2 dm , treatment with metformin for 3 - 4 months exerted conflicting effects on serum hscrp , il-6 , tnf , sicam-1 , svcam-1 , and soluble e - selectin levels ( table 1 ) [ 138141 ] . in one study where metformin was administered only for 1 month , no significant change was observed in hscrp levels . metformin is also used in the management of patients with polycystic ovary syndrome ( pcos ) , where it has been shown to ameliorate reproductive abnormalities , restore ovulation and regular menses , increase pregnancy rates , and reduce androgen production . in 3 studies in women with pcos , treatment with metformin for 6 months also had contradictory effects on hscrp , il-6 , tnf- , sicam-1 , svcam-1 , and soluble e - selectin levels ( table 1 ) [ 144146 ] . in addition to the unclear effects of metformin on inflammatory markers , it should be emphasized that metformin is not currently recommended for the management of obesity . moreover , a recent study in nondiabetic obese women showed that sibutramine monotherapy reduced hscrp levels and that adding metformin to sibutramine did not result in any additional decrease in hscrp levels . bariatric surgery is classified in restrictive ( laparoscopic adjustable gastric banding and vertical banded gastroplasty ) , malabsorptive ( biliopancreatic diversion with or without duodenal switch ) , and combination procedures ( roux - en - y gastric bypass ) . all forms of bariatric surgery result in loss of more than 50% of excess weight . in addition , hypertension , t2 dm , hyperlipidemia , and obstructive sleep apnea resolve in the majority of patients who undergo bariatric surgery . more importantly , bariatric surgery was associated with reduced all - cause mortality in several retrospective studies [ 150152 ] and in a prospective study . malabsorptive or combination procedures induce greater weight loss [ 152 , 154157 ] and appear to be more effective in the resolution of obesity - associated comorbidities [ 149 , 157 ] . however , they are also associated with higher complication rates compared with restrictive procedures [ 149 , 151 , 154 ] . several studies reported a reduction in serum hscrp levels after roux - en - y gastric bypass ( table 1 ) [ 158166 ] . this decrease in hscrp levels is greater in patients who lost more weight after surgery [ 164 , 166 ] . a decline in hscrp levels also occurs in patients undergoing gastric banding ( table 1 ) [ 158 , 167175 ] . the decrease in hscrp levels correlated with the fall in bmi in some of these studies . only one study did not report a significant change of hscrp levels after gastric banding ; however , the decrease in hscrp levels in this report was significant 6 months after surgery ( p = .05 ) and there was a trend for significant decrease also at 12 months ( p = .08 ) . in a recent study , patients who underwent gastric bypass lost more weight than those who underwent gastric banding but the fall in hscrp levels was similar in both groups . other less frequently performed bariatric procedures , such as biliopancreatic diversion , laparoscopic sleeve gastrectomy , and gastric partition , also result in a decrease in hscrp levels that in some cases correlates with the reduction in bmi . the reduction in hscrp levels in almost all studies of all types of bariatric surgery , in contradiction to the equivocal results of lifestyle changes and antiobesity agents , further supports the notion that weight loss per se is the main factor that causes the fall in hscrp levels , since bariatric surgery results in substantially greater weight loss than lifestyle changes or antiobesity agents . the lack of correlation between weight loss and hscrp fall might be attributed to the small number of patients included in these studies , which limits their statistical power . other markers of inflammation , including il-6 [ 160 , 162 , 163 , 165 ] , il-18 , mcp-1 , and soluble e - selectin also fall after gastric bypass ( table 1 ) . only 1 small study ( n = 26 ) did not report significant changes in il-6 , tnf , stnfr-2 , sicam-1 , and svcam-1 levels following gastric bypass . however , serum hscrp levels , which are a more sensitive and specific marker of inflammation , decreased in this study . serum levels of il-6 [ 171 , 172 ] , il-18 , stnfr-2 [ 167 , 170 ] , mcp-1 , soluble e - selectin [ 37 , 176 , 181 , 182 ] , sicam-1 [ 37 , 181 , 182 ] , and svcam-1 levels also fall after gastric banding ( table 1 ) . the change in soluble e - selectin and sicam-1 levels correlated with the reduction in bmi [ 37 , 182 ] . however , il-6 , svcam-1 , and sicam-1 levels did not change significantly in other studies . in addition , serum tnf levels [ 172 , 174 ] were not affected by gastric banding . again , serum hscrp levels fell in most studies that did not record a significant change in other inflammatory markers [ 172 , 174 ] , suggesting that the latter might be less sensitive in identifying a clinically significant anti - inflammatory effect of bariatric surgery . in contrast to bariatric surgery , removal of abdominal subcutaneous fat by liposuction does not appear to reduce hscrp , il-6 , or tnf levels ( table 1 ) [ 183 , 184 ] . however , one study reported a reduction in serum hscrp , il-6 , il-18 , and tnf levels after liposuction . in the latter study , the amount of fat aspirate correlated with the reduction in tnf levels . even though the data on the effect of liposuction on inflammatory markers are limited , the predominantly negative findings are concordant with the concept that visceral adipose tissue is the main factor inducing the inflammatory milieu of obese patients whereas subcutaneous adipose tissue is less important . existing evidence suggests that lifestyle measures , antiobesity agents , and bariatric surgery reduce serum levels of inflammatory markers in obese patients . the lack of significant change in some studies appears to be due to small sample size and/or small weight loss achieved . it appears that the reduction in inflammation is primarily driven by weight loss . in a recent meta - analysis , a linear relation was observed between weight loss following lifestyle changes or bariatric surgery and the fall in hscrp levels , which declined by 0.13 mg / what remains to be established is whether reducing inflammatory markers in obese patients with lifestyle measures or ( where indicated ) with bariatric surgery or drugs will translate in lower vascular risk . in this context , a beneficial effect of suppressing inflammation has been reported during statin treatment in patients with or without established vascular disease , even though it was not assessed whether obese patients benefit more or less [ 187189 ] .

overweight and obesity are highly prevalent in developed countries and are also becoming more frequent in the developing world . overweight and obese patients have elevated levels of several inflammatory markers and this inflammatory state might contribute to their increased vascular risk . we summarize the effects of lifestyle changes , antiobesity agents , and bariatric surgery on serological inflammatory markers in overweight and obese patients . most studies showed a decrease in inflammation with all 3 interventions . however , it remains to be established whether the decrease in inflammatory markers induced by lifestyle changes or ( where indicated ) with antiobesity agents or bariatric surgery will translate into reduced vascular morbidity and mortality in overweight and obese patients .

1. Introduction 2. Lifestyle Measures 3. Antiobesity Agents 4. Bariatric Surgery 5. Conclusions

this epidemic of overweight and obesity has major implications , since both overweight and obesity are independently associated with increased mortality [ 35 ] . adipose tissue is not metabolically inert but synthesizes and secretes proinflammatory mediators , including interleukin 6 ( il-6 ) [ 69 ] , tumor necrosis factor ( tnf ) [ 7 , 10 , 11 ] , il-8 , and monocyte chemoattractant [ protein 1 ] ( mcp-1 ) [ 12 , 13 ] . several studies reported that high sensitivity c - reactive protein levels are elevated in overweight and obese patients [ 1426 ] . compared with lean subjects , overweight and obese patients also have higher levels of il-6 [ 9 , 19 , 24 , 2628 ] , tnf [ 24 , 2630 ] , mcp-1 , as well as of other markers of inflammation , including il-8 [ 31 , 32 ] , il-18 , soluble tnf receptor-2 ( stnfr2 ) [ 27 , 34 , 35 ] , soluble e - selectin [ 26 , 36 , 37 ] , soluble intercellular adhesion molecule-1 ( sicam-1 ) [ 18 , 26 , 27 , 34 , 3638 ] , and soluble vascular cell adhesion molecule-1 ( svcam-1 ) [ 26 , 36 ] . in obese patients , inflammatory markers correlate with visceral adipose tissue mass [ 20 , 21 , 27 , 33 , 34 , 3941 ] and with waist circumference , an index of visceral adiposity [ 17 , 2123 , 27 , 32 , 35 , 39 ] . the activation of inflammation that is present in obesity might contribute to the increased vascular morbidity and mortality of these patients , since elevated levels of inflammatory markers are associated with increased vascular risk [ 4246 ] . in this review , we summarize the effects of lifestyle changes , antiobesity agents , and bariatric surgery on serological inflammatory markers in obese patients . in randomized controlled studies , diet and exercise reduced the risk of type 2 diabetes mellitus ( t2 dm ) in overweight patients with impaired glucose tolerance ( igt ) [ 48 , 49 ] . in observational studies , weight loss achieved with lifestyle changes was associated with reduced incidence of coronary heart disease ( chd ) and with lower all - cause mortality . several studies evaluated the effects of diet and exercise alone or in combination on inflammatory markers in obese patients . several small studies ( n = 11100 ) assessed the effects of low - calorie , very low - calorie , low - fat , or low - carbohydrate diets on inflammatory markers in obese patients ( table 1 ) [ 20 , 22 , 28 , 5275 ] . the majority of studies noted a significant decrease in crp after weight loss induced by dietary modification in otherwise healthy overweight or obese adults [ 20 , 22 , 52 , 54 , 5660 , 62 , 6468 , 70 , 7275 ] , in obese patients with the metabolic syndrome , in overweight patients with raised triglycerides ( tg ) , in severely obese patients , or in obese patients with t2 dm . in some studies , the magnitude of decrease in hscrp levels was linearly correlated to the amount of weight loss [ 20 , 22 , 67 ] . furthermore , several studies compared the effects of different diets on inflammatory markers [ 5254 , 56 , 57 , 59 , 62 , 6568 , 7073 ] . reported a significant 24% decrease in serum hscrp levels after 3 months of replacement of high - fat foods with reduced - fat alternatives . in another study , a carbohydrate restricted diet ( crd ) with daily intake of eggs decreased hscrp levels compared with a crd without eggs , indicating that eggs significantly contribute to the anti - inflammatory effects of crd possibly due to the antioxidant lutein which is present in eggs and modulates certain inflammatory responses . however , most studies comparing diets of different composition reported a similar decrease in hscrp concentration among groups , suggesting that the weight loss per se rather than the dietary composition is the primary determinant of the fall in inflammatory markers during diet [ 5254 , 56 , 57 , 6567 , 7173 ] . regarding the effects of diet on il-8 , a significant reduction , increase , or no change has been reported . in addition , caloric restriction did not affect circulating levels of the anti - inflammatory cytokine il-10 . however , limited data suggest that diet - induced weight loss is associated with a proportional reduction in il-18 and il-20 levels . the conflicting results mentioned above could be attributed to the differences in baseline characteristics of the studied population that might influence inflammatory markers ( i.e. interestingly , recent studies demonstrated that brief periods of reduced food intake can increase resistance to ischemia reperfusion injury in rodents and modify circulating levels of pro - inflammatory agents in humans [ 81 , 82 ] . the effects of short - term interventions on inflammation appear to be mostly due to the calorie restriction and negative energy balance whereas in long - term interventions weight loss and adipose mass reduction also contribute to the suppression of inflammation . however , existing studies do not show a major difference between the effects of short and long terms of calorie restriction on the levels of inflammatory markers . a number of small studies ( n = 8199 ) evaluated the effects of exercise training , resistance training , or aerobic exercise training for 212 months on inflammatory markers ( table 1 ) [ 35 , 8393 ] . some [ 8387 ] but not all [ 8890 ] studies reported a significant reduction in serum hscrp levels . the concentration of il-10 , an anti - inflammatory cytokine , increased after 6 months of aerobic exercise in overweight patients with t2 dm and serum il-18 concentration decreased in the same study , but remained unchanged in another . in a recent controlled exercise training intervention , 189 overweight and mildly obese patients were randomized to 6 months of inactivity or 1 of 3 types of aerobic exercise training regimens : low - amount / moderate intensity , low - amount / vigorous intensity , and high - amount / vigorous intensity . differences in the type , frequency , intensity , and duration of exercise , in the amount of achieved weight loss , and/or in the duration of the study may partly explain the observed discrepancies in the effects of exercise on markers of inflammation . finally , it has been reported that both strenuous exercise ( which can lead to muscle damage ) and overtraining ( which might induce oxidative stress ) might act as an inflammatory stimulus and raise inflammatory markers . more data are available on the effects of combining both diet and exercise on inflammatory markers ( table 1 ) [ 26 , 29 , 40 , 94114 ] . all studies ( n = 12190 , duration 4 weeks to 2 years ) reported a significant decrease in bmi in overweight [ 94 , 111 , 112 ] , obese [ 26 , 29 , 40 , 95107 , 109 , 111 , 112 ] and morbidly obese patients [ 110 , 113 , 114 ] . interestingly , hscrp levels significantly decreased in all but 1 of these studies [ 26 , 29 , 40 , 9496 , 98114 ] ; the remaining study reported a nonsignificant fall in hscrp concentration ( p = .71 ) . the majority of studies also showed a significant decrease in serum il-6 concentration after a multidisciplinary program including diet and exercise [ 26 , 94 , 97 , 98 , 100103 , 107 , 110113 ] and only few studies did not [ 95 , 96 , 109 ] . furthermore , all studies that evaluated the effects of caloric restriction and exercise on serum il-18 levels observed a significant decrease [ 100102 , 106 , 111 ] . limited data also suggest that diet and increased physical activity decrease il-7 , il-8 , sicam-1 [ 26 , 104 ] , and svcam-1 levels . in contrast , the effects of diet and exercise on serum tnf levels were less consistent with both significant decreases [ 26 , 40 , 101 , 105 ] and no change reported [ 94 , 95 , 97 , 109 , 112 ] . finally , 4 studies assessed the separate and combined effects of exercise and diet intervention on several cytokines . nicklas et al . reported that an 18-month dietary intervention resulted in significant reductions in hscrp and il-6 concentrations ( but not in tnf levels ) whereas exercise training had no effect ; however , weight loss was observed only in patients on diet . randomly allocated 188 men with vascular risk factors to 4 groups : diet , exercise , combined diet and exercise , and control . serum tnf levels increased significantly in the 3 intervention groups but the increase was smaller in the combined diet and exercise group and probably represents a chance finding . serum hscrp levels decreased by approximately 15% with all interventions even though weight loss was observed only in patients assigned to diet alone or with exercise . the conflicting effects of the combination of diet and exercise on inflammatory markers are probably due to the short duration of the majority of the studies , the small number of participants , or the varying weight loss achieved . the lack of a decrease in serum tnf concentration may be a result of its transient production and short half - life . similar to the studies that assessed dietary modifications alone , a weight loss of at least 10% resulted in the greatest improvements in serological markers of inflammation in studies where diet was combined with exercise . overall , diet and exercise alone or in combination have favourable effects on serum inflammatory markers . the inconsistent findings in some studies may be due to ( apart from the reasons already mentioned ) differences in the studied population ( i.e. , overweight , obese , patients with diabetes ) , in the baseline concentration of cytokines and inflammatory status of each patient , as well as in the laboratory methods used for the measurement of cytokines . in addition , orlistat lowers low density lipoprotein cholesterol ( ldl - c ) levels , reduces blood pressure ( bp ) , and improves glycemic control in diabetic patients [ 120 , 121 ] . more importantly , in xenical in the prevention of diabetes in obese subjects ( xendos ) study , orlistat reduced the risk of developing t2 dm in obese patients with impaired glucose tolerance . a limited number of studies evaluated the effects of orlistat on inflammatory markers in obese patients ( table 1 ) . however , in other studies the fall in crp levels during orlistat treatment did not reach statistical significance [ 126 , 127 ] . in addition , in a placebo - controlled study that evaluated the effects of long - term ( 3 years ) treatment with orlistat , hscrp levels decreased significantly only in patients who lost more than 11% of their body weight . it is of interest to point out that all 3 studies that reported a decrease in hscrp levels during orlistat treatment [ 123125 ] lasted for 6 months or more and hscrp levels were measured . moreover , baseline crp levels were within the normal range in both negative studies and this might have limited their statistical power to show a significant decrease in crp levels [ 126 , 127 ] . finally , both negative studies had shorter follow - up than the studies that reported a decrease in hscrp levels during orlistat treatment ( 3 and 4 months , resp . ) interestingly , the decrease in hscrp and tnf in some studies was greater in the diet plus orlistat group compared with patients who were only on diet even after correcting for the greater weight loss observed in the orlistat group [ 123 , 125 , 129 ] . overall , it appears that 6 months or more of treatment with orlistat and a weight loss greater than 10% are required for a significant reduction in inflammatory markers with this agent . sibutramine also lowers triglyceride ( tg ) levels but increases bp and pulse rate [ 120 , 130 ] . in some studies , sibutramine reduced serum levels of hscrp [ 132 , 133 ] , il-6 , and tnf and increased serum levels of il-10 when given for 36 months ( table 1 ) . in another study , treatment with sibutramine for 6 months did not affect hscrp levels ; however , the weight loss achieved in this study was substantially smaller ( 2.4 kg or 2.9% ) . therefore , similarly with orlistat , weight loss appears to be the major factor that determines the anti - inflammatory effect of sibutramine . however , weight loss was greater in the sibutramine group compared with the orlistat group ( 5.4% versus 2.5% , resp . ) , suggesting again that the degree of weight loss correlates with the anti - inflammatory action of these antiobesity agents . the food and drug administration also stated that sibutramine is not to be used in patients with a history of coronary heart disease , stroke or transient ischemic attack , heart failure , heart arrhythmias , peripheral arterial disease , or uncontrolled hypertension ( http://www.fda.gov/safety/medwatch/safetyinformation/safetyalertsforhumanmedicalproducts/ucm198221.htm ; accessed 25/1/2010 ) . metformin enhances insulin sensitivity , lowers hepatic glucose production , and is a first - line treatment for t2 dm [ 136 , 137 ] . in 4 studies in patients with t2 dm , treatment with metformin for 3 - 4 months exerted conflicting effects on serum hscrp , il-6 , tnf , sicam-1 , svcam-1 , and soluble e - selectin levels ( table 1 ) [ 138141 ] . metformin is also used in the management of patients with polycystic ovary syndrome ( pcos ) , where it has been shown to ameliorate reproductive abnormalities , restore ovulation and regular menses , increase pregnancy rates , and reduce androgen production . in 3 studies in women with pcos , treatment with metformin for 6 months also had contradictory effects on hscrp , il-6 , tnf- , sicam-1 , svcam-1 , and soluble e - selectin levels ( table 1 ) [ 144146 ] . in addition to the unclear effects of metformin on inflammatory markers , it should be emphasized that metformin is not currently recommended for the management of obesity . moreover , a recent study in nondiabetic obese women showed that sibutramine monotherapy reduced hscrp levels and that adding metformin to sibutramine did not result in any additional decrease in hscrp levels . bariatric surgery is classified in restrictive ( laparoscopic adjustable gastric banding and vertical banded gastroplasty ) , malabsorptive ( biliopancreatic diversion with or without duodenal switch ) , and combination procedures ( roux - en - y gastric bypass ) . all forms of bariatric surgery result in loss of more than 50% of excess weight . in addition , hypertension , t2 dm , hyperlipidemia , and obstructive sleep apnea resolve in the majority of patients who undergo bariatric surgery . more importantly , bariatric surgery was associated with reduced all - cause mortality in several retrospective studies [ 150152 ] and in a prospective study . malabsorptive or combination procedures induce greater weight loss [ 152 , 154157 ] and appear to be more effective in the resolution of obesity - associated comorbidities [ 149 , 157 ] . however , they are also associated with higher complication rates compared with restrictive procedures [ 149 , 151 , 154 ] . this decrease in hscrp levels is greater in patients who lost more weight after surgery [ 164 , 166 ] . the decrease in hscrp levels correlated with the fall in bmi in some of these studies . only one study did not report a significant change of hscrp levels after gastric banding ; however , the decrease in hscrp levels in this report was significant 6 months after surgery ( p = .05 ) and there was a trend for significant decrease also at 12 months ( p = .08 ) . other less frequently performed bariatric procedures , such as biliopancreatic diversion , laparoscopic sleeve gastrectomy , and gastric partition , also result in a decrease in hscrp levels that in some cases correlates with the reduction in bmi . the reduction in hscrp levels in almost all studies of all types of bariatric surgery , in contradiction to the equivocal results of lifestyle changes and antiobesity agents , further supports the notion that weight loss per se is the main factor that causes the fall in hscrp levels , since bariatric surgery results in substantially greater weight loss than lifestyle changes or antiobesity agents . other markers of inflammation , including il-6 [ 160 , 162 , 163 , 165 ] , il-18 , mcp-1 , and soluble e - selectin also fall after gastric bypass ( table 1 ) . only 1 small study ( n = 26 ) did not report significant changes in il-6 , tnf , stnfr-2 , sicam-1 , and svcam-1 levels following gastric bypass . however , serum hscrp levels , which are a more sensitive and specific marker of inflammation , decreased in this study . serum levels of il-6 [ 171 , 172 ] , il-18 , stnfr-2 [ 167 , 170 ] , mcp-1 , soluble e - selectin [ 37 , 176 , 181 , 182 ] , sicam-1 [ 37 , 181 , 182 ] , and svcam-1 levels also fall after gastric banding ( table 1 ) . however , il-6 , svcam-1 , and sicam-1 levels did not change significantly in other studies . again , serum hscrp levels fell in most studies that did not record a significant change in other inflammatory markers [ 172 , 174 ] , suggesting that the latter might be less sensitive in identifying a clinically significant anti - inflammatory effect of bariatric surgery . in contrast to bariatric surgery , removal of abdominal subcutaneous fat by liposuction does not appear to reduce hscrp , il-6 , or tnf levels ( table 1 ) [ 183 , 184 ] . however , one study reported a reduction in serum hscrp , il-6 , il-18 , and tnf levels after liposuction . in the latter study , the amount of fat aspirate correlated with the reduction in tnf levels . even though the data on the effect of liposuction on inflammatory markers are limited , the predominantly negative findings are concordant with the concept that visceral adipose tissue is the main factor inducing the inflammatory milieu of obese patients whereas subcutaneous adipose tissue is less important . existing evidence suggests that lifestyle measures , antiobesity agents , and bariatric surgery reduce serum levels of inflammatory markers in obese patients . the lack of significant change in some studies appears to be due to small sample size and/or small weight loss achieved . it appears that the reduction in inflammation is primarily driven by weight loss . in a recent meta - analysis , a linear relation was observed between weight loss following lifestyle changes or bariatric surgery and the fall in hscrp levels , which declined by 0.13 mg / what remains to be established is whether reducing inflammatory markers in obese patients with lifestyle measures or ( where indicated ) with bariatric surgery or drugs will translate in lower vascular risk . in this context , a beneficial effect of suppressing inflammation has been reported during statin treatment in patients with or without established vascular disease , even though it was not assessed whether obese patients benefit more or less [ 187189 ] .

strategies to interrupt transmission of hiv are urgently needed , especially in generalized epidemic settings with extremely high hiv prevalence in the general population , high hiv - related disease burden and limited resources to identify , engage and retain infected individuals in care . in the long term , serving populations at highest risk of transmitting or acquiring hiv is necessary to reduce the total number of people who will require lifelong treatment , helping to close the gap between global treatment targets and present - day , substantially lower levels of hiv diagnosis , treatment , and viral suppression . in generalized epidemic settings , there is not yet an accepted strategy for identifying these sub - populations , but the patterns of generalized hiv epidemics provide clues about their characteristics . a distinct characteristic of generalized epidemics is sustained high hiv incidence in young women , leading epidemiologists to hypothesize that the age patterns of hiv transmission are central to fueling the epidemic and may be vulnerable to interventions that collapse the chains of hiv transmission . we have previously used mathematical modeling to investigate the age patterns of those most at risk of transmitting hiv to another individual , and further , those most likely to be part of an ongoing chain of transmission ( as opposed to a building upon those investigations , the current study explores whether age - targeted intensification of primary and/or secondary hiv prevention could collapse chains of transmission in generalized epidemic settings . the targeting approach explored here draws inspiration from the epidemiological ring - fencing methods used to control the spread of smallpox since the 1870s up until the final eradication activities in the 1970s . rather than the quarantine and vaccination approaches used to create a shield to prevent the spread of smallpox , intensified hiv treatment and prevention services would be used to create a longer - term demographic we explore a strategy of intensified hiv services for a specific age range , forming a static age band that separates older from younger individuals ( figure 1a ) . alternatively , we explore intensification to an aging birth cohort consisting of individuals born between two specified dates . if begun when these individuals are mostly hiv - unexposed , such a band could sweep through the population , potentially leaving behind a future generation protected from hiv . figure 1.schematic of age - based targeting and cohort - based targeting of outreach . teal regions show the age range of the ( a ) age or ( b ) birth cohort receiving intensified hiv services . people can age in and out of the target group with age targeting , but not with birth cohort targeting . teal regions show the age range of the ( a ) age or ( b ) birth cohort receiving intensified hiv services . people can age in and out of the target group with age targeting , but not with birth cohort targeting . the concept of a demographic shield to protect future generations is relatively unexplored even among available age - structured models of hiv transmission . because the penetration and effectiveness of a hypothetical intervention can not be predicted in silico , the study presented here is exploratory in nature , scanning over a range of model assumptions and hypothetical combinations of novel or highly improved interventions to estimate the maximum potential impact of a given strategy . we used emod - hiv v0.8 , an age - structured , individual - based network model of hiv in south africa , to model the impact of intensifying primary and/or secondary hiv prevention services for a specified age range ( figure 1a ) or birth cohort ( figure 1b ) . primary prevention is defined as a service targeted to an uninfected individual to prevent acquisition of disease , whereas secondary prevention is targeted to an infected individual to prevent transmission of disease . the parameters , projections , and sensitivities of the baseline model projections ( to which the interventions are compared ) have been described previously and a detailed model description , user tutorials , model installer , and source code are available for download at http://idmod.org/software . briefly , emod - hiv is an individual - based model that simulates transmission using an explicitly defined network of relationships that are formed according to preference patterns and dissolve according to age - dependent durations ( younger individuals tending to form shorter - term relationships ) . the age patterns of sexual mixing were configured to match those observed in a rural , hiv - hyperendemic region of kwazulu - natal , south africa . recently , a validation study showed that self - reported partner ages in this setting to be relatively accurate , with 72% of self - reported estimates falling within two years of the partner 's actual date of birth . the model replicates the demographic patterns of south africa , explicitly simulating 1/200th of the south african population with age - dependent fertility and age / gender - dependent mortality . to ensure that the configured relationship preference patterns are realized given the underlying demographics , the rates of relationship formation are adjusted by a feed - forward algorithm to match the desired age patterns between couples . transmission rates within relationships depend on hiv disease stage , male circumcision , condom usage , co - infections , and antiretroviral therapy the latter causing the transmission rate to decline linearly over the first 6 months of therapy until viral suppression is achieved . viral suppression is assumed to reduce transmission by 92% in our more conservative scenarios an estimate based on observational data in which outside partnerships could have contributed to hiv acquisition or by as much as 100% in our most optimistic scenarios . the model includes a configurable health care system module , which we have configured to follow trends in antiretroviral therapy ( art ) expansion in south africa . treatment begins with voluntary counseling and testing ( vct ) , antenatal and infant testing , symptom - driven testing , and low level of couples testing . the model includes loss to follow - up between diagnosis and staging , between staging and linkage to art or pre - art care , and during art or pre - art care . the model 's projections of baseline treatment expansion in south africa predict gradual incidence declines without elimination , so that hiv remains endemic through 2050 . the cost and hiv burden in the targeted treatment scenarios were compared to the baseline scenario using a unit cost and disability - adjusted life year ( daly ) model developed by the hiv modelling consortium for the 2013 who revision of hiv treatment guidelines . primary prevention was implemented as a decrease in the probability that an individual becomes infected , up to a 100% decrease corresponding to full protection . secondary prevention was assumed to consist of hiv testing at a rate of once per year , with art ) initiation regardless of cd4 count for those testing positive . the intensified hiv testing was assumed to replace vct for those in the target group . these assumptions about the target group match those of a recent collaborative analysis of hypothetical universal test - and - treat for the entire population of south africa . in our analysis , we provide the full - population test - and - treat scenario as a reference point to compare to age - targeted test - and - treat . this provides a hypothetical maximum for cost and impact , and also provides a point of reference where our model has been compared to 11 other independently developed mathematical models . unlike birth cohort targeting , age range targeting would allow people to enter and leave the target group as they cross the lower and upper age thresholds . before entering and after leaving the target group , individuals exhibited eligibility , testing , and linkage rates identical to those used for the baseline scenario . however , those aging out of the target group continued art if they were initiated as part of the targeting intervention . presentation for antenatal , infant , couples , or symptom - driven testing was assumed to occur regardless of outreach , although the need for symptom - driven late presentation would decline for those who initiated care earlier than the onset of aids symptoms . a broad view of the model results is presented by scanning over starting and ending ages or years for a hypothetical age range ( figure 1a ) or birth cohort ( figure 1b ) , maintaining a range of two , five , 10 or 20 years from the youngest to the oldest age included in the target group . figure 2 shows the hiv disease burden relative to baseline , infections averted relative to baseline , incidence rate in the total population , and cost relative to baseline , assuming 80% penetration of outreach to the target group . infections , cost , and burden were accumulated over the first 20 years with a 3% annual discount rate , while incidence was reported for the 20th year ( 2035 ) . figure 2 also shows a maximum - impact scenario in which the whole population received the intensified testing and treatment intervention , providing a reference point for a population - wide intervention without age - specific targeting . figure 2.effect of age - targeted treatment expansion on hiv incidence , cumulative infections averted , cumulative disability - adjusted life years ( dalys ) averted , and cumulative program cost . effects of the interventions are represented on the y - axis , with x - axes showing the middle of the age group being targeted ( panels a the inclusion criteria of the targeted groups span 20 ( red ) , 10 ( green ) , 5 ( magenta ) or 2 ( blue ) years . incidence is shown at 20 years after the intervention begins , and other outcomes are accumulated over the first 20 years with a 3% annual discount rate . at the left and right extremes of each plot , the curves converge to black lines showing the baseline ( no intervention ) scenario , which projects current guidelines and trends in hiv treatment . the opposite black line shows universal treatment expansion with 80% coverage of annual testing for all individuals . dots in panels b and f show the strategy with the minimum cost per infection averted over 20 years . dots in panels c and g show the strategy with the mimumum cost per daly averted . effect of age - targeted treatment expansion on hiv incidence , cumulative infections averted , cumulative disability - adjusted life years ( dalys ) averted , and cumulative program cost . effects of the interventions are represented on the y - axis , with x - axes showing the middle of the age group being targeted ( panels a the inclusion criteria of the targeted groups span 20 ( red ) , 10 ( green ) , 5 ( magenta ) or 2 ( blue ) years . incidence is shown at 20 years after the intervention begins , and other outcomes are accumulated over the first 20 years with a 3% annual discount rate . at the left and right extremes of each plot , the curves converge to black lines showing the baseline ( no intervention ) scenario , which projects current guidelines and trends in hiv treatment . the opposite black line shows universal treatment expansion with 80% coverage of annual testing for all individuals . dots in panels b and f show the strategy with the minimum cost per infection averted over 20 years . dots in panels c and g show the strategy with the mimumum cost per daly averted . for interventions involving the scale - up of treatment , we used a unit cost model to approximate the cost and cost - effectiveness of universal and targeted treatment for prevention . the modeled universal treatment expansion intervention produced an additional five - fold reduction in incidence over what would be achieved with current trends in treatment , at an additional discounted cost of us$26 billion over twenty years ( figure 2a and 2e , gray lines ) . as expected , whole - population targeting was the most costly and highest - impact strategy , followed by 20-year age ranges spanning the highest - prevalence age groups . focusing on maximizing the cumulative infections averted , the dots in figure 2b show the most cost - effective ranges to target : ages 1030 at us$6238 per infection averted , ages 2030 at us$5031 per infection averted , ages 2227 at us$4279 per infection averted , and ages 2527 at us$3967 per infection averted . similarly , for infections averted by birth cohort targeting shown in figure 2f , the most cost - effective birth year ranges are marked at 19852005 at us$6826 per infection averted , 19871997 at us$5856 per infection averted , and 19901995 at us$5686 per infection averted . universal expansion to all ages / cohorts cost us$10 812 per infection averted . compared to universal expansion , targeting the 10-year age range of 2030 cost us$5.4 billion over 20 years and provided more than double the number of infections averted per us$ invested . however , the cumulative infections averted by targeting the 2030 age group was only 45% of the number possible with universal expansion . for cumulative dalys averted by age targeting shown in figure 2c , dots show the most cost - effective ranges to target : ages 2444 at us$1489 per daly averted , 2434 at us$1441 per daly averted , 2631 at us$1372 per daly averted , and 2729 at us$1285 per daly averted . similarly , for birth cohort targeting in figure 2 g , the most cost - effective birth year ranges are marked at 19611981 at us$1396 per infection averted , 19681978 at us$1374 per infection averted , and 19721977 at us$1373 per infection averted . the efficiency gained by age targeting was more modest for dalys than for infections averted . the most cost - effective 10-year age range for infections averted ( 2030 ) more than halved the cost per daly averted , whereas the most cost - effective 10-year age range for dalys averted ( 2434 ) reduced the cost per daly averted by only 17% . the most efficient age range for averting dalys was older than for averting infections , because dalys are averted by preventing new infections in the long run , but also by providing a direct health benefit to those receiving treatment in the short run . the health benefit is greatest for those who are most likely to progress quickly from asymptomatic hiv infection to symptomatic aids : older individuals , who experience faster disease progression and are more likely to have been infected for a longer period of time . having identified the 2030 age group as an efficient target for prevention , we then selected this age range as a hypothetical target group , and examined the age pattern of hiv incidence in the broader population to determine whether efficient targeting of treatment expansion to ages 2030 had the potential to interrupt hiv transmission to younger individuals . figure 3a shows the age pattern of incidence for baseline trends in hiv treatment . our model predicted a slow decline and slight aging of the pattern of hiv incidence , with hiv remaining endemic in the year 2050 . figure 3.age distribution of hiv incidence for the baseline simulation ( a ) , targeted treatment outreach ( b ) , and targeted complete prevention of hiv infection ( c ) . incidence is shown just before implementation of the intervention , and at 5 , 10 , 20 and 35 years after implementation of the intervention . shaded areas show the age range of the target group throughout the intervention ( left ) or for a birth cohort in the year of implementation ( start ) and 35 years after implementation ( end ) . age - dependent incidence per 100 uninfected person - years ( py ) was averaged across 20 stochastic simulation runs . age distribution of hiv incidence for the baseline simulation ( a ) , targeted treatment outreach ( b ) , and targeted complete prevention of hiv infection ( c ) . incidence is shown just before implementation of the intervention , and at 5 , 10 , 20 and 35 years after implementation of the intervention . shaded areas show the age range of the target group throughout the intervention ( left ) or for a birth cohort in the year of implementation ( start ) and 35 years after implementation ( end ) . age - dependent incidence per 100 uninfected person - years ( py ) was averaged across 20 stochastic simulation runs . when individuals aged 2030 were targeted with hiv treatment , even at an optimistic penetration level of 80% for outreach with testing and linkage to care , incidence declined across all age groups but fell short of completely eliminating hiv by 2050 ( figure 3b , left panel ) . by 2030 , incidence in the overall population was 37% lower than that of the baseline simulation , while incidence in those under age 20 was 60% lower than in the baseline scenario . by 2050 , the overall and under-20 incidence rates were 59% and 78% lower than the baseline scenario , respectively . one possible explanation for reduced but continuing incidence among teenagers is self - sustaining transmission among youth . thus , we next tested a strategy of birth cohort targeting for to those born in the years 2000 through 2010 . the targeted individuals would begin reaching sexual debut as the intervention was launched , and the 10-year targeted band would age through the population , with the intention of using a ring - fence like approach to the model results showed the cohort - based targeting approach to be less effective than age targeting ( figure 3b , right panel ) . by the year 2030 ( the year in which the target group reached 2030 years of age ) , incidence among those under 20 years of age was only 29% lower than in the baseline scenario , and incidence in the overall population was only 11% lower than in the baseline scenario . we further augmented the simulations of age - targeted treatment with a hypothetical prevention intervention to the same target group ( figure 3c ) . the extreme assumption of 100% coverage with perfect hiv prevention for those aged 2030 still did not eliminate hiv infections among those younger than age 20 , although incidence was greatly diminished . sweep out hiv from the population ( figure 3c , right panel ) . by the year 2030 , incidence emerged in young females at a rate of 005015% per year , and similar levels later emerged in young males . finally we addressed the question of how hiv the epidemic reached youth , even with a complete one - decade gap in hiv transmission . we considered four possible routes by which hiv could have entered the younger generation , illustrated in figure 4a . first , individuals older than the target group could infect those younger than the target group , essentially hopping the fence that was meant to protect younger generations . second , individuals in the target group could transmit to younger generations due to imperfect treatment coverage or efficacy . even when coupled with aggressive prevention interventions , some individuals in the target group could already be infected at the start of the intervention . third , the younger generation could sustain an epidemic on its own , unfettered by treatment or prevention in older generations . fourth , imperfect coverage and effectiveness of interventions to prevent mother - to - child transmission could permit some children to become infected and , with art , survive until sexual maturity . in figure 4a , these modalities are illustrated in blue , green , yellow , and red , respectively . figure 4.sources of new hiv infections in the generation younger than the target group . the diagram in panel ( a ) shows how infections can enter the younger generation from those older than the target group ( blue ) , those in the target group ( teal ) , those already infected in the younger generation itself ( yellow ) , or through mother - to - child transmission from any age group ( maroon ) . the contribution of each of these sources to incidence in those younger than the target group is shown in panel ( b ) for age targeting and panel ( c ) for birth cohort targeting . the denominator for incidence in ( b ) is the uninfected population ages 0 through 19 . in ( c ) , the denominator is the population of uninfected individuals with a birthdate after 2010 , which grows over time as new individuals are born . the diagram in panel ( a ) shows how infections can enter the younger generation from those older than the target group ( blue ) , those in the target group ( teal ) , those already infected in the younger generation itself ( yellow ) , or through mother - to - child transmission from any age group ( maroon ) . the contribution of each of these sources to incidence in those younger than the target group is shown in panel ( b ) for age targeting and panel ( c ) for birth cohort targeting . the denominator for incidence in ( b ) , the denominator is the population of uninfected individuals with a birthdate after 2010 , which grows over time as new individuals are born . the relative contribution from each of these four mechanisms was estimated by categorizing transmission events in the model according to their origin : infection by a sex partner older than , in , or younger than the target group , or mother - to - child transmission . figures 4b and 4c illustrate the relative contribution of each component over time in the targeted treatment scenarios . the baseline scenario ( without any targeted intervention ) , the dominant source of infections was proposed the target group . age or cohort targeting of hiv treatment greatly reduced the target group 's contribution to transmission . in the case of cohort targeting , the age range of those younger than the target group expanded as the group aged . in 2015 , the younger generation consisted of children under age five , and mother - to - child transmission was the only source of new infections . after reaching sexual maturity , they became infected by individuals in or older than the target group , before eventually reaching an age and prevalence level at which transmission within the generation became dominant . this explains the time - dependent sources of incidence in younger generations , shown in figure 4c . finally , we examined how hypothetical improvements to hiv programs might influence the ability to isolate hiv from future generations using the age targeting approach . in figure 5 and supplementary figure 1 , the incidence rates among individuals younger than the target group are shown in response to a variety of improvements to age - targeted or cohort - targeted treatment for prevention . to test the extent to which tools must be improved , we pushed the assumptions to their extreme , assuming that it would be possible to eliminate imperfections in already high - efficacy and high - coverage interventions . figure 5.impact of biomedical or programmatic improvements on the sources of new hiv infections in the generation younger than the target group . stacked bar charts show the rate of new hiv infections in future generations , defined as those younger than the targeted ( a ) age range or ( b ) birth cohort . new infections are color - coded by their source , with possible contributors being those older than the target group ( blue ) , those in the target group ( teal ) , those already infected in the younger generation itself ( yellow ) , and mother - to - child transmission from any age group ( maroon ) . the projections are shown for general art roll - out without any specific intervention for the target group ( 0 ) , achieving 80% coverage of annual testing and art for all new diagnoses in the target group ( 1 ) , further programmatic improvements in treatment as prevention the target group ( 2 ) , further biomedical improvements ( 3 ) , and a combination of programmatic and biomedical improvements ( 4 ) . the individual components of programmatic and biomedical improvements are not shown here , but are compared in supplementary figure 1 . impact of biomedical or programmatic improvements on the sources of new hiv infections in the generation younger than the target group . stacked bar charts show the rate of new hiv infections in future generations , defined as those younger than the targeted ( a ) age range or ( b ) birth cohort . new infections are color - coded by their source , with possible contributors being those older than the target group ( blue ) , those in the target group ( teal ) , those already infected in the younger generation itself ( yellow ) , and mother - to - child transmission from any age group ( maroon ) . the projections are shown for general art roll - out without any specific intervention for the target group ( 0 ) , achieving 80% coverage of annual testing and art for all new diagnoses in the target group ( 1 ) , further programmatic improvements in treatment as prevention the target group ( 2 ) , further biomedical improvements ( 3 ) , and a combination of programmatic and biomedical improvements ( 4 ) . the individual components of programmatic and biomedical improvements are not shown here , but are compared in supplementary figure 1 . for improved biomedical technologies , we first examined increasing the sensitivity of hiv diagnostic testing from 98 to 100% . we then examined increasing the effectiveness of antivirals for prevention of mother - to - child transmission from 90 to 100% . this reduced , but did not eliminate , mother - to - child transmission due to imperfect coverage . finally , we examined increasing the effectiveness of art at preventing transmission in couples from 92 to 100% . of all technological improvements , the prevention efficacy of art had the largest effect of protecting younger generations . as expected , the combination of improved testing and antiviral drugs had a greater impact than any intervention alone . we next examined hypothetical programmatic improvements . increasing the penetration of outreach in the target group from 80 to 100% reduced incidence in youth , as did eliminating delays between infection and treatment initiation . as with technological improvements , the two together reduced incidence in the younger generation more than each one did alone . no single improvement to a technology or program was sufficient to fully interrupt transmission in youth , nor were combinations of purely programmatic or purely technological solutions sufficient . the combination of all of the interventions discussed , both biomedical and programmatic , enabled the elimination of hiv in young generations by 2050 when targeting an age range of 2030 year olds . despite vigorous policy debate about whether an aids - free generation is within reach and the availability of models that include age patterns of hiv transmission , the concept of a demographic shield to protect future generations is relatively unexplored . we have used a network model to demonstrate how age - based targeting could as much as double cost - effectiveness of treatment for prevention , but would be insufficient to prevent hiv transmission to future generations until biomedical tools and programs are dramatically and simultaneously improved . the efficiency of targeting individuals in their twenties was consistent with our prior analysis of hiv transmission chains , which showed that this age group is vital for transmitting hiv from older to younger subsets of the hiv transmission network . sweep out hiv is less effective than simply shutting down transmission in a high - incidence age band . second , treatment as prevention is perhaps an even more powerful tool than primary hiv prevention for interrupting transmission , if used optimally . in figure 3 , we saw that perfect prevention in the 2030 age range was insufficient to protect those under age 20 from acquiring hiv . in contrast , in figure 5 we showed how an idealized treatment intervention would be able to do so , in large part because prevention alone would allow some transmissions to occur from individuals who were already infected when entering the target group . these include school - based programs , workplace wellness programs , youth centers and programs leveraging health care utilization such as antenatal care and sexually transmitted disease treatment . age targeting considerations could potentially help to prioritize and justify the expansion of outreach methods that can reach young adults . an important limitation of this modeling exercise is the assumption that the age- or birthdate - based targeting intervention had a precise cutoff date for eligibility , whereas realistic programs that use age to prioritize outreach would of undoubtedly have spillover effects into other age groups . we would therefore expect that the realistic efficiency gains from age targeting would be more modest than what is predicted by a model of ideal age targeting . an additional shortcoming of the model used for this exercise is the lack of consideration for outreach cost , which could differ for age - based targeting compared to universal expansion of treatment . this too would depend on the program used to access the age group of interest . future modeling studies could potentially explore the tradeoff between coverage , precision of targeting , and cost - effectiveness of outreach by different modalities . lastly , it is important to consider the broader uncertainty of model projects of hiv epidemics . the underlying drivers of generalized epidemics what causes hiv to spread in the general population in some settings and not others are still not well - understood . there is evidence of spatial and sociodemographic heterogeneities in hiv risk , implying hidden sub - epidemics embedded within generalized epidemics . these are still poorly characterized , and thus unlikely to be appropriately captured in existing mathematical models . data on past trends in partnership age gaps are limited and not necessarily predictive of future behavior . the model scenarios presented here were scaled to represent the demographics and baseline treatment program in south africa , but the age patterns represented in the model were measured in a small , rural hyperendemic region of kwazulu - natal , and not necessarily representative of mixing patterns at the national level . although a recent validation study found the self - reported mixing patterns to be relatively accurate , there may be types of relationships that are less likely to be reported and more difficult to validate through demographic surveillance . further , trends in past hiv incidence and self - reported partner choice do not necessarily predict future trends in partner choice . if age patterns of partnerships shift in the future , so too would the optimum age groups to target with intensified hiv services . more broadly , over the time horizons discussed in this analysis , model projections will need to be updated to reflect changes in the arrival of new biomedical tools and new information about the course of the hiv epidemic . as uninfected adolescents reach sexual maturity , primary hiv prevention for adolescents could be compounded with early treatment targeted to the most likely sources of future infections . our modeling exploration revealed that age - based targeting could double the cost - effectiveness of outreach with hiv treatment as prevention , provided that the cost to access the age groups of interest would be similar to that of reaching the general population . however , age - targeted outreach with current tools would be insufficient to fully protect future generations from hiv infection . dramatic but isolated improvements , such as reliable treatment regimens , full coverage in age groups of interest , or rapid detection of new hiv infections , would be insufficient to protect future generations from acquiring hiv . a combination of biomedical and programmatic improvements , applied consistently for more than three decades , would be required to interrupt transmission . these findings highlight the need for diverse investments in both quality and coverage of hiv treatment , as well as potential epidemiological efficiencies of programs targeting diverse populations , such as school - based programs , workplace wellness programs , youth centers and programs leveraging health care utilization such as antenatal care and treatment clinics for sexually transmitted diseases . as uninfected adolescents reach sexual maturity , primary hiv prevention for adolescents could be compounded with early treatment targeted to the most likely sources of future infections . our modeling exploration revealed that age - based targeting could double the cost - effectiveness of outreach with hiv treatment as prevention , provided that the cost to access the age groups of interest would be similar to that of reaching the general population . however , age - targeted outreach with current tools would be insufficient to fully protect future generations from hiv infection . dramatic but isolated improvements , such as reliable treatment regimens , full coverage in age groups of interest , or rapid detection of new hiv infections , would be insufficient to protect future generations from acquiring hiv . a combination of biomedical and programmatic improvements , applied consistently for more than three decades , would be required to interrupt transmission . these findings highlight the need for diverse investments in both quality and coverage of hiv treatment , as well as potential epidemiological efficiencies of programs targeting diverse populations , such as school - based programs , workplace wellness programs , youth centers and programs leveraging health care utilization such as antenatal care and treatment clinics for sexually transmitted diseases .

backgroundgeneralized hiv epidemics propagate to future generations according to the age patterns of transmission . we hypothesized that future generations could be protected from infection using age - targeted prevention , analogous to the ring - fencing strategies used to control the spread of smallpox.methodswe modeled age - targeted or cohort - targeted outreach with hiv treatment and/or prevention using emod - hiv v08 , an individual - based network model of hiv transmission in south africa.resultstargeting ages 20 to 30 with intensified outreach , linkage , and eligibility for antiretroviral therapy ( art ) averted 45% as many infections as universal outreach for approximately one - fifth the cost beyond existing hiv services . though cost - effective , targeting failed to eliminate all infections to those under 20 due to vertical and inter - generational transmission . cost - effectiveness of optimal prevention strategies included us$6238 per infection averted targeting ages 1030 , us$5031 targeting 2030 , us$4279 targeting 2227 , and us$3967 targeting 2527 , compared to us$10 812 for full - population test - and - treat . minimizing burden ( disability - adjusted life years [ dalys ] ) rather than infections resulted in older target age ranges because older adults were more likely to receive a direct health benefit from treatment.conclusionsage-targeted treatment for hiv prevention is unlikely to eliminate hiv epidemics , but is an efficient strategy for reducing new infections in generalized epidemics settings .

Introduction Methods Results Discussion Conclusions Supplementary Material

strategies to interrupt transmission of hiv are urgently needed , especially in generalized epidemic settings with extremely high hiv prevalence in the general population , high hiv - related disease burden and limited resources to identify , engage and retain infected individuals in care . in the long term , serving populations at highest risk of transmitting or acquiring hiv is necessary to reduce the total number of people who will require lifelong treatment , helping to close the gap between global treatment targets and present - day , substantially lower levels of hiv diagnosis , treatment , and viral suppression . in generalized epidemic settings , there is not yet an accepted strategy for identifying these sub - populations , but the patterns of generalized hiv epidemics provide clues about their characteristics . a distinct characteristic of generalized epidemics is sustained high hiv incidence in young women , leading epidemiologists to hypothesize that the age patterns of hiv transmission are central to fueling the epidemic and may be vulnerable to interventions that collapse the chains of hiv transmission . we have previously used mathematical modeling to investigate the age patterns of those most at risk of transmitting hiv to another individual , and further , those most likely to be part of an ongoing chain of transmission ( as opposed to a building upon those investigations , the current study explores whether age - targeted intensification of primary and/or secondary hiv prevention could collapse chains of transmission in generalized epidemic settings . the targeting approach explored here draws inspiration from the epidemiological ring - fencing methods used to control the spread of smallpox since the 1870s up until the final eradication activities in the 1970s . rather than the quarantine and vaccination approaches used to create a shield to prevent the spread of smallpox , intensified hiv treatment and prevention services would be used to create a longer - term demographic we explore a strategy of intensified hiv services for a specific age range , forming a static age band that separates older from younger individuals ( figure 1a ) . figure 1.schematic of age - based targeting and cohort - based targeting of outreach . teal regions show the age range of the ( a ) age or ( b ) birth cohort receiving intensified hiv services . people can age in and out of the target group with age targeting , but not with birth cohort targeting . teal regions show the age range of the ( a ) age or ( b ) birth cohort receiving intensified hiv services . the concept of a demographic shield to protect future generations is relatively unexplored even among available age - structured models of hiv transmission . we used emod - hiv v0.8 , an age - structured , individual - based network model of hiv in south africa , to model the impact of intensifying primary and/or secondary hiv prevention services for a specified age range ( figure 1a ) or birth cohort ( figure 1b ) . primary prevention is defined as a service targeted to an uninfected individual to prevent acquisition of disease , whereas secondary prevention is targeted to an infected individual to prevent transmission of disease . briefly , emod - hiv is an individual - based model that simulates transmission using an explicitly defined network of relationships that are formed according to preference patterns and dissolve according to age - dependent durations ( younger individuals tending to form shorter - term relationships ) . the age patterns of sexual mixing were configured to match those observed in a rural , hiv - hyperendemic region of kwazulu - natal , south africa . the model replicates the demographic patterns of south africa , explicitly simulating 1/200th of the south african population with age - dependent fertility and age / gender - dependent mortality . transmission rates within relationships depend on hiv disease stage , male circumcision , condom usage , co - infections , and antiretroviral therapy the latter causing the transmission rate to decline linearly over the first 6 months of therapy until viral suppression is achieved . the model includes a configurable health care system module , which we have configured to follow trends in antiretroviral therapy ( art ) expansion in south africa . treatment begins with voluntary counseling and testing ( vct ) , antenatal and infant testing , symptom - driven testing , and low level of couples testing . the model 's projections of baseline treatment expansion in south africa predict gradual incidence declines without elimination , so that hiv remains endemic through 2050 . the cost and hiv burden in the targeted treatment scenarios were compared to the baseline scenario using a unit cost and disability - adjusted life year ( daly ) model developed by the hiv modelling consortium for the 2013 who revision of hiv treatment guidelines . primary prevention was implemented as a decrease in the probability that an individual becomes infected , up to a 100% decrease corresponding to full protection . secondary prevention was assumed to consist of hiv testing at a rate of once per year , with art ) initiation regardless of cd4 count for those testing positive . these assumptions about the target group match those of a recent collaborative analysis of hypothetical universal test - and - treat for the entire population of south africa . in our analysis , we provide the full - population test - and - treat scenario as a reference point to compare to age - targeted test - and - treat . this provides a hypothetical maximum for cost and impact , and also provides a point of reference where our model has been compared to 11 other independently developed mathematical models . before entering and after leaving the target group , individuals exhibited eligibility , testing , and linkage rates identical to those used for the baseline scenario . a broad view of the model results is presented by scanning over starting and ending ages or years for a hypothetical age range ( figure 1a ) or birth cohort ( figure 1b ) , maintaining a range of two , five , 10 or 20 years from the youngest to the oldest age included in the target group . figure 2 shows the hiv disease burden relative to baseline , infections averted relative to baseline , incidence rate in the total population , and cost relative to baseline , assuming 80% penetration of outreach to the target group . figure 2 also shows a maximum - impact scenario in which the whole population received the intensified testing and treatment intervention , providing a reference point for a population - wide intervention without age - specific targeting . figure 2.effect of age - targeted treatment expansion on hiv incidence , cumulative infections averted , cumulative disability - adjusted life years ( dalys ) averted , and cumulative program cost . dots in panels b and f show the strategy with the minimum cost per infection averted over 20 years . effect of age - targeted treatment expansion on hiv incidence , cumulative infections averted , cumulative disability - adjusted life years ( dalys ) averted , and cumulative program cost . incidence is shown at 20 years after the intervention begins , and other outcomes are accumulated over the first 20 years with a 3% annual discount rate . at the left and right extremes of each plot , the curves converge to black lines showing the baseline ( no intervention ) scenario , which projects current guidelines and trends in hiv treatment . dots in panels b and f show the strategy with the minimum cost per infection averted over 20 years . for interventions involving the scale - up of treatment , we used a unit cost model to approximate the cost and cost - effectiveness of universal and targeted treatment for prevention . as expected , whole - population targeting was the most costly and highest - impact strategy , followed by 20-year age ranges spanning the highest - prevalence age groups . focusing on maximizing the cumulative infections averted , the dots in figure 2b show the most cost - effective ranges to target : ages 1030 at us$6238 per infection averted , ages 2030 at us$5031 per infection averted , ages 2227 at us$4279 per infection averted , and ages 2527 at us$3967 per infection averted . similarly , for infections averted by birth cohort targeting shown in figure 2f , the most cost - effective birth year ranges are marked at 19852005 at us$6826 per infection averted , 19871997 at us$5856 per infection averted , and 19901995 at us$5686 per infection averted . universal expansion to all ages / cohorts cost us$10 812 per infection averted . compared to universal expansion , targeting the 10-year age range of 2030 cost us$5.4 billion over 20 years and provided more than double the number of infections averted per us$ invested . for cumulative dalys averted by age targeting shown in figure 2c , dots show the most cost - effective ranges to target : ages 2444 at us$1489 per daly averted , 2434 at us$1441 per daly averted , 2631 at us$1372 per daly averted , and 2729 at us$1285 per daly averted . similarly , for birth cohort targeting in figure 2 g , the most cost - effective birth year ranges are marked at 19611981 at us$1396 per infection averted , 19681978 at us$1374 per infection averted , and 19721977 at us$1373 per infection averted . the most cost - effective 10-year age range for infections averted ( 2030 ) more than halved the cost per daly averted , whereas the most cost - effective 10-year age range for dalys averted ( 2434 ) reduced the cost per daly averted by only 17% . the most efficient age range for averting dalys was older than for averting infections , because dalys are averted by preventing new infections in the long run , but also by providing a direct health benefit to those receiving treatment in the short run . the health benefit is greatest for those who are most likely to progress quickly from asymptomatic hiv infection to symptomatic aids : older individuals , who experience faster disease progression and are more likely to have been infected for a longer period of time . having identified the 2030 age group as an efficient target for prevention , we then selected this age range as a hypothetical target group , and examined the age pattern of hiv incidence in the broader population to determine whether efficient targeting of treatment expansion to ages 2030 had the potential to interrupt hiv transmission to younger individuals . figure 3a shows the age pattern of incidence for baseline trends in hiv treatment . our model predicted a slow decline and slight aging of the pattern of hiv incidence , with hiv remaining endemic in the year 2050 . figure 3.age distribution of hiv incidence for the baseline simulation ( a ) , targeted treatment outreach ( b ) , and targeted complete prevention of hiv infection ( c ) . shaded areas show the age range of the target group throughout the intervention ( left ) or for a birth cohort in the year of implementation ( start ) and 35 years after implementation ( end ) . age distribution of hiv incidence for the baseline simulation ( a ) , targeted treatment outreach ( b ) , and targeted complete prevention of hiv infection ( c ) . incidence is shown just before implementation of the intervention , and at 5 , 10 , 20 and 35 years after implementation of the intervention . when individuals aged 2030 were targeted with hiv treatment , even at an optimistic penetration level of 80% for outreach with testing and linkage to care , incidence declined across all age groups but fell short of completely eliminating hiv by 2050 ( figure 3b , left panel ) . by 2030 , incidence in the overall population was 37% lower than that of the baseline simulation , while incidence in those under age 20 was 60% lower than in the baseline scenario . thus , we next tested a strategy of birth cohort targeting for to those born in the years 2000 through 2010 . the targeted individuals would begin reaching sexual debut as the intervention was launched , and the 10-year targeted band would age through the population , with the intention of using a ring - fence like approach to the model results showed the cohort - based targeting approach to be less effective than age targeting ( figure 3b , right panel ) . by the year 2030 ( the year in which the target group reached 2030 years of age ) , incidence among those under 20 years of age was only 29% lower than in the baseline scenario , and incidence in the overall population was only 11% lower than in the baseline scenario . we further augmented the simulations of age - targeted treatment with a hypothetical prevention intervention to the same target group ( figure 3c ) . the extreme assumption of 100% coverage with perfect hiv prevention for those aged 2030 still did not eliminate hiv infections among those younger than age 20 , although incidence was greatly diminished . by the year 2030 , incidence emerged in young females at a rate of 005015% per year , and similar levels later emerged in young males . finally we addressed the question of how hiv the epidemic reached youth , even with a complete one - decade gap in hiv transmission . in figure 4a , these modalities are illustrated in blue , green , yellow , and red , respectively . figure 4.sources of new hiv infections in the generation younger than the target group . the relative contribution from each of these four mechanisms was estimated by categorizing transmission events in the model according to their origin : infection by a sex partner older than , in , or younger than the target group , or mother - to - child transmission . age or cohort targeting of hiv treatment greatly reduced the target group 's contribution to transmission . in 2015 , the younger generation consisted of children under age five , and mother - to - child transmission was the only source of new infections . finally , we examined how hypothetical improvements to hiv programs might influence the ability to isolate hiv from future generations using the age targeting approach . in figure 5 and supplementary figure 1 , the incidence rates among individuals younger than the target group are shown in response to a variety of improvements to age - targeted or cohort - targeted treatment for prevention . figure 5.impact of biomedical or programmatic improvements on the sources of new hiv infections in the generation younger than the target group . stacked bar charts show the rate of new hiv infections in future generations , defined as those younger than the targeted ( a ) age range or ( b ) birth cohort . new infections are color - coded by their source , with possible contributors being those older than the target group ( blue ) , those in the target group ( teal ) , those already infected in the younger generation itself ( yellow ) , and mother - to - child transmission from any age group ( maroon ) . the projections are shown for general art roll - out without any specific intervention for the target group ( 0 ) , achieving 80% coverage of annual testing and art for all new diagnoses in the target group ( 1 ) , further programmatic improvements in treatment as prevention the target group ( 2 ) , further biomedical improvements ( 3 ) , and a combination of programmatic and biomedical improvements ( 4 ) . impact of biomedical or programmatic improvements on the sources of new hiv infections in the generation younger than the target group . stacked bar charts show the rate of new hiv infections in future generations , defined as those younger than the targeted ( a ) age range or ( b ) birth cohort . new infections are color - coded by their source , with possible contributors being those older than the target group ( blue ) , those in the target group ( teal ) , those already infected in the younger generation itself ( yellow ) , and mother - to - child transmission from any age group ( maroon ) . the projections are shown for general art roll - out without any specific intervention for the target group ( 0 ) , achieving 80% coverage of annual testing and art for all new diagnoses in the target group ( 1 ) , further programmatic improvements in treatment as prevention the target group ( 2 ) , further biomedical improvements ( 3 ) , and a combination of programmatic and biomedical improvements ( 4 ) . for improved biomedical technologies , we first examined increasing the sensitivity of hiv diagnostic testing from 98 to 100% . this reduced , but did not eliminate , mother - to - child transmission due to imperfect coverage . finally , we examined increasing the effectiveness of art at preventing transmission in couples from 92 to 100% . despite vigorous policy debate about whether an aids - free generation is within reach and the availability of models that include age patterns of hiv transmission , the concept of a demographic shield to protect future generations is relatively unexplored . we have used a network model to demonstrate how age - based targeting could as much as double cost - effectiveness of treatment for prevention , but would be insufficient to prevent hiv transmission to future generations until biomedical tools and programs are dramatically and simultaneously improved . the efficiency of targeting individuals in their twenties was consistent with our prior analysis of hiv transmission chains , which showed that this age group is vital for transmitting hiv from older to younger subsets of the hiv transmission network . second , treatment as prevention is perhaps an even more powerful tool than primary hiv prevention for interrupting transmission , if used optimally . in figure 3 , we saw that perfect prevention in the 2030 age range was insufficient to protect those under age 20 from acquiring hiv . an important limitation of this modeling exercise is the assumption that the age- or birthdate - based targeting intervention had a precise cutoff date for eligibility , whereas realistic programs that use age to prioritize outreach would of undoubtedly have spillover effects into other age groups . an additional shortcoming of the model used for this exercise is the lack of consideration for outreach cost , which could differ for age - based targeting compared to universal expansion of treatment . this too would depend on the program used to access the age group of interest . future modeling studies could potentially explore the tradeoff between coverage , precision of targeting , and cost - effectiveness of outreach by different modalities . lastly , it is important to consider the broader uncertainty of model projects of hiv epidemics . these are still poorly characterized , and thus unlikely to be appropriately captured in existing mathematical models . the model scenarios presented here were scaled to represent the demographics and baseline treatment program in south africa , but the age patterns represented in the model were measured in a small , rural hyperendemic region of kwazulu - natal , and not necessarily representative of mixing patterns at the national level . if age patterns of partnerships shift in the future , so too would the optimum age groups to target with intensified hiv services . as uninfected adolescents reach sexual maturity , primary hiv prevention for adolescents could be compounded with early treatment targeted to the most likely sources of future infections . our modeling exploration revealed that age - based targeting could double the cost - effectiveness of outreach with hiv treatment as prevention , provided that the cost to access the age groups of interest would be similar to that of reaching the general population . however , age - targeted outreach with current tools would be insufficient to fully protect future generations from hiv infection . dramatic but isolated improvements , such as reliable treatment regimens , full coverage in age groups of interest , or rapid detection of new hiv infections , would be insufficient to protect future generations from acquiring hiv . these findings highlight the need for diverse investments in both quality and coverage of hiv treatment , as well as potential epidemiological efficiencies of programs targeting diverse populations , such as school - based programs , workplace wellness programs , youth centers and programs leveraging health care utilization such as antenatal care and treatment clinics for sexually transmitted diseases . as uninfected adolescents reach sexual maturity , primary hiv prevention for adolescents could be compounded with early treatment targeted to the most likely sources of future infections . our modeling exploration revealed that age - based targeting could double the cost - effectiveness of outreach with hiv treatment as prevention , provided that the cost to access the age groups of interest would be similar to that of reaching the general population . however , age - targeted outreach with current tools would be insufficient to fully protect future generations from hiv infection . these findings highlight the need for diverse investments in both quality and coverage of hiv treatment , as well as potential epidemiological efficiencies of programs targeting diverse populations , such as school - based programs , workplace wellness programs , youth centers and programs leveraging health care utilization such as antenatal care and treatment clinics for sexually transmitted diseases .

recent estimates suggest that 15.4% of us women aged 50 years or older have osteoporosis ( op ) and an additional 51.4% have evidence of low bone density.1 an increased risk of fracture is the most significant consequence of op and low bone density . there were an estimated 2 million op - related fractures in the usa in 2005 and the number of fractures is projected to increase to more than 3 million annually by 2025.2 pharmacologic treatment for op can reduce fracture risk , and is appropriate for many patients with op . a previous study concluded that 30% of us women aged 50 years or older warrant consideration for op treatment , based on their risk for fracture.3 currently , several therapies are approved for op treatment by the us food and drug administration to reduce fracture risk , including bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) , calcitonin , raloxifene , teriparatide , and denosumab.4 despite the availability of multiple treatment options , recent studies indicate a substantial proportion of us women at risk of fracture remain untreated . in the us cohort of the global longitudinal study of women , only 65% of women with a self - reported diagnosis of op reported taking anti - resorptive treatment.5 studies based on electronic medical records or administrative claims documentation of pharmacologic op treatment initiation suggest under - treatment is considerably greater.6,7 in a pennsylvania study , less than 20% of women who had experienced a fracture were receiving oral bisphosphonate in the year following the fracture , and only 49% of women with an op diagnosis or t - score indicative of op received bisphosphonate treatment within 1 year of their diagnosis or bone mineral density test result.6 a study of a national us claims database reported that only 36% of women had evidence of receipt of any op treatment in the first year following their op diagnosis.7 the barriers to treatment initiation are not well understood . certain patient characteristics appear to decrease the likelihood of treatment , including younger age , lower education level , higher bone mineral density t - score , higher body mass index , and lack of concomitant corticosteroid use.6,8,9 patient concern over op medication side effects has also been linked with nontreatment initiation.10 physicians may also be unwilling to prescribe op treatment for their patients with op due to concurrent medical conditions . bisphosphonates are the most widely prescribed therapy and contraindicated for patients with severe renal insufficiency . yet , a study of electronic medical records found that the most common reason ( 28% ) for not prescribing bisphosphonates to patients diagnosed with op was a preexisting gastrointestinal ( gi ) diagnosis.11 among patients who do initiate op treatment , gi intolerance has also been cited as a common cause for op treatment discontinuation.1214 however , the risk of gi events while on op treatment is also a function of preexisting gi conditions.15 the relationship between preexisting gi events and treatment initiation has not been well explored in a managed care population in the usa . the objective of this study was to examine the association between preexisting gi events and likelihood of treatment initiation among women diagnosed with op within a managed care setting in the usa . a retrospective cohort study was conducted using a large national claims database known as the optum insight clinformatics data mart . this database was designed to primarily support health care outcomes research and other research initiatives . the database includes demographic , medical , and pharmacy claims for more than 80 million managed care enrollees in more than 45 health plans throughout the usa . over 3 million patients with op were included in the database during the study period from january 2001 to december 2010 . all study data were de - identified and accessed using techniques compliant with the health insurance portability and accountability act ; therefore , institutional review board approval was not required . within the study period january 1 , 2001 to december 31 , 2010 an index date was assigned during january 1 , 2002 to december 31 , 2009 . the 1-year period before the index date represented the pre - index period , while the post - index period began the day after the index date and lasted for 12 months or until the date that op medications were initiated , if less than 12 months . therefore , patients who did not initiate op treatment had a full 12 months of follow - up , while those who initiated treatment had less than 12 months of follow - up . women aged 55 years or older on the index date with a primary or secondary diagnosis of op ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 733.0x ) were included . patients were required to be continuously enrolled in the health plan for at least 1 year before and 1 year after the index date , and to be op treatment - nave , defined as no claims for op medications prior to the index date . patients were excluded if they had evidence of estrogen therapy in the pre - index period , a diagnosis of malignant neoplasm ( icd-9-cm codes 140170 , 173208 , 230239 ) in the pre - index period or in the 1 year after the index date , or any claim for paget s disease ( icd-9-cm code 731.0 ) ever in the claims history . the occurrence of a gi event was defined by the presence of a claim with a gi - related icd-9-cm diagnostic or current procedural terminology code ( see table s1 ) , including esophagitis , esophageal reflux , ulcer , stricture , perforation , hemorrhage of esophagus , gastric ulcer , duodenal ulcer , peptic ulcer , acute gastritis , duodenitis , gi hemorrhage , nausea or vomiting , and dysphagia . gi events were separately captured for the pre - index and post - index periods . patients who received at least one op medication during the post - index period were characterized as treated , while all others were considered to be untreated . op medications were identified based on national drug codes and healthcare common procedure coding system codes , and included all forms ( oral , injectable , infused ) of bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) and all forms of non - bisphosphonates ( calcitonin , raloxifene , and teriparitide ) . denosumab was excluded because it was not approved until june 2010 , near the end of the study period . patient age was determined on the index date . the pre - index charlson comorbidity index ( cci ) score was computed.16 the cci is used to predict the mortality of a patient based on the occurrence of 19 predefined comorbid conditions . the presence of comorbid conditions , fracture history , and medication use ( gastroprotective agents , nonsteroidal anti - inflammatory drugs [ nsaids ] , and glucocorticoids ) were also determined during the pre - index period . fractures during the pre - index period were identified from icd-9-cm codes ( table s1 ) . patient characteristics were summarized for the entire cohort as well as by the presence or absence of a pre - index gi event . for continuous variables ( age and cci score ) , means and standard deviations were calculated . the number and proportion of patients with pre - index comorbid conditions , pre - index fractures , pre - index medication use , and pre - index and post - index gi events were tabulated . comparisons of baseline characteristics between patients with and without a pre - index gi event were performed using tests for categorical variables and t - tests for continuous variables . to determine the timing and type of op treatment initiation , prescription claims after the index date were searched , and used to classify patients as receiving no treatment , bisphosphonates , or the proportion of patients in each classification was calculated and compared between patients with and without post - index gi events . tests were used to compare the treatment distributions of patients with and without gi events . time - varying cox regression ( which takes into consideration the timing of the post - index gi events before treatment initiation ) was used to estimate the likelihood of treatment initiation for any op treatment , stratified by the presence of pre - index gi events . for treated patients , a discrete choice model was used to estimate the likelihood of receiving a bisphosphonate versus a non - bisphosphonate . in the cox model and the discrete choice model , the independent variable of interest was the presence of a post - index gi event ; models were also adjusted for the presence of pre - index gi events ( discrete choice model only ) , age group , pre - index medication use ( gastroprotective agents , nsaids , glucocorticosteroids ) , pre - index cci , and selected pre - index comorbidities ( chronic inflammatory bowel disease , chronic inflammatory joint disease , celiac disease , diabetes , depression , chronic kidney failure , hypertension , gi mucositis and urination problems , hyperparathyroidism , vitamin d deficiency , and fatigue ) . the cox regression model separately quantified the effects of a post - index gi event for those with and without pre - index gi events . a retrospective cohort study was conducted using a large national claims database known as the optum insight clinformatics data mart . this database was designed to primarily support health care outcomes research and other research initiatives . the database includes demographic , medical , and pharmacy claims for more than 80 million managed care enrollees in more than 45 health plans throughout the usa . over 3 million patients with op were included in the database during the study period from january 2001 to december 2010 . all study data were de - identified and accessed using techniques compliant with the health insurance portability and accountability act ; therefore , institutional review board approval was not required . within the study period january 1 , 2001 to december 31 , 2010 an index date was assigned during january 1 , 2002 to december 31 , 2009 . the 1-year period before the index date represented the pre - index period , while the post - index period began the day after the index date and lasted for 12 months or until the date that op medications were initiated , if less than 12 months . therefore , patients who did not initiate op treatment had a full 12 months of follow - up , while those who initiated treatment had less than 12 months of follow - up . women aged 55 years or older on the index date with a primary or secondary diagnosis of op ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 733.0x ) were included . patients were required to be continuously enrolled in the health plan for at least 1 year before and 1 year after the index date , and to be op treatment - nave , defined as no claims for op medications prior to the index date . patients were excluded if they had evidence of estrogen therapy in the pre - index period , a diagnosis of malignant neoplasm ( icd-9-cm codes 140170 , 173208 , 230239 ) in the pre - index period or in the 1 year after the index date , or any claim for paget s disease ( icd-9-cm code 731.0 ) ever in the claims history . the occurrence of a gi event was defined by the presence of a claim with a gi - related icd-9-cm diagnostic or current procedural terminology code ( see table s1 ) , including esophagitis , esophageal reflux , ulcer , stricture , perforation , hemorrhage of esophagus , gastric ulcer , duodenal ulcer , peptic ulcer , acute gastritis , duodenitis , gi hemorrhage , nausea or vomiting , and dysphagia . gi events were separately captured for the pre - index and post - index periods . patients who received at least one op medication during the post - index period were characterized as treated , while all others were considered to be untreated . op medications were identified based on national drug codes and healthcare common procedure coding system codes , and included all forms ( oral , injectable , infused ) of bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) and all forms of non - bisphosphonates ( calcitonin , raloxifene , and teriparitide ) . denosumab was excluded because it was not approved until june 2010 , near the end of the study period . patient age was determined on the index date . the pre - index charlson comorbidity index ( cci ) score was computed.16 the cci is used to predict the mortality of a patient based on the occurrence of 19 predefined comorbid conditions . the presence of comorbid conditions , fracture history , and medication use ( gastroprotective agents , nonsteroidal anti - inflammatory drugs [ nsaids ] , and glucocorticoids ) were also determined during the pre - index period . fractures during the pre - index period were identified from icd-9-cm codes ( table s1 ) . patient characteristics were summarized for the entire cohort as well as by the presence or absence of a pre - index gi event . for continuous variables ( age and cci score ) , means and standard deviations were calculated . the number and proportion of patients with pre - index comorbid conditions , pre - index fractures , pre - index medication use , and pre - index and post - index gi events were tabulated . comparisons of baseline characteristics between patients with and without a pre - index gi event were performed using tests for categorical variables and t - tests for continuous variables . to determine the timing and type of op treatment initiation , prescription claims after the index date were searched , and used to classify patients as receiving no treatment , bisphosphonates , or the proportion of patients in each classification was calculated and compared between patients with and without post - index gi events . tests were used to compare the treatment distributions of patients with and without gi events . time - varying cox regression ( which takes into consideration the timing of the post - index gi events before treatment initiation ) was used to estimate the likelihood of treatment initiation for any op treatment , stratified by the presence of pre - index gi events . for treated patients , a discrete choice model was used to estimate the likelihood of receiving a bisphosphonate versus a non - bisphosphonate . in the cox model and the discrete choice model , the independent variable of interest was the presence of a post - index gi event ; models were also adjusted for the presence of pre - index gi events ( discrete choice model only ) , age group , pre - index medication use ( gastroprotective agents , nsaids , glucocorticosteroids ) , pre - index cci , and selected pre - index comorbidities ( chronic inflammatory bowel disease , chronic inflammatory joint disease , celiac disease , diabetes , depression , chronic kidney failure , hypertension , gi mucositis and urination problems , hyperparathyroidism , vitamin d deficiency , and fatigue ) . the cox regression model separately quantified the effects of a post - index gi event for those with and without pre - index gi events . the average age at diagnosis was 65.7 years , and 17,828 patients ( 27.3% ) experienced a gi event during the pre - index period ( table 1 ) . among all patients , the most common comorbid conditions were hypertension ( 48% ) , chronic inflammatory joint disease ( 21% ) , and fatigue ( 16% ) . patients with a pre - index gi event were more likely to have each of the comorbid conditions evaluated than patients without a pre - index gi event ( p<0.001 ) . patients with a pre - index gi event were more likely to use gastro - protective agents ( 37.8% versus 7.3% ) , nsaids ( 26.4% versus 19.8% ) , and glucocorticoids ( 18.8% versus 11.7% ) than patients without a pre - index gi event ( p<0.001 ) . the occurrence of a gi event during the pre - index gi event was also associated with a higher mean cci score ( 1.01 versus 0.52 , p<0.001 ) and a higher rate of fractures ( 9.0% versus 6.0% , p<0.001 ) . the frequency of op treatment within this cohort has been previously reported.7 briefly , 42,033 ( 64.3% ) patients received no op medication within the first year following the op diagnosis . a total of 23,311 ( 35.7% ) patients received treatment : 20,200 ( 30.9% ) with a bisphosphonate and 3,111 ( 4.9% ) with a non - bisphosphonate.7 overall , 24% of all patients experienced a gi event in the post - index period , although gi events were more common among those who had experienced a pre - index gi event ( 43.4% versus 16.2% , table 2 ) . the rate of pre - index gi events was similar among patients who did and did not initiate treatment ( bisphosphonate or non - bisphosphonate , figure 2a ) . however , the occurrence of post - index gi events appeared to be associated with op treatment initiation ( figure 2b ) . the proportion of patients receiving no op treatment was 85.1% among patients with a post - index gi event versus 57.9% among patients with no post - index gi event . among treated patients , 11.7% of patients with a post - index gi event initiated treatment with a bisphosphonate compared with 36.9% of patients without a post - index gi event . the results of the time - varying cox regression model for the association between post - index gi events and any op treatment ( bisphosphonate or non - bisphosphonate ) initiation are shown in table 3 . among all covariates included in the analysis , the occurrence of post - index gi events had the greatest impact on op treatment initiation . patients who experienced post - index gi events were 75% less likely to initiate op treatment than patients without post - index gi events ( hazard ratio 0.25 ; 95% confidence interval [ ci ] 0.240.26 ; p<0.0001 ) . hypertension , chronic inflammatory joint disease , and diabetes were also significantly associated with a lower likelihood of op treatment initiation ( p0.04 ) , while depression and use of gastroprotective agents , nsaids , and glucocorticoids were associated with a higher likelihood of treatment initiation ( p<0.001 ) . the results of the discrete choice model among patients who initiated op treatment are shown in table 4 . patients with a post - index gi event were 39% less likely to receive a bisphosphonate than a non - bisphosphonate compared with those without a post - index gi event ( odds ratio 0.61 ; 95% ci 0.540.68 ; p<0.0001 ) . a pre - index gi event , older age , and use of gastroprotective agents also significantly decreased the likelihood of bisphosphonate treatment initiation but to a lesser extent than post - index gi events . comorbid diabetes predicted a slightly higher likelihood of treatment initiation with a bisphosphonate , and gi mucositis and urinary problems were associated with a lower likelihood of treatment initiation with a bisphosphonate . the average age at diagnosis was 65.7 years , and 17,828 patients ( 27.3% ) experienced a gi event during the pre - index period ( table 1 ) . among all patients , the most common comorbid conditions were hypertension ( 48% ) , chronic inflammatory joint disease ( 21% ) , and fatigue ( 16% ) . patients with a pre - index gi event were more likely to have each of the comorbid conditions evaluated than patients without a pre - index gi event ( p<0.001 ) . patients with a pre - index gi event were more likely to use gastro - protective agents ( 37.8% versus 7.3% ) , nsaids ( 26.4% versus 19.8% ) , and glucocorticoids ( 18.8% versus 11.7% ) than patients without a pre - index gi event ( p<0.001 ) . the occurrence of a gi event during the pre - index gi event was also associated with a higher mean cci score ( 1.01 versus 0.52 , p<0.001 ) and a higher rate of fractures ( 9.0% versus 6.0% , p<0.001 ) . the frequency of op treatment within this cohort has been previously reported.7 briefly , 42,033 ( 64.3% ) patients received no op medication within the first year following the op diagnosis . a total of 23,311 ( 35.7% ) patients received treatment : 20,200 ( 30.9% ) with a bisphosphonate and 3,111 ( 4.9% ) with a non - bisphosphonate.7 overall , 24% of all patients experienced a gi event in the post - index period , although gi events were more common among those who had experienced a pre - index gi event ( 43.4% versus 16.2% , table 2 ) . the rate of pre - index gi events was similar among patients who did and did not initiate treatment ( bisphosphonate or non - bisphosphonate , figure 2a ) . however , the occurrence of post - index gi events appeared to be associated with op treatment initiation ( figure 2b ) . the proportion of patients receiving no op treatment was 85.1% among patients with a post - index gi event versus 57.9% among patients with no post - index gi event . among treated patients , 11.7% of patients with a post - index gi event initiated treatment with a bisphosphonate compared with 36.9% of patients without a post - index gi event . the results of the time - varying cox regression model for the association between post - index gi events and any op treatment ( bisphosphonate or non - bisphosphonate ) initiation are shown in table 3 . among all covariates included in the analysis , the occurrence of post - index gi events had the greatest impact on op treatment initiation . patients who experienced post - index gi events were 75% less likely to initiate op treatment than patients without post - index gi events ( hazard ratio 0.25 ; 95% confidence interval [ ci ] 0.240.26 ; p<0.0001 ) . hypertension , chronic inflammatory joint disease , and diabetes were also significantly associated with a lower likelihood of op treatment initiation ( p0.04 ) , while depression and use of gastroprotective agents , nsaids , and glucocorticoids were associated with a higher likelihood of treatment initiation ( p<0.001 ) . the results of the discrete choice model among patients who initiated op treatment are shown in table 4 . patients with a post - index gi event were 39% less likely to receive a bisphosphonate than a non - bisphosphonate compared with those without a post - index gi event ( odds ratio 0.61 ; 95% ci 0.540.68 ; p<0.0001 ) . a pre - index gi event , older age , and use of gastroprotective agents also significantly decreased the likelihood of bisphosphonate treatment initiation but to a lesser extent than post - index gi events . comorbid diabetes predicted a slightly higher likelihood of treatment initiation with a bisphosphonate , and gi mucositis and urinary problems were associated with a lower likelihood of treatment initiation with a bisphosphonate . in this population of us women diagnosed with op , a majority ( 64.3% ) received no op medication in the year following their op diagnosis . gi events in the post - index period strongly decreased the likelihood of initiating any treatment for op . among patients who initiated treatment , the presence of a gi event in the post - index period also decreased the likelihood of treatment with a bisphosphonate versus a non - bisphosphonate . while published data regarding the association of gi events and op treatment is limited , some research suggests a possible relationship . in a study of elderly women who had recently experienced a fracture , preexisting gi disease was associated with a modest but nonsignificantly lower odds of receiving treatment.17 foster et al reported that certain pre existing gi conditions increased the likelihood of treatment with raloxifene versus bisphosphonates in a commercial / medicare cohort ( but not a medicaid cohort).18 however , the proportion of patients who experienced a post - index gi event prior to treatment initiation in our study was significantly higher than that observed by foster et al ( 24% versus 1%3% ) . in addition , the definition of what constituted a gi event differed , as we used a more comprehensive series of codes and examined the collective impact of all gi events on choice of op treatment , rather than individual categories of gi events ( eg , peptic ulcer and gastric ulcer ) . our results indicate that baseline gi events did not appear to impact treatment initiation , but gi events that more closely coincided with op diagnosis ( ie , post - index gi events ) strongly decreased the likelihood of treatment initiation . these results suggest that the clinical management of patients diagnosed with op should take into account not only gi event history but also gi events that may occur after op diagnosis . twenty - seven percentage of patients in this study had a gi event prior to their op diagnosis and 23.7% experienced a gi event after their op diagnosis ( but before op treatment initiation ) . in the possible - us study , approximately 20% of patients initiating op medication reported gi symptoms at study entry.19 the higher rate of gi events we observed may be a function of the length of the observation period for gi events and the definition of gi events . we captured baseline gi events during the full 12-month period prior to diagnosis and post - op diagnosis events up until treatment initiation or 12 months for patients who did not initiate treatment . in contrast , the possible - us study identified gi symptoms at study entry and was based on patient report . irrespective of the methodologic differences , the results of both studies indicate that gi events are common among women with op . we found other patient characteristics also predicted op treatment initiation , but to a lesser extent than post - index gi events . use of gastroprotective agents , nsaids , and glucocorticoids prior to op diagnosis were each significantly associated with an increased likelihood of receiving op treatment . previous studies have noted a similar association between glucocorticoid or corticosteroid use and treatment initiation.6,17,20 although a higher comorbidity score was not associated with lower likelihood of treatment initiation , the presence of specific comorbid conditions ( hypertension , chronic inflammatory disease , and diabetes ) independently predicted lower likelihood of op treatment initiation . considering that diabetes and hypertension are common in the us population21,22 and that diabetes in particular has been linked with an increased risk of fracture in older women,2325 the potential impact of comorbid conditions on treatment initiation warrants further research . the reasons for not initiating treatment may lie with a physician s decision to not prescribe or patients not electing to receive treatment . a study of electronic medical records revealed that gi diagnoses were the most common ( 28% ) reason for not prescribing oral bisphosphonates to patients diagnosed with op , followed by functional status ( 24% ) and renal impairment ( 12%).11 patients who did not initiate op treatment are more likely to report a concern over op medication side effects than patients who begin treatment,10 and the risk of gi side effects in particular may also influence patient treatment preferences.26 in a discrete choice experiment of preferred op treatments , concern over the risk of gi side effects ranked higher than other side effects evaluated.26 patients with preexisting gi disorders prior to treatment also have a high risk of experiencing gi events while on treatment,15 and gi side effects while on treatment are associated with worse adherence to treatment27 and greater likelihood of early treatment discontinuation.19,28,29 our results coupled with previous research suggest that gi events may pose a significant barrier to effective treatment of op . this study is subject to the limitations inherent in a retrospective analysis of a claims database . claims data are collected primarily for payment purposes , not research , and are subject to coding errors . the presence of a claim for a filled prescription does not necessarily indicate that the medication was taken or that it was taken as prescribed . however , different bisphosphonates may have different adverse effect profiles,30 and the timing ( eg , daily versus monthly ) and type ( oral versus intravenous ) of the dosing regimen may be a determinant of these profiles.12 the length of the study period ( 10 years ) includes a span during which clinical patterns of bisphosphonate use may have been changing from daily to weekly regimens.31 from the available data , we are not able to ascertain why op treatment was not initiated ; it may reflect a clinical decision not to prescribe treatment or the failure of a patient to fill a prescription . over - the - counter drugs such as low - dose nsaids and aspirin are not included in this analysis . only gi events that resulted in medical service utilization were captured in this analysis ; therefore , the association observed may reflect more severe gi events . also , bone mineral density test results to support the claims diagnosis of op were not available in the database . finally , there may have been other unmeasured patient and clinician characteristics that influenced op treatment initiation and receipt of a bisphosphonate versus a non - bisphosphonate treatment . this study advances the understanding of how gi events impact treatment of op . in a us managed care population of women with an incident diagnosis of op , approximately one of four patients diagnosed with op had a gi event post diagnosis and before treatment initiation , of whom 85% did not receive any treatment . patients with a post - diagnosis gi event were 75% less likely to initiate treatment ( versus those without a gi event ) . among patients who initiated op treatment , post - diagnosis gi events were associated with a 39% lower likelihood of initiation with a bisphosphonate compared with a non - bisphosphonate therapy . new therapies may be needed to address the unmet need for op treatments for patients with prior and existing gi events . codes used to identify gastrointestinal events , fractures , and use of bisphosphonates and non - bisphosphonates abbreviations : cpt , current procedural terminology ; gi , gastrointestinal ; icd-9-cm dx , international classification of diseases , ninth revision , clinical modification diagnosis codes ; kub , kidney , ureter , and bladder x - ray ; nec , not elsewhere classifiable ; nos , not otherwise specified .

backgroundpreexisting gastrointestinal ( gi ) events may deter the use of pharmacologic treatment in patients diagnosed with osteoporosis ( op ) . the objective of this study was to examine the association between preexisting gi events and op pharmacotherapy initiation among women diagnosed with op.methodsthe study utilized claims data from a large us managed care database to identify women aged 55 years with a diagnosis code for op ( index date ) during 20022009 . patients with a claim for pharmacologic op treatment in the 12-month pre - index period ( baseline ) were excluded . op treatment initiation in the post - index period was defined as a claim for bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) , calcitonin , raloxifene , or teriparatide . during the post - index period ( up to 12 months ) , gi events were identified before treatment initiation . a time - dependent cox regression model was used to investigate the likelihood of initiating any op treatment . among patients initiating op treatment , a discrete choice model was utilized to assess the relationship between post - index gi events and likelihood of initiating with a bisphosphonate versus a non-bisphosphonate.resultsin total , 65,344 patients ( mean age 66 years ) were included ; 23.7% had a gi event post diagnosis and before treatment initiation . post - index gi events were associated with a 75% lower likelihood of any treatment initiation ( hazard ratio 0.25 ; 95% confidence interval 0.240.26 ) . among treated patients ( n=23,311 ) , those with post - index gi events were 39% less likely to receive a bisphosphonate versus a non - bisphosphonate ( odds ratio 0.61 ; 95% confidence interval 0.540.68).conclusiongi events after op diagnosis were associated with a decreased likelihood of op treatment initiation and an increased likelihood of treatment initiation with a non - bisphosphonate versus a bisphosphonate .

Introduction Materials and methods Study design Study sample Measures Statistical analysis Results Patient characteristics Osteoporosis treatment GI events and OP treatment initiation Multivariate analysis for association between GI events and OP treatment initiation Discussion Conclusion Supplementary material

recent estimates suggest that 15.4% of us women aged 50 years or older have osteoporosis ( op ) and an additional 51.4% have evidence of low bone density.1 an increased risk of fracture is the most significant consequence of op and low bone density . a previous study concluded that 30% of us women aged 50 years or older warrant consideration for op treatment , based on their risk for fracture.3 currently , several therapies are approved for op treatment by the us food and drug administration to reduce fracture risk , including bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) , calcitonin , raloxifene , teriparatide , and denosumab.4 despite the availability of multiple treatment options , recent studies indicate a substantial proportion of us women at risk of fracture remain untreated . in the us cohort of the global longitudinal study of women , only 65% of women with a self - reported diagnosis of op reported taking anti - resorptive treatment.5 studies based on electronic medical records or administrative claims documentation of pharmacologic op treatment initiation suggest under - treatment is considerably greater.6,7 in a pennsylvania study , less than 20% of women who had experienced a fracture were receiving oral bisphosphonate in the year following the fracture , and only 49% of women with an op diagnosis or t - score indicative of op received bisphosphonate treatment within 1 year of their diagnosis or bone mineral density test result.6 a study of a national us claims database reported that only 36% of women had evidence of receipt of any op treatment in the first year following their op diagnosis.7 the barriers to treatment initiation are not well understood . yet , a study of electronic medical records found that the most common reason ( 28% ) for not prescribing bisphosphonates to patients diagnosed with op was a preexisting gastrointestinal ( gi ) diagnosis.11 among patients who do initiate op treatment , gi intolerance has also been cited as a common cause for op treatment discontinuation.1214 however , the risk of gi events while on op treatment is also a function of preexisting gi conditions.15 the relationship between preexisting gi events and treatment initiation has not been well explored in a managed care population in the usa . the objective of this study was to examine the association between preexisting gi events and likelihood of treatment initiation among women diagnosed with op within a managed care setting in the usa . the 1-year period before the index date represented the pre - index period , while the post - index period began the day after the index date and lasted for 12 months or until the date that op medications were initiated , if less than 12 months . women aged 55 years or older on the index date with a primary or secondary diagnosis of op ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 733.0x ) were included . patients were excluded if they had evidence of estrogen therapy in the pre - index period , a diagnosis of malignant neoplasm ( icd-9-cm codes 140170 , 173208 , 230239 ) in the pre - index period or in the 1 year after the index date , or any claim for paget s disease ( icd-9-cm code 731.0 ) ever in the claims history . the occurrence of a gi event was defined by the presence of a claim with a gi - related icd-9-cm diagnostic or current procedural terminology code ( see table s1 ) , including esophagitis , esophageal reflux , ulcer , stricture , perforation , hemorrhage of esophagus , gastric ulcer , duodenal ulcer , peptic ulcer , acute gastritis , duodenitis , gi hemorrhage , nausea or vomiting , and dysphagia . op medications were identified based on national drug codes and healthcare common procedure coding system codes , and included all forms ( oral , injectable , infused ) of bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) and all forms of non - bisphosphonates ( calcitonin , raloxifene , and teriparitide ) . the number and proportion of patients with pre - index comorbid conditions , pre - index fractures , pre - index medication use , and pre - index and post - index gi events were tabulated . to determine the timing and type of op treatment initiation , prescription claims after the index date were searched , and used to classify patients as receiving no treatment , bisphosphonates , or the proportion of patients in each classification was calculated and compared between patients with and without post - index gi events . time - varying cox regression ( which takes into consideration the timing of the post - index gi events before treatment initiation ) was used to estimate the likelihood of treatment initiation for any op treatment , stratified by the presence of pre - index gi events . for treated patients , a discrete choice model was used to estimate the likelihood of receiving a bisphosphonate versus a non - bisphosphonate . in the cox model and the discrete choice model , the independent variable of interest was the presence of a post - index gi event ; models were also adjusted for the presence of pre - index gi events ( discrete choice model only ) , age group , pre - index medication use ( gastroprotective agents , nsaids , glucocorticosteroids ) , pre - index cci , and selected pre - index comorbidities ( chronic inflammatory bowel disease , chronic inflammatory joint disease , celiac disease , diabetes , depression , chronic kidney failure , hypertension , gi mucositis and urination problems , hyperparathyroidism , vitamin d deficiency , and fatigue ) . the cox regression model separately quantified the effects of a post - index gi event for those with and without pre - index gi events . the 1-year period before the index date represented the pre - index period , while the post - index period began the day after the index date and lasted for 12 months or until the date that op medications were initiated , if less than 12 months . women aged 55 years or older on the index date with a primary or secondary diagnosis of op ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] code 733.0x ) were included . patients were excluded if they had evidence of estrogen therapy in the pre - index period , a diagnosis of malignant neoplasm ( icd-9-cm codes 140170 , 173208 , 230239 ) in the pre - index period or in the 1 year after the index date , or any claim for paget s disease ( icd-9-cm code 731.0 ) ever in the claims history . op medications were identified based on national drug codes and healthcare common procedure coding system codes , and included all forms ( oral , injectable , infused ) of bisphosphonates ( alendronate , ibandronate , risedronate , zoledronic acid ) and all forms of non - bisphosphonates ( calcitonin , raloxifene , and teriparitide ) . to determine the timing and type of op treatment initiation , prescription claims after the index date were searched , and used to classify patients as receiving no treatment , bisphosphonates , or the proportion of patients in each classification was calculated and compared between patients with and without post - index gi events . time - varying cox regression ( which takes into consideration the timing of the post - index gi events before treatment initiation ) was used to estimate the likelihood of treatment initiation for any op treatment , stratified by the presence of pre - index gi events . for treated patients , a discrete choice model was used to estimate the likelihood of receiving a bisphosphonate versus a non - bisphosphonate . in the cox model and the discrete choice model , the independent variable of interest was the presence of a post - index gi event ; models were also adjusted for the presence of pre - index gi events ( discrete choice model only ) , age group , pre - index medication use ( gastroprotective agents , nsaids , glucocorticosteroids ) , pre - index cci , and selected pre - index comorbidities ( chronic inflammatory bowel disease , chronic inflammatory joint disease , celiac disease , diabetes , depression , chronic kidney failure , hypertension , gi mucositis and urination problems , hyperparathyroidism , vitamin d deficiency , and fatigue ) . the cox regression model separately quantified the effects of a post - index gi event for those with and without pre - index gi events . the average age at diagnosis was 65.7 years , and 17,828 patients ( 27.3% ) experienced a gi event during the pre - index period ( table 1 ) . patients with a pre - index gi event were more likely to have each of the comorbid conditions evaluated than patients without a pre - index gi event ( p<0.001 ) . patients with a pre - index gi event were more likely to use gastro - protective agents ( 37.8% versus 7.3% ) , nsaids ( 26.4% versus 19.8% ) , and glucocorticoids ( 18.8% versus 11.7% ) than patients without a pre - index gi event ( p<0.001 ) . the occurrence of a gi event during the pre - index gi event was also associated with a higher mean cci score ( 1.01 versus 0.52 , p<0.001 ) and a higher rate of fractures ( 9.0% versus 6.0% , p<0.001 ) . a total of 23,311 ( 35.7% ) patients received treatment : 20,200 ( 30.9% ) with a bisphosphonate and 3,111 ( 4.9% ) with a non - bisphosphonate.7 overall , 24% of all patients experienced a gi event in the post - index period , although gi events were more common among those who had experienced a pre - index gi event ( 43.4% versus 16.2% , table 2 ) . however , the occurrence of post - index gi events appeared to be associated with op treatment initiation ( figure 2b ) . the proportion of patients receiving no op treatment was 85.1% among patients with a post - index gi event versus 57.9% among patients with no post - index gi event . among treated patients , 11.7% of patients with a post - index gi event initiated treatment with a bisphosphonate compared with 36.9% of patients without a post - index gi event . the results of the time - varying cox regression model for the association between post - index gi events and any op treatment ( bisphosphonate or non - bisphosphonate ) initiation are shown in table 3 . patients who experienced post - index gi events were 75% less likely to initiate op treatment than patients without post - index gi events ( hazard ratio 0.25 ; 95% confidence interval [ ci ] 0.240.26 ; p<0.0001 ) . hypertension , chronic inflammatory joint disease , and diabetes were also significantly associated with a lower likelihood of op treatment initiation ( p0.04 ) , while depression and use of gastroprotective agents , nsaids , and glucocorticoids were associated with a higher likelihood of treatment initiation ( p<0.001 ) . patients with a post - index gi event were 39% less likely to receive a bisphosphonate than a non - bisphosphonate compared with those without a post - index gi event ( odds ratio 0.61 ; 95% ci 0.540.68 ; p<0.0001 ) . a pre - index gi event , older age , and use of gastroprotective agents also significantly decreased the likelihood of bisphosphonate treatment initiation but to a lesser extent than post - index gi events . comorbid diabetes predicted a slightly higher likelihood of treatment initiation with a bisphosphonate , and gi mucositis and urinary problems were associated with a lower likelihood of treatment initiation with a bisphosphonate . the average age at diagnosis was 65.7 years , and 17,828 patients ( 27.3% ) experienced a gi event during the pre - index period ( table 1 ) . patients with a pre - index gi event were more likely to have each of the comorbid conditions evaluated than patients without a pre - index gi event ( p<0.001 ) . patients with a pre - index gi event were more likely to use gastro - protective agents ( 37.8% versus 7.3% ) , nsaids ( 26.4% versus 19.8% ) , and glucocorticoids ( 18.8% versus 11.7% ) than patients without a pre - index gi event ( p<0.001 ) . the occurrence of a gi event during the pre - index gi event was also associated with a higher mean cci score ( 1.01 versus 0.52 , p<0.001 ) and a higher rate of fractures ( 9.0% versus 6.0% , p<0.001 ) . a total of 23,311 ( 35.7% ) patients received treatment : 20,200 ( 30.9% ) with a bisphosphonate and 3,111 ( 4.9% ) with a non - bisphosphonate.7 overall , 24% of all patients experienced a gi event in the post - index period , although gi events were more common among those who had experienced a pre - index gi event ( 43.4% versus 16.2% , table 2 ) . however , the occurrence of post - index gi events appeared to be associated with op treatment initiation ( figure 2b ) . the proportion of patients receiving no op treatment was 85.1% among patients with a post - index gi event versus 57.9% among patients with no post - index gi event . among treated patients , 11.7% of patients with a post - index gi event initiated treatment with a bisphosphonate compared with 36.9% of patients without a post - index gi event . the results of the time - varying cox regression model for the association between post - index gi events and any op treatment ( bisphosphonate or non - bisphosphonate ) initiation are shown in table 3 . patients who experienced post - index gi events were 75% less likely to initiate op treatment than patients without post - index gi events ( hazard ratio 0.25 ; 95% confidence interval [ ci ] 0.240.26 ; p<0.0001 ) . hypertension , chronic inflammatory joint disease , and diabetes were also significantly associated with a lower likelihood of op treatment initiation ( p0.04 ) , while depression and use of gastroprotective agents , nsaids , and glucocorticoids were associated with a higher likelihood of treatment initiation ( p<0.001 ) . patients with a post - index gi event were 39% less likely to receive a bisphosphonate than a non - bisphosphonate compared with those without a post - index gi event ( odds ratio 0.61 ; 95% ci 0.540.68 ; p<0.0001 ) . a pre - index gi event , older age , and use of gastroprotective agents also significantly decreased the likelihood of bisphosphonate treatment initiation but to a lesser extent than post - index gi events . comorbid diabetes predicted a slightly higher likelihood of treatment initiation with a bisphosphonate , and gi mucositis and urinary problems were associated with a lower likelihood of treatment initiation with a bisphosphonate . gi events in the post - index period strongly decreased the likelihood of initiating any treatment for op . among patients who initiated treatment , the presence of a gi event in the post - index period also decreased the likelihood of treatment with a bisphosphonate versus a non - bisphosphonate . in a study of elderly women who had recently experienced a fracture , preexisting gi disease was associated with a modest but nonsignificantly lower odds of receiving treatment.17 foster et al reported that certain pre existing gi conditions increased the likelihood of treatment with raloxifene versus bisphosphonates in a commercial / medicare cohort ( but not a medicaid cohort).18 however , the proportion of patients who experienced a post - index gi event prior to treatment initiation in our study was significantly higher than that observed by foster et al ( 24% versus 1%3% ) . our results indicate that baseline gi events did not appear to impact treatment initiation , but gi events that more closely coincided with op diagnosis ( ie , post - index gi events ) strongly decreased the likelihood of treatment initiation . twenty - seven percentage of patients in this study had a gi event prior to their op diagnosis and 23.7% experienced a gi event after their op diagnosis ( but before op treatment initiation ) . use of gastroprotective agents , nsaids , and glucocorticoids prior to op diagnosis were each significantly associated with an increased likelihood of receiving op treatment . previous studies have noted a similar association between glucocorticoid or corticosteroid use and treatment initiation.6,17,20 although a higher comorbidity score was not associated with lower likelihood of treatment initiation , the presence of specific comorbid conditions ( hypertension , chronic inflammatory disease , and diabetes ) independently predicted lower likelihood of op treatment initiation . a study of electronic medical records revealed that gi diagnoses were the most common ( 28% ) reason for not prescribing oral bisphosphonates to patients diagnosed with op , followed by functional status ( 24% ) and renal impairment ( 12%).11 patients who did not initiate op treatment are more likely to report a concern over op medication side effects than patients who begin treatment,10 and the risk of gi side effects in particular may also influence patient treatment preferences.26 in a discrete choice experiment of preferred op treatments , concern over the risk of gi side effects ranked higher than other side effects evaluated.26 patients with preexisting gi disorders prior to treatment also have a high risk of experiencing gi events while on treatment,15 and gi side effects while on treatment are associated with worse adherence to treatment27 and greater likelihood of early treatment discontinuation.19,28,29 our results coupled with previous research suggest that gi events may pose a significant barrier to effective treatment of op . finally , there may have been other unmeasured patient and clinician characteristics that influenced op treatment initiation and receipt of a bisphosphonate versus a non - bisphosphonate treatment . in a us managed care population of women with an incident diagnosis of op , approximately one of four patients diagnosed with op had a gi event post diagnosis and before treatment initiation , of whom 85% did not receive any treatment . patients with a post - diagnosis gi event were 75% less likely to initiate treatment ( versus those without a gi event ) . among patients who initiated op treatment , post - diagnosis gi events were associated with a 39% lower likelihood of initiation with a bisphosphonate compared with a non - bisphosphonate therapy .

dysphagia ( swallowing impairment ) is a serious condition of increasing burden and importance in our ageing society ( gonzalez - fernandez & daniels , 2008 ; humbert & robbins , 2008 ; kikawada , iwamoto , & takasaki , 2005 ; pikus , levine , yang , rubesin , katzka , & laufer , 2003 ) . it is estimated that 3778% of the 750,000 americans who suffer a stroke each year will experience dysphagia ( martino , foley , bhogal , diamant , speechley , & teasell , 2005 ; www.stroke.org ) and that 4354% of these individuals will experience aspiration ( entry of foreign material into the airway ) ( daniels , brailey , priestly , herrington , weisberg , & foundas , 1998 ; horner , massey , riski , lathrop , & chase , 1988 ) . stroke patients who aspirate face 11.56-times the risk of developing pneumonia , compared to those without dysphagia ( martino et al . , 2005 ) . in the acquired brain injury ( abi ) population , dysphagia has a similar reported incidence of 4161% ( halper , cherney , cichowski & zhang , 1999 ; winstein , 1983 ) , with videofluoroscopically - confirmed aspiration incidence reported at 38% ( lazarus & logemann , 1987 ) . impaired tongue function is a prominent component in neurogenic dysphagia ; the ability to generate tongue - pressure plays a primary role in controlling the flow of liquids through the mouth and pharynx ( pouderoux & kahrilas , 1995 ) . the tongue is also responsible for initiating the driving forces that propel liquids and foods through the oropharynx into the esophagus ( kahrilas , lin , logemann , ergun , & facchini , 1993 ) . when tongue - pressure generation is impaired , liquids may spill into the pharynx before the airway is protected , or bolus clearance may be impaired , leaving residue in the pharynx . in both of these situations , there is a heightened risk of aspiration ( marik & kaplan , 2003 ; martino et al . , 2005 ; pikus et al . , palate pressures during the performance of effortful swallows enhances the generation of increased pharyngeal pressures and speeds up propagation of the pharyngeal pressure wave ( huckabee & steele , 2006 ; steele & huckabee , 2007 ) . palate pressure generation plays a pivotal role in establishing the overall strength of a swallow , and establishes a theoretical basis for using tongue the most common intervention for managing aspiration risk in oropharyngeal dysphagia is to prescribe thickened liquids and texture - modified foods ( garcia , chambers , & molander , 2005 ) . unfortunately , texture - modifications are reported to be unpalatable and patients are prone to dehydration and inadequate nutritional intake ( colodny , 2005 ; robbins , gensler , hind , logemann , lindblad , & brandt , 2008 ; sharpe , ward , cichero , sopade , & halley , 2007 ) . consequently , it is important that dysphagia researchers explore treatments that facilitate the return of functional swallowing without requiring texture modification . in this study , we describe treatment outcomes for a tongue - pressure resistance training intervention . over the past decade , the field of dysphagia intervention research has turned to using principles of exercise physiology in treatment design . robbins developed an 8-week tongue - pressure resistance training protocol , which she applied in healthy seniors and individuals with dysphagia post - stroke ( robbins , gangnon , theis , kays , hewitt , & hind , 2005 ; robbins , kays , gangnon , hind , hewitt , & gentry , 2007 ) . the robbins protocol employs elements of exercise load , intensity , and treatment duration , intended to support physiological changes in tongue muscle strength and capacity . patients practice repeated tongue - pressure tasks , with targets set between 6080% of their maximum capacity , on a schedule requiring 6090 task repetitions on 3 non - consecutive days of the week . . effectively demonstrated that tongue - pressure resistance training builds tongue strength . in a group of 10 patients with stroke - related dysphagia of up to 3 months duration , anterior maximum isometric tongue pressures ( mips ) , i.e. , the highest pressure achieved during a maximum effort task while compressing a palatal pressure sensor with the tongue , improved by 44% vs baseline , while posterior mips increased by 83% ( robbins et al . , 2007 ) . unfortunately , there was less convincing evidence that functional swallowing improvements accompanied these strength changes . post - treatment videofluoroscopy showed a significant groupwise improvement in aspiration , but there were no systematic changes in pharyngeal residues . quite why this pattern of change was observed remains unclear , but the possibility that spontaneous recovery played some role in these outcomes can not be dismissed . swallowing is recognized to be a sub - maximal pressure task ( nicosia , hind , roecker , carnes , doyle , & dengel , 2000 ) . although a decline in mips is seen with ageing in healthy individuals , this is not seen in the context of swallowing ( nicosia et al . , 2000 ; given this situation , we speculated that the task of trying to hit variable amplitude targets precisely , without over - shoot or under - shoot , might involve skills similar to those involved in varying tongue pressures for liquid bolus control in swallowing . we designed a modification to the robbins protocol , emphasizing amplitude accuracy targets in the 2090% of mip range in addition to strength targets ( tongue - pressure strength and accuracy training , or tpsat ) . we hoped that the modification of including variable target practice would still facilitate strength gains , while achieving improved functional outcomes for safe swallowing and residue clearance . this manuscript describes a preliminary prospective case series of six patients with dysphagia secondary to acquired brain injury who completed this protocol . we were interested in determining whether the tpsat protocol would yield improvements in three areas : ( 1 ) tongue strength for mips and saliva swallows ; ( 2 ) swallowing safety ; and ( 3 ) swallowing efficiency . we enrolled six consenting participants with dysphagia secondary to acquired brain injury following a motor vehicle accident , with injuries sustained either as a driver , passenger or pedestrian . table i provides summary information about these individuals , including the available medical history information regarding the nature of their brain injuries . all participants were at least 5 months post - onset and had no pre - accident history of dysphagia . the inclusion criteria for the study were as follows : ( 1 ) impaired swallowing safety , i.e. , scores 3 on the 8-point penetration - aspiration scale ( rosenbek , robbins , roecker , coyle , & wood , 1996 ) with thin liquids ; and ( 2 ) post - swallow residues in the valleculae or pyriform sinuses with either thin and/or spoon - thick liquids measured using a 4-point ordinal scale described by eisenhuber , schima , schober , pokieser , stadler , and scharitzer ( 2002 ) : 0 = none , 1 = thin coating , 2 = 2550% full , 3 50% full . table ii provides an overview of the videofluoroscopy profiles of each participant at baseline , reflecting the worst scores received by each participant for each bolus consistency on the three parameters of interest . apenetration aspiration was rated using the 8-point penetration aspiration scale ( rosenbek et al . , 1996 ) in which a score of 1 reflects normal swallowing safety , a score of 2 indicates high penetration of the supraglottic space with subsequent ejection , scores of 35 indicate laryngeal penetration , and 68 indicate aspiration of material below the true vocal folds . bresidues in the valleculae and pyriform sinuses were rated using a 4-point ordinal scale described by eisenhuber et al . ( 2002 ) in which 0 = no residue , 1 = thin coating , 2 = 2550% full , and 3 50% full . all participants received 24 sessions of treatment , scheduled twice weekly ( with a third session occasionally scheduled within the same week ) , and spanning a total of 1112 weeks . this schedule was dictated by practical constraints due to the fact that several of the patients were commuting as outpatients to the hospital to participate . figure 1 outlines the design of a treatment session , which involved a total of 60 tongue - pressure tasks , arranged in blocks of six , with individual task repetitions separated by a minimum 10-second rest period . all tasks were performed using the iowa oral performance instrument ( iopi ) , a hand - held manometer connected to a 2.7 millilitre air - filled pressure bulb that was positioned between the tongue and hard palate in either an anterior or posterior oral position . the first two task - blocks in the session were mips ( anterior and posterior ) , from which 20% and 90% of mip values were calculated . for the variable accuracy tasks that followed , the clinician randomly selected variable target values between these 20% and 90% boundaries and instructed the participant to try to hit each target pressure amplitude as accurately as possible . feedback was provided to the participant after each task repetition , by telling them the pressure amplitude shown on the iopi . equal time was spent on strength tasks ( mips ) and target accuracy tasks in each session . additionally , a generalization task of saliva swallows was included with the iopi bulb placed in the anterior position . these swallows were cued saliva swallows with as much time allowed as required between task repetitions . the bulb was removed between each swallow and repositioned , thereby allowing a brief rest and allowing the patient to indicate when they were ready to proceed . single - subject descriptive methods were used to report treatment progress for the tongue pressure parameters , which were probed at each treatment session . a control chart method with effect - size banding ( nourbakhsh & ottenbacher , 1994 ) was used to confirm the presence or absence of change , as follows : ( 1 ) measures of baseline tongue pressure range , by task , were computed using mean and standard deviation values for each participant for all mip tasks across the first three treatment sessions ( n = 36 in the anterior and 36 in the posterior sensor positions ) . ( 2 ) means and standard deviations for tongue - pressure amplitudes were calculated separately by task from the iopi data collected during each session : 12 anterior mips , 12 posterior mips , and 12 non - effortful saliva swallows . these values were plotted on control charts to document change in tongue pressure parameters over time ( see figure 2 ) . palate pressures over the course of therapy . dashed horizontal lines indicate a moderate effect size band around baseline performance , used as a threshold to determine whether there was evidence of change in the form of at least three consecutive data points exceeding the effect size band . ( 3 ) an effect size band of cohen 's d = 0.6 , i.e. , a strong effect ( kotrlik & williams , 2003 ) , was established around these baseline mean values and plotted using dashed lines above and below the baseline reference range on the control charts . ( 4 ) an a priori criterion for concluding that change in mip values had occurred was established as evidence of three or more consecutive data points falling outside the effect - size band boundaries . measures of functional swallowing outcome were derived from post - treatment videofluoroscopies ( compared to baseline ) , performed according to a standard protocol using a 40% weight / volume thin liquid solution of polibar ( bracco imaging s.p.a . , princeton , new jersey ) and water , and a spoon - thick liquid prepared using ez - hd barium power ( bracco ) , mixed in 40% weight / volume ratio with applesauce . the standard protocol included three repetitions of each of these stimuli , all delivered in 5-ml volumes using a spoon and swallowed using a command swallow in head neutral position without any compensations . additional tasks were included at clinician discretion , and included other consistencies ( nectar - thick , honey - thick , or solid consistencies ) and the exploration of compensatory maneouvres , but these were not considered for the purposes of measuring treatment outcome in the research study . the videofluoroscopic measures of interest were the 8-point penetration - aspiration scale ( rosenbek et al . , 1996 ) and 4-point ordinal ratings of residue in the valleculae and pyriform sinuses ( eisenhuber et al . ratings were made by two experienced speech - language pathologists , who had initially been trained to consensus in the scoring of these features using a training set of three videofluoroscopy recordings from patients not involved in this study . the videofluoroscopy recordings of the head - neutral , non - compensated thin liquid and spoon - thick liquid swallows were spliced into bolus - specific clips . these were arranged in random order and divided into two rating sets with 20% overlap between raters for the purposes of measuring inter - rater agreement . using this partial reliability dataset , intra - class coefficients ( icc ) for inter - rater agreement were strong for all measures : penetration - aspiration icc = .937 , 95% ci = .86.97 ; vallecular residue icc = .94 , 95% ci = .86.97 ; pyriform sinus residue , icc = .917 , 95% ci = .81.96 ) . based on this strong performance for the reliability dataset , it was decided that duplicate ratings of the entire dataset across both raters was not necessary . disagreements for individual ratings within the reliability dataset were resolved by consensus . for the purposes of the research study , it was decided a priori that the worst scores seen across all swallows for a given bolus consistency would be used to represent a patient 's functional swallowing status at each time - point ( pre- , post - treatment ) . table iii summarizes mean and standard deviation values for the three tongue - pressure parameters of interest , calculated across the baseline timeframe spanning the first three treatment sessions for each participant . figures 24 provide a graphic summary of the progression of tongue pressure measures for each participant across the course of the study . baseline measures of tongue strength ( means and standard deviations ) calculated over the first three treatment sessions , with 12 repetitions of each task per session . the charts showing anterior ( figure 2 ) and posterior maximum isometric pressures ( figure 3 ) are graphed using a uniform y - axis range of 080 kpa . for the reader 's reference , it may be useful to note that previous studies have suggested that the lower boundary for normal anterior isometric pressures in elderly men falls at 40 kpa ( nicosia et al . , 2000 ) . in the case of this study , it can be appreciated that all participants except participants 2 and 4 began treatment with anterior mips below this threshold . over the course of treatment , increased anterior mips ( sustained above the cohen 's d = 0.6 effect size boundary for at least three consecutive sessions ) can be seen for participants 1 , 2 , 3 , 4 , and 5 . participants 1 and 4 failed to maintain these gains across the entire course of treatment . participant 6 showed an unusual pattern with respect to anterior mips , which increased over the first 10 sessions , but then declined below the d = .6 effect size boundary for the second half of treatment , and displayed much greater variability as seen in the standard deviation error bars shown on the chart . this suggests that some change in the strategy used to perform mips occurred after the first 10 sessions , rendering the initial baseline measures inappropriate as a comparison for the second half of treatment . palate pressures over the course of therapy . dashed horizontal lines indicate a moderate effect size band around baseline performance , used as a threshold to determine whether there was evidence of change in the form of at least three consecutive data points exceeding the effect size band . at baseline , increases in posterior tongue palate pressures , beyond the d = .6 effect size boundary , were seen over at least three consecutive treatment sessions for all six participants ; however , sustained gains were not seen for participant 1 . saliva swallowing pressures are plotted in figure 4 using a uniform y - axis range of 050 kpa , based on evidence that saliva swallow pressures typically fall below 50% of mips ( nicosia et al . , 2000 ) . for the six participants in this study , saliva swallow pressures were at or below 20 kpa in all cases at baseline . the only exception to this was seen in participant 5 , who was unable to produce volitional saliva swallows at baseline . when these emerged at around session 10 , his saliva swallow pressures were in the range of 3040 kpa and similar in amplitude to his mips . increases in saliva swallowing pressures , beyond the effect size boundary over at least three consecutive sessions were seen for participants 1 , 2 , and 6 , but gains were not clearly sustained for participants 2 and 6 . participants 3 , 4 , and 5 failed to demonstrate any notable changes in saliva swallowing pressures . control charts showing progress in saliva swallowing pressures over the course of therapy . dashed horizontal lines indicate a moderate effect size band around baseline performance , used as a threshold to determine whether there was evidence of change in the form of at least three consecutive data points exceeding the effect size band . table iv summarizes treatment outcomes in terms of the functional swallowing measures derived from the videofluoroscopies in this study . when improvement was seen compared to baseline , this is indicated by a black font on a white background ; deterioration is indicated using a white font on a black background , while an absence of change is shown using a black font on a grey background . roman text in the table reflect a functional swallowing improvement in comparison to the pre - treatment videofluoroscopy , while italics reflect no noticeable change and underlined text indicates deterioration in function for that bolus / parameter combination , relative to the baseline evaluation . penetration aspiration was rated using the 8-point penetration aspiration scale ( rosenbek et al . , 1996 ) in which a score of 1 reflects normal swallowing safety , a score of 2 indicates high penetration of the supraglottic space with subsequent ejection , scores of 35 indicate laryngeal penetration , and 68 indicate aspiration of material below the true vocal folds . residues in the valleculae and pyriform sinuses were rated using a 4-point ordinal scale described by eisenhuber et al . ( 2002 ) in which 0 = no residue , 1 = thin coating , 2 = 2550% full , and 3 50% full . improved swallowing safety for thin liquids was seen post - treatment for participants 1 , 2 , 4 , 5 , and 6 , but remained unchanged for participant 3 . impaired swallowing safety for spoon - thick liquids was a concern at baseline in participants 1 , 2 , 3 , and 6 ; post - treatment improvement was seen in all four of these participants . however , it should also be noted that two participants who had shown baseline safety ( scores of 1 ) with spoon - thick liquids showed new evidence of impaired swallowing safety with this consistency on their post - treatment videofluoroscopies ; participant 4 showed high penetration ( score of 2 ) , while participant 5 showed silent aspiration ( score of 8) . vallecular residue with thin liquids was considered problematic at baseline ( i.e. , scores of 2 or higher ) in participants 1 , 4 , and 6 , but remained unchanged post - treatment in all three cases . in participant 3 , baseline evidence of mild vallecular residue ( i.e. , a score of 1 ) with thin liquids actually worsened to moderate residue post - treatment ( i.e. , progression from a score of 1 to 2 ) . with spoon - thick liquids , vallecular residue was a concern at baseline for participants 1 , 3 , 4 , and 6 . scores remained unchanged post - treatment for participants 3 and 6 , but deteriorated by one grade of severity on the eisenhuber rating scale in both participants 1 and 4 . pyriform sinus residue scores of 2 or higher for thin liquids were seen at baseline in participants 1 and 2 . participant 2 showed post - treatment improvement in this measure , while participant 1 showed deterioration by one grade of severity to a post - treatment score of 3 . additionally , post - treatment measures for participant 4 revealed moderate pyriform residue for thin liquids , when only mild residue had been seen at baseline , a worsening from a score of 12 . pyriform sinus residue scores of 2 or higher for spoon - thick liquids at baseline were seen in participants 1 , 2 , and 4 . post - treatment , this feature remained unchanged in participant 2 , while participants 1 and 4 showed a worsening to scores of 3 . overall , these results show improvement in swallowing safety with thin liquids for the majority , and improved safety with spoon - thick liquids in about half of these patients . changes in post - swallow vallecular and pyriform sinus residues are much less apparent , and there is no trend of improvement in this group of patients . indeed , the data suggest that there was worsening of bolus clearance in many of these patients at the end of therapy . our results show that all six participants in this study achieved increases in either anterior and/or posterior tongue strength on mips post - treatment . first of all , improvements were achieved in individuals who had dysphagia of long - standing ( minimum 5 months ) . even the participants with dysphagia of longer than 18 months chronicity demonstrated convincing improvements in both anterior and posterior tongue strength , thereby speaking to the ongoing plasticity of the tongue and its potential to respond to exercise training in this population . secondly , the observed improvements in strength are interesting given that the focus of our treatment was equally distributed between maximum isometric tasks and accuracy tasks requiring the participant to hit sub - maximal pressure targets as precisely as possible . this finding is reminiscent of findings in the limb rehabilitation literature , where it has been shown that strength gains can arise from skill - focused exercise ( monfils & teskey , 2004 ; winstein , rose , tan , lewthwaite , chui , & azen , 2004 ) . taken together with the previous results reported by robbins et al . ( 2007 ) , these data confirm that tongue - pressure resistance exercises , practiced 23-times weekly , with amplitude targets tailored to the patient 's maximum pressure capacity , are an effective method for increasing tongue strength in individuals with neurogenic dysphagia . unfortunately , the types and degree of functional swallowing changes that accompanied these improvements in tongue strength are less clear . in this study five of the six patients displaying aspiration on thin liquids at baseline showed resolution of this impairment at the post - treatment videofluoroscopy . ( 2007 ) , despite the fact that our participants had dysphagia of longer chronicity than the stroke patients in her study . nevertheless , it must be acknowledged that spontaneous recovery may have contributed to the improvements seen in our participants and it remains unclear to what extent the improvement in aspiration can be attributed to tongue - pressure resistance training . in contrast , in the current data set , we did not observe any positive trends with respect to improved bolus clearance and reduced residues in the valleculae and pyriform sinuses . in fact , the videofluoroscopy results appear to show deterioration in bolus clearance function in five of our six participants , and there is no apparent pattern to these results with respect to dysphagia chronicity or severity , in terms of pre - treatment diet tolerance . these results are disappointing and raise challenging questions regarding the possibility that there was some negative impact with this treatment protocol . on the one hand , the lack of improvement in residue clearance adds support to the speculation that spontaneous recovery may have contributed to some extent to the observed improvements in swallowing safety . on the other hand , it must be remembered that the observed results reflect each participant 's worst score across a series of three thin liquid and three spoon - thick liquid boluses at each assessment . the apparent worsening may simply reflect variability in swallowing function within these participants within a limited videofluoroscopy protocol , given the long - standing nature of their dysphagia . we feel that it would be an over - interpretation of these data to conclude that these individuals showed functional deterioration in their swallowing , even in the presence of at least one residue score at the post - treatment videofluoroscopy that was more severe than that observed at baseline . however , the fact that residues appeared to be resistant to change in the presence of increased tongue pressures does raise questions about one of the underlying assumptions of this study , namely that tongue driving force contributes to effective bolus clearance from the pharynx ( dejaeger , pelemans , ponette , & joosten , 1997 ) . our data suggest that the tongue - pressure resistance exercises practiced in this study did not yield generalized improvements in pharyngeal swallowing strength , although we had hoped to see such generalization based on evidence regarding the impact of effortful swallows involving heightened tongue pressure generation on pharyngeal pressure generation and propagation ( huckabee & steele , 2006 ; steele & huckabee , 2007 ) . fortunately , perhaps , we do not appear to be alone in our failure to achieve positive results with respect to bolus clearance through exercise - based approaches to swallowing therapy . ( 2007 ) similarly failed to find convincing evidence of improvements in bolus clearance with their tongue pressure training protocol , even given shorter chronicity of dysphagia and a clearer emphasis on building tongue strength in their protocol . similarly , prior studies of the shaker exercise and neuromuscular electrical stimulation have also failed to show convincing evidence of physiological improvements in bolus clearance in patients with neurogenic dysphagia ( blow , speyer , baijens , woisard , & ekberg , 2008 ; logemann , rademaker , pauloski , kelly , stangl - mcbreen , & antinoja , 2009 ; robbins et al . , 2007 ) . overall , the less - than - satisfactory findings in these six participants suggest a need to better understand the role that tongue pressure generation plays in relation to other functional aspects of swallowing physiology , such as pharyngeal bolus clearance . for example , there may be aspects of the oral phase of swallowing that are more sensitive to the influence of tongue pressure amplitudes than the parameters measured in our study . indeed , our original plan had been to include measures related to oral control of the bolus in this study , but we determined that the literature lacked evidence regarding valid quantitative measures of bolus control that would lend themselves to reflecting the possible influence of treatment . stage transition duration , for example , is a quantitative measure that captures the interval following arrival of the bolus in the pharynx prior to the onset of hyolaryngeal motion associated with the pharyngeal phase of the swallow ( lof & robbins , 1990 ) . while stage transition duration would definitely be prolonged in a situation of impaired oral bolus control resulting in premature spillage of material into the pharynx , there are no clear guidelines for dissociating this type of impairment from delayed pharyngeal swallow response . furthermore , research has shown that there can be considerable variation , even amongst healthy individuals in the position of the head of the bolus at swallow onset ( martin - harris , brodsky , michel , lee , & walters , 2007 ) , making the objective delineation of impairment in liquid bolus control quite challenging . in this study , we report data from six participants in whom bolus clearance measures did not improve , despite measurable improvements in tongue strength first , it must be recognized that neither this treatment protocol , nor the one used by robbins et al . ( 2007 ) , included any swallowing tasks ( saliva or other material ) in which there was a specific instruction to use heightened tongue pressure while executing the swallow . it may be that this sort of specific emphasis on generating swallows involving higher pressure amplitudes would be necessary to support generalization and that tongue - pressure tasks , which are not immediately followed by a swallow , lack specificity for improving swallowing function ( ferguson & rice , 2001 ) . another possibility is that there are aspects of tongue pressure generation in swallowing , other than the amplitudes that are generated , that are relevant for effective bolus propulsion and clearance . recent work has shown that healthy individuals modulate both the amplitude and time - scale of tongue - pressures when swallowing liquids of differing consistency ( steele , bailey , & molfenter , 2010 ) . it may be that treatment protocols need to incorporate this aspect by emphasizing tasks that mimic healthy swallows in their amplitude - time profile in order to achieve improved functional outcomes with respect to bolus control ( steele , bailey , molfenter , yeates , & grace - martin , 2010 ) . a sample of only six participants is clearly under - powered to detect groupwise changes in swallowing . for this reason as previously acknowledged , although participants were at least 5 months post - onset , it is still possible that spontaneous recovery contributed to some of the changes seen . additionally , our decision to take a conservative approach by using the worst scores observed to represent a patient 's function may have contributed to the lack of apparent change . our experience using ordinal rating scales to capture residue severity also suggests that such scales lack resolution for detecting change in statistical analyses . perceptual judgements of the amount of residue occupying the valleculae or pyriform sinuses , categorized in quarterly increments , lack resolution and validation and are not sensitive to subtle changes that may or may not be present with real functional change . finally , it may be that modifications to the schedule and intensity of treatment used in this study might lead to different treatment outcomes . participants in this study attended sessions twice weekly , as a rule , over the course of 1112 weeks . this schedule was found to be feasible , even for patients who were commuting to treatment as outpatients , and could be scheduled in conjunction with any other medical appointments that were required during participation in the study . whether treatment intensification over a different time frame ( either shorter or longer ) would lead to different outcomes a sample of only six participants is clearly under - powered to detect groupwise changes in swallowing . as previously acknowledged , although participants were at least 5 months post - onset , it is still possible that spontaneous recovery contributed to some of the changes seen . additionally , our decision to take a conservative approach by using the worst scores observed to represent a patient 's function may have contributed to the lack of apparent change . our experience using ordinal rating scales to capture residue severity also suggests that such scales lack resolution for detecting change in statistical analyses . perceptual judgements of the amount of residue occupying the valleculae or pyriform sinuses , categorized in quarterly increments , lack resolution and validation and are not sensitive to subtle changes that may or may not be present with real functional change . finally , it may be that modifications to the schedule and intensity of treatment used in this study might lead to different treatment outcomes . participants in this study attended sessions twice weekly , as a rule , over the course of 1112 weeks . this schedule was found to be feasible , even for patients who were commuting to treatment as outpatients , and could be scheduled in conjunction with any other medical appointments that were required during participation in the study . whether treatment intensification over a different time frame ( either shorter or longer ) would lead to different outcomes is a question for future research . in conclusion , this study shows that a tongue - pressure resistance training protocol emphasizing strength and accuracy targets results in improved tongue strength measures , both in swallowing and in isometric tasks , and in improved penetration aspiration scores . however , post - swallow pharyngeal residues did not improve as a result of treatment and , in fact , showed apparent deterioration in some cases .

the purpose of this study was to measure treatment outcomes in a group of six adults with chronic dysphagia following acquired brain injury , who each completed 24 sessions of tongue - pressure resistance training , over a total of 1112 weeks . the treatment protocol emphasized both strength and accuracy . biofeedback was provided using the iowa oral performance instrument . amplitude accuracy targets were set between 2090% of the patient 's maximum isometric pressure capacity . single subject methods were used to track changes in tongue strength ( maximum isometric pressures ) , with functional swallowing outcomes measured using blinded ratings of a standard pre- and post - treatment videofluoroscopy protocol . improvements were seen in post - treatment measures of tongue pressure and penetration aspiration . no improvements were seen in pharyngeal residues , indeed worsening residue was seen in some patients .

Introduction Method Results Discussion Study limitations Conclusions

it is estimated that 3778% of the 750,000 americans who suffer a stroke each year will experience dysphagia ( martino , foley , bhogal , diamant , speechley , & teasell , 2005 ; www.stroke.org ) and that 4354% of these individuals will experience aspiration ( entry of foreign material into the airway ) ( daniels , brailey , priestly , herrington , weisberg , & foundas , 1998 ; horner , massey , riski , lathrop , & chase , 1988 ) . in the acquired brain injury ( abi ) population , dysphagia has a similar reported incidence of 4161% ( halper , cherney , cichowski & zhang , 1999 ; winstein , 1983 ) , with videofluoroscopically - confirmed aspiration incidence reported at 38% ( lazarus & logemann , 1987 ) . impaired tongue function is a prominent component in neurogenic dysphagia ; the ability to generate tongue - pressure plays a primary role in controlling the flow of liquids through the mouth and pharynx ( pouderoux & kahrilas , 1995 ) . when tongue - pressure generation is impaired , liquids may spill into the pharynx before the airway is protected , or bolus clearance may be impaired , leaving residue in the pharynx . in this study , we describe treatment outcomes for a tongue - pressure resistance training intervention . robbins developed an 8-week tongue - pressure resistance training protocol , which she applied in healthy seniors and individuals with dysphagia post - stroke ( robbins , gangnon , theis , kays , hewitt , & hind , 2005 ; robbins , kays , gangnon , hind , hewitt , & gentry , 2007 ) . the robbins protocol employs elements of exercise load , intensity , and treatment duration , intended to support physiological changes in tongue muscle strength and capacity . patients practice repeated tongue - pressure tasks , with targets set between 6080% of their maximum capacity , on a schedule requiring 6090 task repetitions on 3 non - consecutive days of the week . effectively demonstrated that tongue - pressure resistance training builds tongue strength . in a group of 10 patients with stroke - related dysphagia of up to 3 months duration , anterior maximum isometric tongue pressures ( mips ) , i.e. unfortunately , there was less convincing evidence that functional swallowing improvements accompanied these strength changes . post - treatment videofluoroscopy showed a significant groupwise improvement in aspiration , but there were no systematic changes in pharyngeal residues . we designed a modification to the robbins protocol , emphasizing amplitude accuracy targets in the 2090% of mip range in addition to strength targets ( tongue - pressure strength and accuracy training , or tpsat ) . this manuscript describes a preliminary prospective case series of six patients with dysphagia secondary to acquired brain injury who completed this protocol . we enrolled six consenting participants with dysphagia secondary to acquired brain injury following a motor vehicle accident , with injuries sustained either as a driver , passenger or pedestrian . , scores 3 on the 8-point penetration - aspiration scale ( rosenbek , robbins , roecker , coyle , & wood , 1996 ) with thin liquids ; and ( 2 ) post - swallow residues in the valleculae or pyriform sinuses with either thin and/or spoon - thick liquids measured using a 4-point ordinal scale described by eisenhuber , schima , schober , pokieser , stadler , and scharitzer ( 2002 ) : 0 = none , 1 = thin coating , 2 = 2550% full , 3 50% full . apenetration aspiration was rated using the 8-point penetration aspiration scale ( rosenbek et al . all participants received 24 sessions of treatment , scheduled twice weekly ( with a third session occasionally scheduled within the same week ) , and spanning a total of 1112 weeks . figure 1 outlines the design of a treatment session , which involved a total of 60 tongue - pressure tasks , arranged in blocks of six , with individual task repetitions separated by a minimum 10-second rest period . all tasks were performed using the iowa oral performance instrument ( iopi ) , a hand - held manometer connected to a 2.7 millilitre air - filled pressure bulb that was positioned between the tongue and hard palate in either an anterior or posterior oral position . feedback was provided to the participant after each task repetition , by telling them the pressure amplitude shown on the iopi . the bulb was removed between each swallow and repositioned , thereby allowing a brief rest and allowing the patient to indicate when they were ready to proceed . single - subject descriptive methods were used to report treatment progress for the tongue pressure parameters , which were probed at each treatment session . a control chart method with effect - size banding ( nourbakhsh & ottenbacher , 1994 ) was used to confirm the presence or absence of change , as follows : ( 1 ) measures of baseline tongue pressure range , by task , were computed using mean and standard deviation values for each participant for all mip tasks across the first three treatment sessions ( n = 36 in the anterior and 36 in the posterior sensor positions ) . ( 2 ) means and standard deviations for tongue - pressure amplitudes were calculated separately by task from the iopi data collected during each session : 12 anterior mips , 12 posterior mips , and 12 non - effortful saliva swallows . these values were plotted on control charts to document change in tongue pressure parameters over time ( see figure 2 ) . measures of functional swallowing outcome were derived from post - treatment videofluoroscopies ( compared to baseline ) , performed according to a standard protocol using a 40% weight / volume thin liquid solution of polibar ( bracco imaging s.p.a . , princeton , new jersey ) and water , and a spoon - thick liquid prepared using ez - hd barium power ( bracco ) , mixed in 40% weight / volume ratio with applesauce . the videofluoroscopic measures of interest were the 8-point penetration - aspiration scale ( rosenbek et al . , 1996 ) and 4-point ordinal ratings of residue in the valleculae and pyriform sinuses ( eisenhuber et al . ratings were made by two experienced speech - language pathologists , who had initially been trained to consensus in the scoring of these features using a training set of three videofluoroscopy recordings from patients not involved in this study . the videofluoroscopy recordings of the head - neutral , non - compensated thin liquid and spoon - thick liquid swallows were spliced into bolus - specific clips . based on this strong performance for the reliability dataset , it was decided that duplicate ratings of the entire dataset across both raters was not necessary . for the purposes of the research study , it was decided a priori that the worst scores seen across all swallows for a given bolus consistency would be used to represent a patient 's functional swallowing status at each time - point ( pre- , post - treatment ) . table iii summarizes mean and standard deviation values for the three tongue - pressure parameters of interest , calculated across the baseline timeframe spanning the first three treatment sessions for each participant . figures 24 provide a graphic summary of the progression of tongue pressure measures for each participant across the course of the study . baseline measures of tongue strength ( means and standard deviations ) calculated over the first three treatment sessions , with 12 repetitions of each task per session . the charts showing anterior ( figure 2 ) and posterior maximum isometric pressures ( figure 3 ) are graphed using a uniform y - axis range of 080 kpa . for the reader 's reference , it may be useful to note that previous studies have suggested that the lower boundary for normal anterior isometric pressures in elderly men falls at 40 kpa ( nicosia et al . in the case of this study , it can be appreciated that all participants except participants 2 and 4 began treatment with anterior mips below this threshold . participant 6 showed an unusual pattern with respect to anterior mips , which increased over the first 10 sessions , but then declined below the d = .6 effect size boundary for the second half of treatment , and displayed much greater variability as seen in the standard deviation error bars shown on the chart . this suggests that some change in the strategy used to perform mips occurred after the first 10 sessions , rendering the initial baseline measures inappropriate as a comparison for the second half of treatment . for the six participants in this study , saliva swallow pressures were at or below 20 kpa in all cases at baseline . the only exception to this was seen in participant 5 , who was unable to produce volitional saliva swallows at baseline . participants 3 , 4 , and 5 failed to demonstrate any notable changes in saliva swallowing pressures . table iv summarizes treatment outcomes in terms of the functional swallowing measures derived from the videofluoroscopies in this study . when improvement was seen compared to baseline , this is indicated by a black font on a white background ; deterioration is indicated using a white font on a black background , while an absence of change is shown using a black font on a grey background . roman text in the table reflect a functional swallowing improvement in comparison to the pre - treatment videofluoroscopy , while italics reflect no noticeable change and underlined text indicates deterioration in function for that bolus / parameter combination , relative to the baseline evaluation . penetration aspiration was rated using the 8-point penetration aspiration scale ( rosenbek et al . improved swallowing safety for thin liquids was seen post - treatment for participants 1 , 2 , 4 , 5 , and 6 , but remained unchanged for participant 3 . impaired swallowing safety for spoon - thick liquids was a concern at baseline in participants 1 , 2 , 3 , and 6 ; post - treatment improvement was seen in all four of these participants . however , it should also be noted that two participants who had shown baseline safety ( scores of 1 ) with spoon - thick liquids showed new evidence of impaired swallowing safety with this consistency on their post - treatment videofluoroscopies ; participant 4 showed high penetration ( score of 2 ) , while participant 5 showed silent aspiration ( score of 8) . , scores of 2 or higher ) in participants 1 , 4 , and 6 , but remained unchanged post - treatment in all three cases . , a score of 1 ) with thin liquids actually worsened to moderate residue post - treatment ( i.e. with spoon - thick liquids , vallecular residue was a concern at baseline for participants 1 , 3 , 4 , and 6 . scores remained unchanged post - treatment for participants 3 and 6 , but deteriorated by one grade of severity on the eisenhuber rating scale in both participants 1 and 4 . participant 2 showed post - treatment improvement in this measure , while participant 1 showed deterioration by one grade of severity to a post - treatment score of 3 . additionally , post - treatment measures for participant 4 revealed moderate pyriform residue for thin liquids , when only mild residue had been seen at baseline , a worsening from a score of 12 . pyriform sinus residue scores of 2 or higher for spoon - thick liquids at baseline were seen in participants 1 , 2 , and 4 . post - treatment , this feature remained unchanged in participant 2 , while participants 1 and 4 showed a worsening to scores of 3 . changes in post - swallow vallecular and pyriform sinus residues are much less apparent , and there is no trend of improvement in this group of patients . our results show that all six participants in this study achieved increases in either anterior and/or posterior tongue strength on mips post - treatment . first of all , improvements were achieved in individuals who had dysphagia of long - standing ( minimum 5 months ) . even the participants with dysphagia of longer than 18 months chronicity demonstrated convincing improvements in both anterior and posterior tongue strength , thereby speaking to the ongoing plasticity of the tongue and its potential to respond to exercise training in this population . secondly , the observed improvements in strength are interesting given that the focus of our treatment was equally distributed between maximum isometric tasks and accuracy tasks requiring the participant to hit sub - maximal pressure targets as precisely as possible . ( 2007 ) , these data confirm that tongue - pressure resistance exercises , practiced 23-times weekly , with amplitude targets tailored to the patient 's maximum pressure capacity , are an effective method for increasing tongue strength in individuals with neurogenic dysphagia . unfortunately , the types and degree of functional swallowing changes that accompanied these improvements in tongue strength are less clear . in this study five of the six patients displaying aspiration on thin liquids at baseline showed resolution of this impairment at the post - treatment videofluoroscopy . nevertheless , it must be acknowledged that spontaneous recovery may have contributed to the improvements seen in our participants and it remains unclear to what extent the improvement in aspiration can be attributed to tongue - pressure resistance training . in fact , the videofluoroscopy results appear to show deterioration in bolus clearance function in five of our six participants , and there is no apparent pattern to these results with respect to dysphagia chronicity or severity , in terms of pre - treatment diet tolerance . these results are disappointing and raise challenging questions regarding the possibility that there was some negative impact with this treatment protocol . the apparent worsening may simply reflect variability in swallowing function within these participants within a limited videofluoroscopy protocol , given the long - standing nature of their dysphagia . we feel that it would be an over - interpretation of these data to conclude that these individuals showed functional deterioration in their swallowing , even in the presence of at least one residue score at the post - treatment videofluoroscopy that was more severe than that observed at baseline . however , the fact that residues appeared to be resistant to change in the presence of increased tongue pressures does raise questions about one of the underlying assumptions of this study , namely that tongue driving force contributes to effective bolus clearance from the pharynx ( dejaeger , pelemans , ponette , & joosten , 1997 ) . our data suggest that the tongue - pressure resistance exercises practiced in this study did not yield generalized improvements in pharyngeal swallowing strength , although we had hoped to see such generalization based on evidence regarding the impact of effortful swallows involving heightened tongue pressure generation on pharyngeal pressure generation and propagation ( huckabee & steele , 2006 ; steele & huckabee , 2007 ) . ( 2007 ) similarly failed to find convincing evidence of improvements in bolus clearance with their tongue pressure training protocol , even given shorter chronicity of dysphagia and a clearer emphasis on building tongue strength in their protocol . similarly , prior studies of the shaker exercise and neuromuscular electrical stimulation have also failed to show convincing evidence of physiological improvements in bolus clearance in patients with neurogenic dysphagia ( blow , speyer , baijens , woisard , & ekberg , 2008 ; logemann , rademaker , pauloski , kelly , stangl - mcbreen , & antinoja , 2009 ; robbins et al . for example , there may be aspects of the oral phase of swallowing that are more sensitive to the influence of tongue pressure amplitudes than the parameters measured in our study . indeed , our original plan had been to include measures related to oral control of the bolus in this study , but we determined that the literature lacked evidence regarding valid quantitative measures of bolus control that would lend themselves to reflecting the possible influence of treatment . stage transition duration , for example , is a quantitative measure that captures the interval following arrival of the bolus in the pharynx prior to the onset of hyolaryngeal motion associated with the pharyngeal phase of the swallow ( lof & robbins , 1990 ) . while stage transition duration would definitely be prolonged in a situation of impaired oral bolus control resulting in premature spillage of material into the pharynx , there are no clear guidelines for dissociating this type of impairment from delayed pharyngeal swallow response . furthermore , research has shown that there can be considerable variation , even amongst healthy individuals in the position of the head of the bolus at swallow onset ( martin - harris , brodsky , michel , lee , & walters , 2007 ) , making the objective delineation of impairment in liquid bolus control quite challenging . in this study , we report data from six participants in whom bolus clearance measures did not improve , despite measurable improvements in tongue strength first , it must be recognized that neither this treatment protocol , nor the one used by robbins et al . ( 2007 ) , included any swallowing tasks ( saliva or other material ) in which there was a specific instruction to use heightened tongue pressure while executing the swallow . it may be that this sort of specific emphasis on generating swallows involving higher pressure amplitudes would be necessary to support generalization and that tongue - pressure tasks , which are not immediately followed by a swallow , lack specificity for improving swallowing function ( ferguson & rice , 2001 ) . another possibility is that there are aspects of tongue pressure generation in swallowing , other than the amplitudes that are generated , that are relevant for effective bolus propulsion and clearance . recent work has shown that healthy individuals modulate both the amplitude and time - scale of tongue - pressures when swallowing liquids of differing consistency ( steele , bailey , & molfenter , 2010 ) . a sample of only six participants is clearly under - powered to detect groupwise changes in swallowing . for this reason as previously acknowledged , although participants were at least 5 months post - onset , it is still possible that spontaneous recovery contributed to some of the changes seen . additionally , our decision to take a conservative approach by using the worst scores observed to represent a patient 's function may have contributed to the lack of apparent change . perceptual judgements of the amount of residue occupying the valleculae or pyriform sinuses , categorized in quarterly increments , lack resolution and validation and are not sensitive to subtle changes that may or may not be present with real functional change . finally , it may be that modifications to the schedule and intensity of treatment used in this study might lead to different treatment outcomes . participants in this study attended sessions twice weekly , as a rule , over the course of 1112 weeks . whether treatment intensification over a different time frame ( either shorter or longer ) would lead to different outcomes a sample of only six participants is clearly under - powered to detect groupwise changes in swallowing . as previously acknowledged , although participants were at least 5 months post - onset , it is still possible that spontaneous recovery contributed to some of the changes seen . additionally , our decision to take a conservative approach by using the worst scores observed to represent a patient 's function may have contributed to the lack of apparent change . perceptual judgements of the amount of residue occupying the valleculae or pyriform sinuses , categorized in quarterly increments , lack resolution and validation and are not sensitive to subtle changes that may or may not be present with real functional change . finally , it may be that modifications to the schedule and intensity of treatment used in this study might lead to different treatment outcomes . participants in this study attended sessions twice weekly , as a rule , over the course of 1112 weeks . whether treatment intensification over a different time frame ( either shorter or longer ) would lead to different outcomes is a question for future research . in conclusion , this study shows that a tongue - pressure resistance training protocol emphasizing strength and accuracy targets results in improved tongue strength measures , both in swallowing and in isometric tasks , and in improved penetration aspiration scores . however , post - swallow pharyngeal residues did not improve as a result of treatment and , in fact , showed apparent deterioration in some cases .

pelvic organ prolapse ( pop ) is a significant health issue in females worldwide [ 1 , 2 ] . there are approximately 250,000 procedures annually in the usa for pop , with as many as 29% of women having to undergo repeat surgery . traditional anterior repair of cystocele or anterior compartment prolapse utilizing the patient s own tissue is a compensatory procedure that utilizes weakened and/or damaged tissue and has reported failure rates in the range of 4060% . additionally , plication or colporrhaphy techniques address only midline defects in the anterior compartment and do not provide any apical support which may also contribute to the failure rates seen with this type of repair . abdominal y - mesh sacralcolpopexy has the highest cure rates in the literature for vault prolapse , and the benefit of utilizing mesh in this particular repair seems to outweigh the risks . it results in anatomic repair with minimal tension and does not rely on the patients weakened tissue to maintain support . with the success of apical graft use , more recently , graft augmentation of prolapse repair has been utilized via the vaginal route . a recent cochrane review has confirmed that mesh use in the anterior compartment has a lower failure rate versus traditional repair [ 5 , 6 ] . mesh trocar - based kits such as perigee and prolift were developed to try to simplify the placement of mesh , and initial studies have shown cure rates in the range of 8796% [ 79 ] . these first - generation kits all required needles to be passed through the groins and obturator space with mesh arms subsequently pulled through this same space to attach the body of the graft to the levator muscles . although many studies have shown successful cure rates when used by experienced surgeons in the right patient population , complications resulting from the external needle passes such as visceral and vascular injury as well as post - operative mesh complications have been concerning [ 911 ] . vaginal or pelvic pain from the mesh arms being too tight , as well as issues such as rates of mesh extrusion as high as 15% , have been reported [ 1214 ] . additionally , a shortfall of most of these first - generation kits , is that they did not offer true level i or apical support in the anterior compartment , which may have led to apical failure . in an attempt to both benefit from the advantages of mesh use in the anterior compartment and also decrease some of the associated risks seen in the first - generation kits , a new single - incision vaginal approach was developed that utilizes a lighter and less - dense type i polpypropylene mesh ( intepro lite , ams ) and eliminates all blind external needle passes . it also obtains true level i support through an anterior approach , by attaching the mesh , in an adjustable , tension - free matter to the sacrospinous ligaments via a novel and very minimally invasive fixation technique involving small self - fixating tips attached to the apical mesh arms . the purpose of the current study is to report the outcomes on a group of patients that underwent this procedure for treatment of symptomatic anerior compartment and/or apical prolapse at our specialty urogynecology center . this study is a descriptive retrospective case series of 60 consecutive women with symptomatic stage 2 or greater anterior compartment prolapse ( cystocele ) that underwent anterior repair with mesh graft augmentation and vaginal vault suspension through a single transvaginal incision utilizing the anterior elevate by ams ( american medical systems , minnetonka , mn , usa ) system over a consecutive 12-month period at our center . comprehensive pre - operative urogynecologic exams were completed including prolapse quantification utilizing the international continence society pelvic organ prolapse quantification ( pop - q ) staging system . inclusion criteria were patients with symptomatic anterior compartment prolapse stage 2 that received the anterior elevate procedure over a consecutive 12-month timeframe at our center . exclusion criteria included any patient that had any other vault suspension procedure at the time of the surgery . additional testing included complex urodynamic testing to evaluate for the presence of concomitant stress urinary incontinence ( sui ) ( or detrusor instability ) with and without the patient s prolapse reduced . if sui was documented on urodynamic testing with or without the prolapse reduced , the patient was scheduled for a sub - urethral mesh tape sling procedure at the time of surgery . a sling was not placed prophylactically in any patient if sui was not seen on testing . the procedure is begun with injection of a hydrodissection solution ( 25 cc of 1% lidocaine with epinephrine 1:200,000 diluted in 250 cc of saline ) into the anterior vaginal wall . a 3 to 4-cm vertical incision is then made in the anterior vaginal wall starting at the level of the bladder neck towards the vaginal vault or the cervix . the incision is kept as small as possible and is not taken all the way to the apex of vagina . if the uterus was in situ , it was left in place in the current trial . a full - thickness dissection is then completed in the relatively avascular vesico - vaginal space out to the pelvic sidewalls and then up to the ischial spines bilaterally . once the spine is reached , a medial sweep with the index and/or middle finger is completed to isolate the sacropinous ligament ( ssl ) . it should be noted that the retropubic space does not need to be entered , nor does the ligament need to be visualized . the bladder neck arms of the graft are placed into the obturator internus muscle at the level of the bladder neck with their small self - fixating tips . the separate apical arms are then placed in the ssls bilaterally approximately 2 cm medial to the ischial spine ( fig . 1 ) . the apical portion of the graft is then fed over the arms attached to the ssls and the vault adjusted into place in a tension free manner with an adjusting tool provided . two 20 pds sutures are used to attach the graft in the midline to the pericervical ring or the vaginal cuff . once the final adjustment is reached , the graft is locked in place with small locking eyelets and the excess mesh cut off of the apical arms . minimal to no vaginal epithelium is excised and the incision closed with a 20 delayed absorbable suture in a running - locked fashion . 1apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . ( reproduced with permission from ams ) apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . ( reproduced with permission from ams ) if an incontinence procedure was completed concomitantly , a separate sub - urethral incision was made and the tension - free sling placed utilizing standard technique . vaginal estrogen cream was started 1 week post - operatively and used every other day . patients were evaluated in the office at 4 weeks , 3 months , 6 months and then every 6 months thereafter . ics pop - q staging was completed as well as subjective assessment of prolapse ( feeling or seeing a bulge ) , incontinence and urinary urgency and frequency symptoms . quality of life questionnaires utilized were the incontinence impact questionnaire - short form ( iiq-7 ) and the urogenital distress inventory - short form ( udi-6 ) . objective cure was defined if the midline anterior vaginal wall ( points aa and ba ) was < 1.0 1.0 cm inside the hymenal ring and the apex was also less than or equal to stage i. the procedure is begun with injection of a hydrodissection solution ( 25 cc of 1% lidocaine with epinephrine 1:200,000 diluted in 250 cc of saline ) into the anterior vaginal wall . typically , 4060 cc of this solution is utilized . a 3 to 4-cm vertical incision is then made in the anterior vaginal wall starting at the level of the bladder neck towards the vaginal vault or the cervix . the incision is kept as small as possible and is not taken all the way to the apex of vagina . if the uterus was in situ , it was left in place in the current trial . a full - thickness dissection is then completed in the relatively avascular vesico - vaginal space out to the pelvic sidewalls and then up to the ischial spines bilaterally . once the spine is reached , a medial sweep with the index and/or middle finger is completed to isolate the sacropinous ligament ( ssl ) . it should be noted that the retropubic space does not need to be entered , nor does the ligament need to be visualized . the bladder neck arms of the graft are placed into the obturator internus muscle at the level of the bladder neck with their small self - fixating tips . the separate apical arms are then placed in the ssls bilaterally approximately 2 cm medial to the ischial spine ( fig . 1 ) . the apical portion of the graft is then fed over the arms attached to the ssls and the vault adjusted into place in a tension free manner with an adjusting tool provided . two 20 pds sutures are used to attach the graft in the midline to the pericervical ring or the vaginal cuff . once the final adjustment is reached , the graft is locked in place with small locking eyelets and the excess mesh cut off of the apical arms . minimal to no vaginal epithelium is excised and the incision closed with a 20 delayed absorbable suture in a running - locked fashion . 1apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . ( reproduced with permission from ams ) apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . ( reproduced with permission from ams ) if an incontinence procedure was completed concomitantly , a separate sub - urethral incision was made and the tension - free sling placed utilizing standard technique . vaginal estrogen cream was started 1 week post - operatively and used every other day . patients were evaluated in the office at 4 weeks , 3 months , 6 months and then every 6 months thereafter . ics pop - q staging was completed as well as subjective assessment of prolapse ( feeling or seeing a bulge ) , incontinence and urinary urgency and frequency symptoms . quality of life questionnaires utilized were the incontinence impact questionnaire - short form ( iiq-7 ) and the urogenital distress inventory - short form ( udi-6 ) . objective cure was defined if the midline anterior vaginal wall ( points aa and ba ) was < 1.0 1.0 cm inside the hymenal ring and the apex was also less than or equal to stage i. patient demographics are presented in table 1 . of the 60 patients , 11 ( 18.4% ) had stage 2 prolapse , 43(71.6% ) stage 3 , and 6 ( 10% ) had stage 4 on pre - operative pelvic examination and pop - q scoring . the mean ba value ( sd ) was + 2.04 1.3 cm outside the vaginal opening , average c value ( sd ) was 2.7 2.9 . sixteen ( 26.6% ) patients had previous anterior vaginal wall repairs and had recurrent cystoceles . sui was present in 61.6% of patients and tension - free tape slings were placed at the time of their surgeries . twenty - six patients ( 43.3% ) presented with sui and 11 ( 18.3% ) were discovered to have occult stress leakage with their prolapse reduced during urodynamic testing . concomitant procedures at time of surgery included , 26/60 patients ( 43.3% ) with posterior repair ( 16 with mesh grafts , 10 no graft used ) . no patient had any other vault support besides the anterior elevate and no patient underwent hysterectomy ( 27% of patients had their uterus in situ ) ( table 2 ) . nineteen patients ( 31.6% ) were sexually active prior to surgery.table 1demographicsn = 60age ( years sd)70.8 6.1 ( range 5181)parity2.8 1.6 ( range 08)previous hysterectomyn = 44 ( 73%)menopausaln = 59 ( 98.4%)estrogen usen = 19 ( 31.6%)previous repairn = 16 ( 26.6%)>1 previous repairn = 5 ( 8.3%)prolapse stageiin = 11 ( 18.4%)iiin = 43 ( 71.6%)ivn = 6 ( 10.0%)table 2concomitant proceduresproceduren%hysterectomy00sling mini - sling3355 tot23.3 tvt23.3posterior repair no graft1016.6 mesh graft1626.6 concomitant procedures average blood loss was 47 cc ( range 25125 cc ) and was deemed minimal . there were no post - operative bleeds or hematomas and no patient had to be taken back to the o.r . for bleeding or pain . there was one midline cystotomy above the trigone that occurred during the dissection of the anterior wall . this was closed in a two - layer fashion with absorbable sutures and the mesh still placed and ureteral patency confirmed . the average hospital stay was 23 h ( range 18 h to 2 days ) . foley catheters were taken out on post - operative day 1 and voiding trial attempted . if patient did not pass the voiding trial , she was sent home with an indwelling catheter and voiding trial re - attempted on post - operative day 3 . twelve patients required catheterization for more than 3 days with ten of these having sling procedure at time of surgery . one patient who had the complication of cystotomy had her catheter for 10 days and had no sequalae from the injury . four patients suffered from post - operative urinary tract infections ( uti ) ; however , one of these had a history of recurrent utis . only one patient has symptoms of recurrent prolapse for a subjective cure rate of 98.4% . objective / anatomic cure rate was 91.7% using a definition of ba < 1.0 ( stage i ) and no patients have required repeat surgery for prolapse . mean ba value was 2.45 0.9 , point c was 8.3 0.9 , and tvl was 9.1 0.3 cm . mean vaginal length did not change statistically from pre - operative values ( table 3).table 3pre - operative versus post - operative pop - q measurements ( mean)preoperativepostoperativep value ( t test)mean pop - q measurementspoint aa ( cm)+1.4 1.42.4 0.8<0.001point ba ( cm)+2.0 1.32.5 0.9<0.001point c ( cm ) , cervix2.7 2.98.3 0.9<0.001point ap1.1 1.32.4 0.6<0.001point bp0.9 1.62.3 0.6<0.001total vaginal length ( cm)9.09 0.59.16 0.30.343 pre - operative versus post - operative pop - q measurements ( mean ) there have been no patients that reported any significant buttock or leg pain that would be consistent with a pudendal or obturator type of neuropathy . three patients suffered from post - operative levator myalgia or vaginal pain that required short - term treatment with nsaids and/or physical therapy with resolution of all patients pain . in this predominantly elderly post - menopausal patient group , 31% were sexually active pre - operatively with one of these patients reporting clinically significant dyspareunia that is being treated with conservative measures ( physical therapy ) and is improving . thirty - two patients ( 53% ) complained of significant urge symptoms pre - operatively , and 20 of these patients ( 62% ) had resolution of these symptoms post - operatively following their surgery . one patient ( 1.6% ) developed de - novo urge symptoms post - operatively requiring treatment with anticholinergic agents . overall iiq-7 and udi-6 scores improved significantly from baseline to follow - up ( table 4 ) . the udi-6 subdomains of urge , stress , and obstructive symptoms were also evaluated and a significant improvement in each domain was also seen in the overall group ( table 4 ) . patients that had a concomitant sling procedure or that had a previous repair did not statistically differ in improvement in these qol indices compared to those that did not ( tables 5 and 6 ) . the udi-6 subdomain scores were also evaluated in the patients that had a concomitant sling or not and these results can be seen in table 6.table 4overall udi-6 and iiq-7 scores ( pre - operative vs. post - operative ) and udi-6 subscalesvariable ( n = 58)baseline mean sdfollow - up mean sdfollow - up vs. baselinep value ( t test)udi-642.9 26.922.4 24.420.6 30.8<0.001iiq-731.5 25.715.4 23.416.1 26.9<0.001udi subscalesirritative ( 56)52.4 31.726.9 32.324.4 38.5<0.001stress ( 56)44.6 29.825.4 30.420.2 36.5<0.001obstructive ( 58)40.4 30.415.5 22.724.1 32.9<0.00158/60 patients filled out both pre- and post - operative questionnairestable 5iiq-7/udi-6 scores in patients with previous anterior compartment repairs compared to those that did notprevious repairiiq-7 preiiq-7 postiiq-7 changeudi-6 preudi postudi-6 changeno = 54mean29.310.618.727.713.614.1stdev26.016.825.019.015.323.4yes = 16mean36.428.18.343.525.617.9stdev25.732.931.319.723.423.2 t test ( between groups)0.350.010.190.010.020.58table 6iiq-7/udi-6 scores ( and udi-6 domain scores ) in patients with and without concomitant sling at time of surgeryquestionnaire ( n)pre - operative scorepost - operative scoremean improvementp valueiiq-7sling ( 37)33.2 26.016.6 24.616.6 28.80.001no sling ( 21)28.1 26.013.6 22.114.5 24.30.013 t test(between groups)0.480.650.72udi-6sling ( 38)45.1 23.626.0 27.019.0 28.6<0.001no sling ( 22)38.5 32.216.4 19.022.1 35.10.008 t test(between groups)0.370.150.72udi subscalesirritativesling ( 37)53.2 31.426.8 33.526.6 37.6<0.001no sling ( 21)48.4 32.927.3 31.120.6 40.80.031stresssling ( 36)49.1 27.028.4 31.619.9 38.40.004no sling ( 20)36.7 33.620.5 28.120.8 33.70.012obstructivesling ( 35)44.0 33.617.6 24.825.7 29.0<0.001no sling ( 21)34.1 35.512.1 18.721.4 39.10.021 overall udi-6 and iiq-7 scores ( pre - operative vs. post - operative ) and udi-6 subscales 58/60 patients filled out both pre- and post - operative questionnaires iiq-7/udi-6 scores in patients with previous anterior compartment repairs compared to those that did not iiq-7/udi-6 scores ( and udi-6 domain scores ) in patients with and without concomitant sling at time of surgery three patients suffered from significant sui post - operatively . one had a concomitant tension - free sling ( tvt type ) at time of surgery that failed and the other two did not have a concomitant sling as they did not suffer from sui pre - operatively and tested negative on udts . the patient that had the sling already placed is scheduled for treatment with injectable bulking agents and the other two are being scheduled for outpatient sling procedures . there were no post - operative infections of the mesh and no mesh had to be removed secondary to infection or pain or allergic reaction . no patients had to be taken back to the operating room for revision of the mesh or release of any of the mesh attachment points for pain or dyspareunia . the current series utilizing the anterior elevate system , showed excellent anatomic results and an objective cure rate of 92% with up to 24 months of f / u ( mean f / u 13.4 months ) . subjectively , only one patient complained of symptomatic prolapse ( 98.4% subjective cure rate ) and no patient has had prolapse of the anterior wall outside of the introitus . these results are consistent with other series utilizing synthetic mesh for anterior compartment repair ; however , we believe the anterior elevate procedure to be less invasive and a more simplified technique to place an anterior wall graft . additionally , it allows for concomitant apical support at the time of anterior compartment repair . showed the anterior approach to ssl to result in anatomic restoration of the vaginal vault with cure rates consistent with the posterior approach which our anatomic results support this as well . the anterior elevate system utilizes small self - fixating tips for attachment of the graft to the obturator internus muscle at the level of the bladder neck and into the ssls at the apex ( figs . 2 and 3 ) . the attachment of the graft to the ssls involves minimal dissection with placement of the small tip into the mid - portion of the ligament . secondary to this , risk of nerve injury or post - operative pain pudendal nerve type pain syndromes is minimal and this is supported in the current trial with no patients developing a post - operative pudendal nerve pain type syndrome and no patient requiring any surgical intervention for post - operative pain or dyspareunia . the elimination of lateral mesh arms penetrating through and through the levators also should help reduce the risk of vaginal or pelvic pain from the arms being placed too tight and banding in the vagina . this and/or mesh bunching seems to be the cause of most cases of dyspareunia with the first generation trocar - based mesh kits [ 12 , 13 , 16 ] . we did have three patients develop post - operative pain remote from surgery that required treatment ; however , it was all deemed muscular and responded to pelvic floor physical therapy and did not need surgical intervention . one does need to be careful ; however , in the adjustment of the graft at the apex as if too much tension is placed then this could potentially cause pain that may require release.fig . 2close - up view of the small self - fixating tip attaching into the ligament ( reproduced with permission from ams)fig . the bladder neck portion of the graft has been fixated to the levators and the apical portion of the graft is slid up the arms in a tension - free manner to elevate the anterior wall and vault . ( reproduced with permission from ams ) close - up view of the small self - fixating tip attaching into the ligament ( reproduced with permission from ams ) final adjustment of the graft into place . the bladder neck portion of the graft has been fixated to the levators and the apical portion of the graft is slid up the arms in a tension - free manner to elevate the anterior wall and vault . ( reproduced with permission from ams ) concerning the safety of the procedure , bleeding was minimal in most cases with average blood loss at 47 cc . one patient suffered a midline cystotomy during the procedure ; however , this was during the dissection of the anterior wall and was not related to the mesh or needle passes . we repaired the cystotomy with a double - layered closure , still placed the mesh , and she recovered without sequelae . overall , with the elimination of blind external needle passes the risk of intra - operative bladder or vascular injury is reduced . our mesh extrusion rate in the current trial was very low , and we attribute that to two variables : mesh quality and surgical technique . the mesh used in the current trial ( intepro lite ) is a type i macroporous polypropylene mesh that is 50% less dense and lighter than the first generation type i mesh ( intepro ) used in apogee / perigee . in a previously published trial from our center , we reported mesh extrusion rate of 6.5% with the use of the perigee procedure for treatment of cystocele in a similar group of patients , which is consistent with other reports in the literature . utilizing a less dense and softer mesh , results in a smaller mesh load and , most likely , less inflammatory response during healing which may reduce mesh extrusion rates . additionally , our surgical technique of utilizing a deeper dissection plane , i.e. , a full - thickness dissection utilizing hydrodissection in a more avascular space , allows for less bleeding during the procedure , less risk of post - operative hematoma , and a thicker vascular flap healing over the mesh . all of these factors may contribute to the lower extrusion rate seen in our trial . other lighter meshes have also been introduced , however , have not shown this low of extrusion rate . prolift - m utilizes a soft polypropylene mesh that has a similar density to intepro - lite however is 50% monocryl , and therefore , 50% of the mesh is absorbable . the thought was that this would decrease vaginal exposure rate ; however , in a recent study , cosson et al . reported a relatively high extrusion rate of 10.5% . sixteen patients ( 26.6% ) had previous repairs and would be considered higher risk for failure ; however , their cure rate was consistent with patients that had not had prior repair and there was no difference in complications seen in these patients either . in our opinion , this is a group of patients that benefit the greatest from a graft , given a previous failure using their own tissue , and the results of these patients in our series is very encouraging . the current study is limited by its retrospective nature and its medium - term follow - up . another limitation , again inherent to a retrospective single - center trial , is surgeon bias in evaluating their own surgical outcomes . a limitation of any surgical trial that also has to be considered is surgeon experience with the particular procedure or similar procedures as well as the anatomy of the dissection involved . complications in the current trial may have been kept to a minimum secondary to this variable . in conclusion , we have found the vaginal repair of anterior / apical wall prolapse utilizing a lightweight soft type i anterior wall mesh placed via the elevate system a safe , minimally invasive , and effective procedure for the treatment of anterior wall prolapse in this subset of mostly post - menopausal patients . we feel that the role of mesh in vaginal repairs is in its infancy , and much study still needs to be done to determine the ideal material to be utilized and the optimal way to place and attach the graft vaginally and the proper patient to utilize it in ; however , it can be expected that improvements in technology and techniques will continue to improve outcomes . the small self - fixating tips on the arms of the elevate procedure that hold the mesh in place are one of the least invasive approaches to fixate a mesh graft in place vaginally to date . the elimination of trocar - based , blind needle passes through the groins also seems to decrease the risk of complications . we do recommend further prospective studies with longer - term follow - up to further help delineate its role in clinical practice .

introduction and hypothesisthe safety and early efficacy of a new technique to treat cystocele and/or concomitant apical prolapse through a single vaginal incision with a lightweight mesh anchored apically bilaterally to the sacrospinous ligaments is reported.methodswomen with anterior compartment and/or apical prolapse stage ii underwent repair through a single anterior vaginal wall incision with the anterior elevate system ( aes ) . the technique utilizes a lightweight ( 24 g / m2 ) type i mesh anchored to the sacrospinous ligaments via two mesh arms with small self - fixating tips . the bladder neck portion of the graft is anchored to the obturator internus with similar self - fixating tips . the apical portion of the graft is adjustable to vaginal length prior to locking in place . outcome measures included prolapse degree at last follow - up visit , intra / post - operative complications , and qol assessments.resultssixty patients were implanted with average follow - up of 13.4 months ( range 324 months ) . mean pre - op ba was + 2.04 1.3 and c 2.7 2.9 . average blood loss was 47 cc and average hospital stay was 23 h. sixty - two percent of patients had concomitant sling for sui . mean post - op ba is 2.45 0.9 and c 8.3 0.9 . there was no statistical difference in pre- to post - op tvl . objective cure rate at current follow - up is 91.7% ( stage 1 ) . to date , there have been no mesh extrusions . no patients have reported significant buttock or leg pain . no patients have required surgical revision for any reason.conclusionthe aes is a minimally invasive technique to treat anterior compartment and/or apical prolapse through a single vaginal incision . initial results show the procedure to be safe and early efficacy is promising . longer - term follow - up is ongoing .

Introduction Materials and methods Procedure technique Results Discussion Conclusion

additionally , plication or colporrhaphy techniques address only midline defects in the anterior compartment and do not provide any apical support which may also contribute to the failure rates seen with this type of repair . these first - generation kits all required needles to be passed through the groins and obturator space with mesh arms subsequently pulled through this same space to attach the body of the graft to the levator muscles . although many studies have shown successful cure rates when used by experienced surgeons in the right patient population , complications resulting from the external needle passes such as visceral and vascular injury as well as post - operative mesh complications have been concerning [ 911 ] . in an attempt to both benefit from the advantages of mesh use in the anterior compartment and also decrease some of the associated risks seen in the first - generation kits , a new single - incision vaginal approach was developed that utilizes a lighter and less - dense type i polpypropylene mesh ( intepro lite , ams ) and eliminates all blind external needle passes . it also obtains true level i support through an anterior approach , by attaching the mesh , in an adjustable , tension - free matter to the sacrospinous ligaments via a novel and very minimally invasive fixation technique involving small self - fixating tips attached to the apical mesh arms . the purpose of the current study is to report the outcomes on a group of patients that underwent this procedure for treatment of symptomatic anerior compartment and/or apical prolapse at our specialty urogynecology center . this study is a descriptive retrospective case series of 60 consecutive women with symptomatic stage 2 or greater anterior compartment prolapse ( cystocele ) that underwent anterior repair with mesh graft augmentation and vaginal vault suspension through a single transvaginal incision utilizing the anterior elevate by ams ( american medical systems , minnetonka , mn , usa ) system over a consecutive 12-month period at our center . comprehensive pre - operative urogynecologic exams were completed including prolapse quantification utilizing the international continence society pelvic organ prolapse quantification ( pop - q ) staging system . inclusion criteria were patients with symptomatic anterior compartment prolapse stage 2 that received the anterior elevate procedure over a consecutive 12-month timeframe at our center . the procedure is begun with injection of a hydrodissection solution ( 25 cc of 1% lidocaine with epinephrine 1:200,000 diluted in 250 cc of saline ) into the anterior vaginal wall . a 3 to 4-cm vertical incision is then made in the anterior vaginal wall starting at the level of the bladder neck towards the vaginal vault or the cervix . the bladder neck arms of the graft are placed into the obturator internus muscle at the level of the bladder neck with their small self - fixating tips . the apical portion of the graft is then fed over the arms attached to the ssls and the vault adjusted into place in a tension free manner with an adjusting tool provided . once the final adjustment is reached , the graft is locked in place with small locking eyelets and the excess mesh cut off of the apical arms . 1apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . ( reproduced with permission from ams ) apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . objective cure was defined if the midline anterior vaginal wall ( points aa and ba ) was < 1.0 1.0 cm inside the hymenal ring and the apex was also less than or equal to stage i. the procedure is begun with injection of a hydrodissection solution ( 25 cc of 1% lidocaine with epinephrine 1:200,000 diluted in 250 cc of saline ) into the anterior vaginal wall . a 3 to 4-cm vertical incision is then made in the anterior vaginal wall starting at the level of the bladder neck towards the vaginal vault or the cervix . the bladder neck arms of the graft are placed into the obturator internus muscle at the level of the bladder neck with their small self - fixating tips . the apical portion of the graft is then fed over the arms attached to the ssls and the vault adjusted into place in a tension free manner with an adjusting tool provided . once the final adjustment is reached , the graft is locked in place with small locking eyelets and the excess mesh cut off of the apical arms . 1apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . ( reproduced with permission from ams ) apical arm of the elevate system placed into the sacropinous ligament 2 cm medial to the ischial spine . objective cure was defined if the midline anterior vaginal wall ( points aa and ba ) was < 1.0 1.0 cm inside the hymenal ring and the apex was also less than or equal to stage i. patient demographics are presented in table 1 . of the 60 patients , 11 ( 18.4% ) had stage 2 prolapse , 43(71.6% ) stage 3 , and 6 ( 10% ) had stage 4 on pre - operative pelvic examination and pop - q scoring . the mean ba value ( sd ) was + 2.04 1.3 cm outside the vaginal opening , average c value ( sd ) was 2.7 2.9 . sixteen ( 26.6% ) patients had previous anterior vaginal wall repairs and had recurrent cystoceles . no patient had any other vault support besides the anterior elevate and no patient underwent hysterectomy ( 27% of patients had their uterus in situ ) ( table 2 ) . nineteen patients ( 31.6% ) were sexually active prior to surgery.table 1demographicsn = 60age ( years sd)70.8 6.1 ( range 5181)parity2.8 1.6 ( range 08)previous hysterectomyn = 44 ( 73%)menopausaln = 59 ( 98.4%)estrogen usen = 19 ( 31.6%)previous repairn = 16 ( 26.6%)>1 previous repairn = 5 ( 8.3%)prolapse stageiin = 11 ( 18.4%)iiin = 43 ( 71.6%)ivn = 6 ( 10.0%)table 2concomitant proceduresproceduren%hysterectomy00sling mini - sling3355 tot23.3 tvt23.3posterior repair no graft1016.6 mesh graft1626.6 concomitant procedures average blood loss was 47 cc ( range 25125 cc ) and was deemed minimal . there were no post - operative bleeds or hematomas and no patient had to be taken back to the o.r . there was one midline cystotomy above the trigone that occurred during the dissection of the anterior wall . the average hospital stay was 23 h ( range 18 h to 2 days ) . objective / anatomic cure rate was 91.7% using a definition of ba < 1.0 ( stage i ) and no patients have required repeat surgery for prolapse . mean ba value was 2.45 0.9 , point c was 8.3 0.9 , and tvl was 9.1 0.3 cm . mean vaginal length did not change statistically from pre - operative values ( table 3).table 3pre - operative versus post - operative pop - q measurements ( mean)preoperativepostoperativep value ( t test)mean pop - q measurementspoint aa ( cm)+1.4 1.42.4 0.8<0.001point ba ( cm)+2.0 1.32.5 0.9<0.001point c ( cm ) , cervix2.7 2.98.3 0.9<0.001point ap1.1 1.32.4 0.6<0.001point bp0.9 1.62.3 0.6<0.001total vaginal length ( cm)9.09 0.59.16 0.30.343 pre - operative versus post - operative pop - q measurements ( mean ) there have been no patients that reported any significant buttock or leg pain that would be consistent with a pudendal or obturator type of neuropathy . three patients suffered from post - operative levator myalgia or vaginal pain that required short - term treatment with nsaids and/or physical therapy with resolution of all patients pain . thirty - two patients ( 53% ) complained of significant urge symptoms pre - operatively , and 20 of these patients ( 62% ) had resolution of these symptoms post - operatively following their surgery . the udi-6 subdomain scores were also evaluated in the patients that had a concomitant sling or not and these results can be seen in table 6.table 4overall udi-6 and iiq-7 scores ( pre - operative vs. post - operative ) and udi-6 subscalesvariable ( n = 58)baseline mean sdfollow - up mean sdfollow - up vs. baselinep value ( t test)udi-642.9 26.922.4 24.420.6 30.8<0.001iiq-731.5 25.715.4 23.416.1 26.9<0.001udi subscalesirritative ( 56)52.4 31.726.9 32.324.4 38.5<0.001stress ( 56)44.6 29.825.4 30.420.2 36.5<0.001obstructive ( 58)40.4 30.415.5 22.724.1 32.9<0.00158/60 patients filled out both pre- and post - operative questionnairestable 5iiq-7/udi-6 scores in patients with previous anterior compartment repairs compared to those that did notprevious repairiiq-7 preiiq-7 postiiq-7 changeudi-6 preudi postudi-6 changeno = 54mean29.310.618.727.713.614.1stdev26.016.825.019.015.323.4yes = 16mean36.428.18.343.525.617.9stdev25.732.931.319.723.423.2 t test ( between groups)0.350.010.190.010.020.58table 6iiq-7/udi-6 scores ( and udi-6 domain scores ) in patients with and without concomitant sling at time of surgeryquestionnaire ( n)pre - operative scorepost - operative scoremean improvementp valueiiq-7sling ( 37)33.2 26.016.6 24.616.6 28.80.001no sling ( 21)28.1 26.013.6 22.114.5 24.30.013 t test(between groups)0.480.650.72udi-6sling ( 38)45.1 23.626.0 27.019.0 28.6<0.001no sling ( 22)38.5 32.216.4 19.022.1 35.10.008 t test(between groups)0.370.150.72udi subscalesirritativesling ( 37)53.2 31.426.8 33.526.6 37.6<0.001no sling ( 21)48.4 32.927.3 31.120.6 40.80.031stresssling ( 36)49.1 27.028.4 31.619.9 38.40.004no sling ( 20)36.7 33.620.5 28.120.8 33.70.012obstructivesling ( 35)44.0 33.617.6 24.825.7 29.0<0.001no sling ( 21)34.1 35.512.1 18.721.4 39.10.021 overall udi-6 and iiq-7 scores ( pre - operative vs. post - operative ) and udi-6 subscales 58/60 patients filled out both pre- and post - operative questionnaires iiq-7/udi-6 scores in patients with previous anterior compartment repairs compared to those that did not iiq-7/udi-6 scores ( and udi-6 domain scores ) in patients with and without concomitant sling at time of surgery three patients suffered from significant sui post - operatively . one had a concomitant tension - free sling ( tvt type ) at time of surgery that failed and the other two did not have a concomitant sling as they did not suffer from sui pre - operatively and tested negative on udts . there were no post - operative infections of the mesh and no mesh had to be removed secondary to infection or pain or allergic reaction . no patients had to be taken back to the operating room for revision of the mesh or release of any of the mesh attachment points for pain or dyspareunia . the current series utilizing the anterior elevate system , showed excellent anatomic results and an objective cure rate of 92% with up to 24 months of f / u ( mean f / u 13.4 months ) . subjectively , only one patient complained of symptomatic prolapse ( 98.4% subjective cure rate ) and no patient has had prolapse of the anterior wall outside of the introitus . these results are consistent with other series utilizing synthetic mesh for anterior compartment repair ; however , we believe the anterior elevate procedure to be less invasive and a more simplified technique to place an anterior wall graft . additionally , it allows for concomitant apical support at the time of anterior compartment repair . showed the anterior approach to ssl to result in anatomic restoration of the vaginal vault with cure rates consistent with the posterior approach which our anatomic results support this as well . the anterior elevate system utilizes small self - fixating tips for attachment of the graft to the obturator internus muscle at the level of the bladder neck and into the ssls at the apex ( figs . the attachment of the graft to the ssls involves minimal dissection with placement of the small tip into the mid - portion of the ligament . secondary to this , risk of nerve injury or post - operative pain pudendal nerve type pain syndromes is minimal and this is supported in the current trial with no patients developing a post - operative pudendal nerve pain type syndrome and no patient requiring any surgical intervention for post - operative pain or dyspareunia . one does need to be careful ; however , in the adjustment of the graft at the apex as if too much tension is placed then this could potentially cause pain that may require release.fig . 2close - up view of the small self - fixating tip attaching into the ligament ( reproduced with permission from ams)fig . the bladder neck portion of the graft has been fixated to the levators and the apical portion of the graft is slid up the arms in a tension - free manner to elevate the anterior wall and vault . ( reproduced with permission from ams ) close - up view of the small self - fixating tip attaching into the ligament ( reproduced with permission from ams ) final adjustment of the graft into place . the bladder neck portion of the graft has been fixated to the levators and the apical portion of the graft is slid up the arms in a tension - free manner to elevate the anterior wall and vault . ( reproduced with permission from ams ) concerning the safety of the procedure , bleeding was minimal in most cases with average blood loss at 47 cc . one patient suffered a midline cystotomy during the procedure ; however , this was during the dissection of the anterior wall and was not related to the mesh or needle passes . overall , with the elimination of blind external needle passes the risk of intra - operative bladder or vascular injury is reduced . the mesh used in the current trial ( intepro lite ) is a type i macroporous polypropylene mesh that is 50% less dense and lighter than the first generation type i mesh ( intepro ) used in apogee / perigee . in a previously published trial from our center , we reported mesh extrusion rate of 6.5% with the use of the perigee procedure for treatment of cystocele in a similar group of patients , which is consistent with other reports in the literature . , a full - thickness dissection utilizing hydrodissection in a more avascular space , allows for less bleeding during the procedure , less risk of post - operative hematoma , and a thicker vascular flap healing over the mesh . prolift - m utilizes a soft polypropylene mesh that has a similar density to intepro - lite however is 50% monocryl , and therefore , 50% of the mesh is absorbable . sixteen patients ( 26.6% ) had previous repairs and would be considered higher risk for failure ; however , their cure rate was consistent with patients that had not had prior repair and there was no difference in complications seen in these patients either . in our opinion , this is a group of patients that benefit the greatest from a graft , given a previous failure using their own tissue , and the results of these patients in our series is very encouraging . the current study is limited by its retrospective nature and its medium - term follow - up . a limitation of any surgical trial that also has to be considered is surgeon experience with the particular procedure or similar procedures as well as the anatomy of the dissection involved . in conclusion , we have found the vaginal repair of anterior / apical wall prolapse utilizing a lightweight soft type i anterior wall mesh placed via the elevate system a safe , minimally invasive , and effective procedure for the treatment of anterior wall prolapse in this subset of mostly post - menopausal patients . we feel that the role of mesh in vaginal repairs is in its infancy , and much study still needs to be done to determine the ideal material to be utilized and the optimal way to place and attach the graft vaginally and the proper patient to utilize it in ; however , it can be expected that improvements in technology and techniques will continue to improve outcomes . the small self - fixating tips on the arms of the elevate procedure that hold the mesh in place are one of the least invasive approaches to fixate a mesh graft in place vaginally to date . we do recommend further prospective studies with longer - term follow - up to further help delineate its role in clinical practice .

cardiac magnetic resonance ( cmr ) offers a unique method of studying left ventricular ( lv ) morphology and function in patients with coronary artery disease ( cad ) . this includes the use of contrast media , in which the late gadolinium enhancement ( lge ) of the lv myocardium is indicative of nonviable tissue and eventually the formation of scar.1,2 irreversible injury of the myocardium following acute coronary artery occlusion is still an important cause of lv dysfunction and heart failure . a significant proportion of cases are clinically silent , ie , without symptoms that cause the patients to seek medical care.3,4 important determinants in the development of heart failure are the extent and transmurality of the infarcted wall and development of adverse lv remodeling.58 further , applying 1.5 t cmr transmurality of lv lge has been shown to predict improvement of the myocardial function after revascularization,9 and thereby provide valuable information with regard to preoperative individualized decision making . bearing in mind that cardiovascular disease is common , accounting for approximately 30% of all deaths in the us,10 the choice of optimized treatment strategy is of considerable importance to health care in general . achieving images at higher magnetic fields , eg , 3 t rather than 1.5 t , provides increased signal - to - noise ratio ( snr ) , which in turn may be translated into improved spatial resolution . although some particular artifacts are increased at high - field scanning compared to scanning at 1.5 t , this is outweighed by the benefits of image quality and reduced imaging time.11 acceptable inter - observer reproducibility for solid - state free precession ( ssfp ) and lge images at 3 t has been shown as well.12 on comparing lge imaging at 3 t versus 1.5 t , the snr has been shown to increase by 1.63.9 times , and the contrast - to - noise ratio by 1.93.3 times between an infarcted and a normal myocardium , depending on various sequences that were applied.13 combined with the high spatial resolution of electrocardiogram ( ecg)-triggered cmr acquisitions , this would be expected to give more detailed information about the localization and extent of the nonviable myocardium as well as lv regional wall remodeling ( rwr ) of the corresponding segments . in the present study , the lv myocardium of patients with suspected ischemic heart disease was prospectively examined by high - field 3 t cmr , whereas coronary angiography was used for demonstrating the extent and distribution of cad . the medical history of each patient was then followed for a mean time of 58 months , and the major adverse cardiovascular events ( mace ) were noted . the study was approved by the regional committee for medical and health research ( rek west ) , and conducted in accordance with the principles of the declaration of helsinki . we hypothesize that there is a positive correlation between the presence of nonviable myocardium and cad , and that the extent of nonviable myocardium is associated with the development of lvrwr and decreased lv ejection fraction ( lvef ) . eighty - seven patients ( 59 9 years ; 13 women ) with suspected stable cad undergoing elective coronary angiography ( n = 72 ) or coronary angiography due to unstable acute coronary syndrome including unstable angina pectoris or non - st - elevation myocardial infarction ( n = 15 ) were included . patients with general contraindications to mr scanning were excluded from the study , and all patients presented with a clinical and hemodynamic stable condition . prior to the procedures , all patients underwent a clinical examination , blood sampling including measurements of creatinine , c - reactive protein ( crp ) , cholesterol , and glucose , and echocardiography . the simpson method was applied for determining lvef ( % ) by echocardiography.14 patients also completed a self - administered questionnaire that provided information about medical history , risk factors , and prior medications . history of hypertension is with reference to subjects currently being treated with antihypertensive drugs , according to clinical criteria . smokers include current smokers and those reporting having quit within the last 4 weeks.15 information from the questionnaires was checked against medical records . coronary angiography was performed by experienced ( > 15 years ) cardiologists , and a total of 16 coronary artery segments were evaluated for possible stenosis in all patients : ie , 15 segments as per the american heart association standardization criteria16 plus the right atrioventricular branch . lesions with a diameter reduction of 30% and < 50% were classified as non - significant cad . significant cad was defined as a diameter of stenosis of 50% in any of the main coronary arteries ( left anterior descending ( lad ) , circumflex ( cx ) , and right coronary artery ( rca ) ) including their main side branches . the extent of significant cad was scored as no cad , one - vessel disease , two - vessel disease , or three - vessel disease , according to the number of main vessels with significant stenosis . presence of left main - stem artery stenosis with no rca stenosis was classified as two - vessel disease or as three - vessel disease if rca was hypoplastic . known coronary stenoses or occlusions from previously performed coronary angiography were also included , even if revascularization with percutaneous coronary intervention ( pci ) or coronary artery bypass grafting ( cabg ) had been done . the cmr examination was performed prior to , on the same day , or a few days following coronary angiography ( median 1 day ( 1 to + 1 days ) ) . cmr was performed and evaluated blindly with regard to the angiographic findings by an experienced ( > 15 years ) cmr reader . the patients were included over a period of 26 months , and the median follow - up time was 58 ( 4562 ) months . information on clinical events was collected from the cause of death registry at statistics norway and from the patient administrative systems at our university hospital , which is the primary hospital for the city of bergen and secondary hospital for western norway . the primary endpoint was mace , and included fatal and nonfatal acute myocardial infarction ( mi ) , re - angiography , and admission for major arrhythmias or heart failure . mi was classified according to the diagnostic criteria of the revised definition published in 2000.17 events occurring within 24 hours after pci or cabg were considered as procedure - related and were not included as endpoints . patients were examined with a 3 t ge signa excite scanner ( milwaukee , wi , usa ) . all images were obtained during breath - hold with ecg - triggering . for evaluation of regional wall thickness during the heart cycle , cine views of the left ventricle were taken . vertical long - axis view and four - chamber view were obtained , as well as consecutive breath - hold short - axis views of the entire left ventricle . the parameters of the ssfp sequence were as follows : field of view ( fov ) 330 330 mm ; slice thickness 8 mm ; matrix size : 192 192 ; flip angle 45. in order to detect lge of the lv myocardium , gadobutrol ( gadovist 1 mmol / ml ; bayer schering pharma , leverkusen , germany ) was administered intravenously at a concentration of 0.2 mmol / kg . twenty minutes later , images positioned correspondingly to that of the cine views were acquired . the parameters of the 2d fast inversion - recovery gradient echo sequence were as follows : fov 350 350 mm ; slice thickness 8 mm ; matrix size 256 128 ; flip angle 20. the time of inversion ( ti ) was optimized for each examination , but commonly 250 milliseconds was used . the presence of lv myocardial lge was defined as nonviable tissue , whereas the absence was defined as viable myocardial tissue . for assessing the distribution of lge , a 17-segment bulls - eye model was applied.18 hence , segments 1 , 2 , 3 , 7 , 8 , 13 , 14 , and 17 corresponded to the myocardium supplied by lad ; segments 5 , 6 , 11 , 12 , and 16 to cx ; and segments 4 , 9 , 10 , and 15 to rca , which then enabled comparison of viability with the angiographic findings of the corresponding coronary arteries . the extent of lge across the wall was noted and allocated into subgroups : subendocardial nonviable myocardium ( comprising < 50% of wall thickness ) ; transmural nonviable myocardium ( comprising 50% of wall thickness ) ; or combined subendocardial and transmural nonviable myocardium . lvrwr was defined as significant thinning of the myocardial wall ( 50% of the wall thickness compared to neighboring segment with viable myocardium).19 this was performed by assessing the ssfp short - axis views of the lv after identifying the end - diastolic phase . the cmr examination was well tolerated by all patients , and neither the presence of steel threads in sternum after cabg nor stents in the coronary stents showed any significant image artifacts with regard to image quality . continuous variables are given as means ( 1 standard deviation ( sd ) ) or medians ( 25th75th percentile ) , and categorical variables as counts ( percentages ) . for comparisons of subgroups of patients , differences in continuous variables were explored using independent student t - test , and proportions were compared by pearson s chi - square test ( cross - tabulations ) . furthermore , correlations between variables were analyzed by simple linear regression or stepwise regression models . logistic regression analysis was applied to calculate the odds ratios ( or ; with 95% confidence intervals ) ( ci ) for risk factors related to mace during follow - up . a multivariable model was applied , adjusting for lvef ( % ) , number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) . p < 0.05 are considered significant , and values > 0.05 are referred to as nonsignificant ( ns ) . all statistical analyses were computed by pasw statistics 18.0 ( spss inc , chicago , il , usa ) . eighty - seven patients ( 59 9 years ; 13 women ) with suspected stable cad undergoing elective coronary angiography ( n = 72 ) or coronary angiography due to unstable acute coronary syndrome including unstable angina pectoris or non - st - elevation myocardial infarction ( n = 15 ) were included . patients with general contraindications to mr scanning were excluded from the study , and all patients presented with a clinical and hemodynamic stable condition . prior to the procedures , all patients underwent a clinical examination , blood sampling including measurements of creatinine , c - reactive protein ( crp ) , cholesterol , and glucose , and echocardiography . the simpson method was applied for determining lvef ( % ) by echocardiography.14 patients also completed a self - administered questionnaire that provided information about medical history , risk factors , and prior medications . history of hypertension is with reference to subjects currently being treated with antihypertensive drugs , according to clinical criteria . smokers include current smokers and those reporting having quit within the last 4 weeks.15 information from the questionnaires was checked against medical records . coronary angiography was performed by experienced ( > 15 years ) cardiologists , and a total of 16 coronary artery segments were evaluated for possible stenosis in all patients : ie , 15 segments as per the american heart association standardization criteria16 plus the right atrioventricular branch . lesions with a diameter reduction of 30% and < 50% were classified as non - significant cad . significant cad was defined as a diameter of stenosis of 50% in any of the main coronary arteries ( left anterior descending ( lad ) , circumflex ( cx ) , and right coronary artery ( rca ) ) including their main side branches . the extent of significant cad was scored as no cad , one - vessel disease , two - vessel disease , or three - vessel disease , according to the number of main vessels with significant stenosis . presence of left main - stem artery stenosis with no rca stenosis was classified as two - vessel disease or as three - vessel disease if rca was hypoplastic . known coronary stenoses or occlusions from previously performed coronary angiography were also included , even if revascularization with percutaneous coronary intervention ( pci ) or coronary artery bypass grafting ( cabg ) had been done . the cmr examination was performed prior to , on the same day , or a few days following coronary angiography ( median 1 day ( 1 to + 1 days ) ) . cmr was performed and evaluated blindly with regard to the angiographic findings by an experienced ( > 15 years ) cmr reader . the patients were included over a period of 26 months , and the median follow - up time was 58 ( 4562 ) months . information on clinical events was collected from the cause of death registry at statistics norway and from the patient administrative systems at our university hospital , which is the primary hospital for the city of bergen and secondary hospital for western norway . the primary endpoint was mace , and included fatal and nonfatal acute myocardial infarction ( mi ) , re - angiography , and admission for major arrhythmias or heart failure . mi was classified according to the diagnostic criteria of the revised definition published in 2000.17 events occurring within 24 hours after pci or cabg were considered as procedure - related and were not included as endpoints . patients were examined with a 3 t ge signa excite scanner ( milwaukee , wi , usa ) . all images were obtained during breath - hold with ecg - triggering . for evaluation of regional wall thickness during the heart cycle , cine views of the left ventricle were taken . vertical long - axis view and four - chamber view were obtained , as well as consecutive breath - hold short - axis views of the entire left ventricle . the parameters of the ssfp sequence were as follows : field of view ( fov ) 330 330 mm ; slice thickness 8 mm ; matrix size : 192 192 ; flip angle 45. in order to detect lge of the lv myocardium , gadobutrol ( gadovist 1 mmol / ml ; bayer schering pharma , leverkusen , germany ) was administered intravenously at a concentration of 0.2 mmol / kg . twenty minutes later , images positioned correspondingly to that of the cine views were acquired . the parameters of the 2d fast inversion - recovery gradient echo sequence were as follows : fov 350 350 mm ; slice thickness 8 mm ; matrix size 256 128 ; flip angle 20. the time of inversion ( ti ) was optimized for each examination , but commonly 250 milliseconds was used . the presence of lv myocardial lge was defined as nonviable tissue , whereas the absence was defined as viable myocardial tissue . for assessing the distribution of lge , a 17-segment bulls - eye model was applied.18 hence , segments 1 , 2 , 3 , 7 , 8 , 13 , 14 , and 17 corresponded to the myocardium supplied by lad ; segments 5 , 6 , 11 , 12 , and 16 to cx ; and segments 4 , 9 , 10 , and 15 to rca , which then enabled comparison of viability with the angiographic findings of the corresponding coronary arteries . the extent of lge across the wall was noted and allocated into subgroups : subendocardial nonviable myocardium ( comprising < 50% of wall thickness ) ; transmural nonviable myocardium ( comprising 50% of wall thickness ) ; or combined subendocardial and transmural nonviable myocardium . lvrwr was defined as significant thinning of the myocardial wall ( 50% of the wall thickness compared to neighboring segment with viable myocardium).19 this was performed by assessing the ssfp short - axis views of the lv after identifying the end - diastolic phase . the cmr examination was well tolerated by all patients , and neither the presence of steel threads in sternum after cabg nor stents in the coronary stents showed any significant image artifacts with regard to image quality . continuous variables are given as means ( 1 standard deviation ( sd ) ) or medians ( 25th75th percentile ) , and categorical variables as counts ( percentages ) . for comparisons of subgroups of patients , differences in continuous variables were explored using independent student t - test , and proportions were compared by pearson s chi - square test ( cross - tabulations ) . furthermore , correlations between variables were analyzed by simple linear regression or stepwise regression models . logistic regression analysis was applied to calculate the odds ratios ( or ; with 95% confidence intervals ) ( ci ) for risk factors related to mace during follow - up . a multivariable model was applied , adjusting for lvef ( % ) , number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) . p < 0.05 are considered significant , and values > 0.05 are referred to as nonsignificant ( ns ) . all statistical analyses were computed by pasw statistics 18.0 ( spss inc , chicago , il , usa ) . lge in the lv wall demonstrating nonviable myocardium was detected in 35 ( 40% ) patients . all nonviable segments were related to the presence of significant stenosis or occlusion as confirmed by coronary angiography ; moreover , nonviable segments were not detected in angiographically open vessels . viable segments , on the other hand , were frequently found in territories with significant cad ( figs . 1 and 2 ) . the characteristics of the study population according to presence or absence of viable myocardium are summarized in table 1 . known chronic mis ( > 3 months old ) , previous cabg , or pci was overrepresented in the nonviable group , and lvef was significantly decreased ( p < 0.05 ) : 50 16% versus 61 8% . a considerable number of the patients underwent pci ( 39% , n = 34 ) , either directly or during a second intervention , whereas 12% ( n = 10 ) were scheduled for succeeding cabg . transmurality as well as the segmental localization and distribution of nonviable myocardium was evaluated and compared to the angiographic findings ( figs . 3 and 4 ) . of the 35 patients with nonviable myocardium , the number of these segments with regard to the corresponding blood supply territories was comparable : lad n = 18 segments , cx n = 17 , and rca n = 21 . further , the frequency of nonviable segments depicting subendocardial , transmural , or combined subendocardial and transmural nonviable myocardium was not statistically different for the three vessel territories . for the lad territory , the presence of subendocardial , transmural , and combined nonviable myocardium was 5% ( n = 1 ) , 67% ( 12 ) , 28% ( 5 ) ; for the cx territory 12% ( n = 2 ) , 47% ( 8) , 41% ( 7 ) ; and for rca territory 0% ( n = 0 ) , 52% ( 11 ) , 48% ( 10 ) . when allocating the patients with nonviable myocardium into three subgroups according to the number of nonviable segments , ie , low 03 segments , medium 47 segments , and high 8 segments , in addition to the group with only viable myocardium , a significant negative correlation to lvef was noted ( fig . moreover , the nonviable myocardium with evidence of lvrwr , 66% ( n = 23 ) , appeared mainly in the groups with medium and high numbers of nonviable segments ( fig . in nonviable hearts with lvrwr , the average number of nonviable segments was 6.0 3.2 , whereas the number of segments was significantly lower ( p < 0.001 ) , 2.6 1.3 , in hearts without lvrwr . further , in hearts with nonviable myocardium and lvrwr , lvef was significantly reduced ( p < 0.001 ) compared to nonviable hearts without lvrwr , ie , 44 17% versus 62 8% . when comparing patients with stable versus unstable angina , a significant difference in the presence of nonviable segments was found , ie , 1.5 2.6 versus 3.9 4.3 ( p = 0.007 ) , respectively . the incidence of mace was significantly higher ( p < 0.05 ) for those with nonviable myocardial segments than the group with only viable myocardial segments , ie , 19 patients out of 35 ( 54% ) versus 15 out of 52 ( 29% ) . by applying logistic regression analysis , we found that in the multivariable model adjusting for lvef ( % ) , number of nonviable segments , lvrwr ( yes versus no ) , and diagnosis , the number of nonviable segments was associated with mace by an or of 1.33 ( 95% ci , 0.981.80 ; p = 0.063 ) , and also diagnosis was borderline significant with an or of 0.27 ( 95% ci , 0.061.28 ; p = 0.098 ) . in a simple regression model , number of nonviable segments by lge correlated with lvef by an r value of 0.66 , p < 0.001 . furthermore , in a multi - variable stepwise regression model with lvef as dependent variable , including number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) , only the number of nonviable segments by lge was found as a significant predictor for lvef with r = 0.66 , b = 2.8 ( 95% ci : 3.5 to 2.1 ; p < 0.0001 ) . lge in the lv wall demonstrating nonviable myocardium was detected in 35 ( 40% ) patients . all nonviable segments were related to the presence of significant stenosis or occlusion as confirmed by coronary angiography ; moreover , nonviable segments were not detected in angiographically open vessels . viable segments , on the other hand , were frequently found in territories with significant cad ( figs . 1 and 2 ) . the characteristics of the study population according to presence or absence of viable myocardium are summarized in table 1 . known chronic mis ( > 3 months old ) , previous cabg , or pci was overrepresented in the nonviable group , and lvef was significantly decreased ( p < 0.05 ) : 50 16% versus 61 8% . a considerable number of the patients underwent pci ( 39% , n = 34 ) , either directly or during a second intervention , whereas 12% ( n = 10 ) were scheduled for succeeding cabg . transmurality as well as the segmental localization and distribution of nonviable myocardium was evaluated and compared to the angiographic findings ( figs . 3 and 4 ) . of the 35 patients with nonviable myocardium , the number of these segments with regard to the corresponding blood supply territories was comparable : lad n = 18 segments , cx n = 17 , and rca n = 21 . further , the frequency of nonviable segments depicting subendocardial , transmural , or combined subendocardial and transmural nonviable myocardium was not statistically different for the three vessel territories . for the lad territory , the presence of subendocardial , transmural , and combined nonviable myocardium was 5% ( n = 1 ) , 67% ( 12 ) , 28% ( 5 ) ; for the cx territory 12% ( n = 2 ) , 47% ( 8) , 41% ( 7 ) ; and for rca territory 0% ( n = 0 ) , 52% ( 11 ) , 48% ( 10 ) . when allocating the patients with nonviable myocardium into three subgroups according to the number of nonviable segments , ie , low 03 segments , medium 47 segments , and high 8 segments , in addition to the group with only viable myocardium , a significant negative correlation to lvef was noted ( fig . moreover , the nonviable myocardium with evidence of lvrwr , 66% ( n = 23 ) , appeared mainly in the groups with medium and high numbers of nonviable segments ( fig . hence , in nonviable hearts with lvrwr , the average number of nonviable segments was 6.0 3.2 , whereas the number of segments was significantly lower ( p < 0.001 ) , 2.6 1.3 , in hearts without lvrwr . further , in hearts with nonviable myocardium and lvrwr , lvef was significantly reduced ( p < 0.001 ) compared to nonviable hearts without lvrwr , ie , 44 17% versus 62 8% . when comparing patients with stable versus unstable angina , a significant difference in the presence of nonviable segments was found , ie , 1.5 2.6 versus 3.9 4.3 ( p = 0.007 ) , respectively . the incidence of mace was significantly higher ( p < 0.05 ) for those with nonviable myocardial segments than the group with only viable myocardial segments , ie , 19 patients out of 35 ( 54% ) versus 15 out of 52 ( 29% ) . by applying logistic regression analysis , we found that in the multivariable model adjusting for lvef ( % ) , number of nonviable segments , lvrwr ( yes versus no ) , and diagnosis , the number of nonviable segments was associated with mace by an or of 1.33 ( 95% ci , 0.981.80 ; p = 0.063 ) , and also diagnosis was borderline significant with an or of 0.27 ( 95% ci , 0.061.28 ; p = 0.098 ) . in a simple regression model , number of nonviable segments by lge correlated with lvef by an r value of 0.66 , p < 0.001 furthermore , in a multi - variable stepwise regression model with lvef as dependent variable , including number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) , only the number of nonviable segments by lge was found as a significant predictor for lvef with r = 0.66 , b = 2.8 ( 95% ci : 3.5 to 2.1 ; p < 0.0001 ) . in the present study , stable patients with suspected cad and referred for coronary angiography were examined with high - field 3 t cmr . being unaware of the angiographic results , the patients were allocated into a nonviable group and a viable group based on the presence or absence of lge of the lv myocardium . in this patient cohort , with a mean age of 59 years , nonviable segments occurred only in territories supplied with vessels confirmed to be either occluded or significantly stenotic . on the other hand , viable segments were frequently noted in territories supplied with vessels showing significant lumen narrowing . probably , sufficient collateral circulation from neighboring territories , which protects against myocardial necrosis , is an important mechanism for maintaining viability . the presence of lge in the myocardium has been shown to predict mace in both ischemic and nonischemic hearts . clinical studies of patients with hypertrophic cardiomyopathies , diabetes mellitus ii , hypertension , and congenital conditions including the senning - corrected systemic right ventricle and the fontan - corrected single ventricle have demonstrated significant prognostic value of lge as well.4,7,2027 hence , replacement of the normal contracting myocardium with fibrotic tissues may present common mechanisms for both ischemic and nonischemic heart diseases with regard to mace . the fibrotic tissues , therefore , may represent a substrate for severe ventricular arrhythmias and may contribute to wall motion abnormalities and thereby to adverse remodeling of the lv . a positive correlation between the amount of scar tissue and increased lv end - diastolic volume has been previously demonstrated.21,28 in the present study , the nonviable segments were directly related to lvrwr . a significant correlation between the number of nonviable segments and the development of lvrwr appeared . most likely , this can be explained by the increased regional wall stress due to the replacement of myocardium with fibrotic tissues characterized by inferior compliance during the diastole and reduced contractile properties at systole . a corresponding theory has been suggested for hypertrophic cardiomyopathies , where increased deposition of fibrotic tissues facilitates an escalation of the condition.27,29 further , in a study on patients treated with primary pci , it was found that hearts exhibiting at least four lv segments with lge was a strong predictor of adverse remodeling.28,30 this correlates well with the present patient cohort , where nonviable hearts with lvrwr had an average of 6.0 nonviable segments , whereas nonviable hearts without lvrwr had an average of 2.3 nonviable segments as defined by the presence of lge . the lvrwr was reflected by a decrease in global lv systolic function , where ef was 44% compared to 62% for the nonviable group not showing evidence of lvrwr . at 1.5 t , it has been shown that in patients with ischemic heart disease and reduced lvef there was a significant association between the extent of lge and increase in mortality and the need of cardiac transplantation.31 as lvef has been widely used a prognostic factor , we analyzed the relationship between lvef and the number of nonviable segments by lge , and found a significant negative correlation . thus , lvef could be used as a surrogate endpoint . on the other hand , in the multivariable logistic regression model , the number of nonviable segments by lge was found to be a stronger predictor for mace . lge was a stronger predictor than unstable coronary syndrome used as a variable in the analysis in spite of significantly more nonviable segments among unstable patients . . however , invasive treatment modifies this risk factor by treating and thereby reducing the significance of unstable coronary plaques , and this probably explains why the nonmodifiable lge remains as the strongest predictor . hence , considering nonviable myocardium of the lv myocardium as a valuable variable for the planning of revascularization and assessment of prognosis , the need for optimal and robust imaging tools is essential . although increased image resolution and snr is an advantage when using 3 t cmr compared to 1.5 t scanners , the increased magnetic field , however , is also potentially related to more image artifacts . this includes increased susceptibility , field inhomogeneity , and specific absorption rate ( sar ) while using 3 t scanners.32 however , these disadvantages have been considerably overcome , and presently 3 t cmr is emerging as a robust diagnostic technique.11 thus , comparison of lge at 3 t and 1.5 t mr systems has shown similar or superior image quality.3335 in this study , the images were assessable for all patients , and it was demonstrated that nonviable myocardium confirmed by lge was prognostically significant with regard to hard endpoints at a 3 t mr system similar to that noted for a number of 1.5 t cmr studies.7,23,24 the main limitations of this observational study are the small sample size and the restriction of the cohort to one large center . this may have influence on the demographics , treatment strategies , and enrolment criteria for being subjected to coronary angiography and cmr . a larger cohort would have enabled subgroup analyses and strengthened the statistical power of the results . patients with implanted devices ( eg , implantable cardioverter defibrillators / cardiac resynchronization devices ) or considerably reduced kidney function were not examined . thus , it is likely that a considerable proportion of those patients with notable advanced ischemic heart disease were excluded . this , in turn , may contribute to a selection bias and thereby influence the robustness of predicting clinical outcomes and survival patients with the most severe mis . the main limitations of this observational study are the small sample size and the restriction of the cohort to one large center . this may have influence on the demographics , treatment strategies , and enrolment criteria for being subjected to coronary angiography and cmr . a larger cohort would have enabled subgroup analyses and strengthened the statistical power of the results . patients with implanted devices ( eg , implantable cardioverter defibrillators / cardiac resynchronization devices ) or considerably reduced kidney function were not examined . thus , it is likely that a considerable proportion of those patients with notable advanced ischemic heart disease were excluded . this , in turn , may contribute to a selection bias and thereby influence the robustness of predicting clinical outcomes and survival patients with the most severe mis . thus , in our study , applying a segment - based comparison of the presence of nonviable myocardium detected by 3 t cmr and significant cad as shown by coronary angiography , a positive correlation was confirmed . the extent of nonviable myocardium correlated with the presence of lvrwr and reduced lv systolic function . although only a limited number of patients were examined and followed up , an increase of mace was found in the group with nonviable myocardium corresponding to that found by others using 1.5 t cmr . hence , this study suggests that 3 t cmr with detection of nonviable myocardium can be used as a noninvasive method for assessing patients with cad , and may contribute to the individualized risk - stratification and treatment strategy of the patient .

backgroundclinical follow - up studies comparing left ventricular ( lv ) function and late gadolinium enhancement ( lge ) by high - field 3 t cardiac magnetic resonance ( cmr ) are of general interest due to the increased use of 3 t scanners . in this study , the occurrence of lge and lv regional wall remodeling ( rwr ) was assessed by 3 t cmr in patients undergoing coronary angiography for suspected stable coronary artery disease ( cad).materials and methodsanalysis of myocardial viability by lge was performed at the segmental level . lvrwr was identified by a significant reduction ( 50% ) of the wall thickness . major adverse cardiovascular events ( mace ) were registered during a median follow - up time of 58 ( 4562 ) months.resultsof the 87 patients ( 59 9 years ; 13 women ) enrolled , nonviable myocardium was detected in 35 ( 40% ) and significant cad in 69 ( 79% ) . nonviable myocardium was correlated to angiographic significant stenosis or occlusion . lvrwr was significantly related to a higher number of nonviable segments compared to those without lvrwr : ie , 6.0 3.2 segments versus 2.6 1.3 ; p < 0.001 . in the nonviable group , lvef was significantly reduced ( p < 0.001 ) compared to the viable group : ie , 50 16% versus 61 8% , and lvef was significantly correlated to the number of nonviable segments ( r = 0.66 , p < 0.001 ) . the number of nonviable segments by lge was significantly associated with mace by an odds ratio of 1.25 ( 95% ci , 1.051.49 ; p = 0.013).conclusionthe presence of nonviable myocardium as detected by lge at 3 t cmr is associated with angiographically significant cad , and is associated with the development of lvrwr and reduced lvef . assessing the extent of nonviable myocardium by both lge and lvrwr at the segmental level may therefore contribute to individualized risk stratification and treatment strategies .

Introduction Methods Patients Follow-up and clinical endpoints CMR technique Analyses of the LV myocardium Statistical analysis Results Patient characteristics Nonviable segments and LVRWR Follow-up data Discussion Study limitations Conclusion

cardiac magnetic resonance ( cmr ) offers a unique method of studying left ventricular ( lv ) morphology and function in patients with coronary artery disease ( cad ) . this includes the use of contrast media , in which the late gadolinium enhancement ( lge ) of the lv myocardium is indicative of nonviable tissue and eventually the formation of scar.1,2 irreversible injury of the myocardium following acute coronary artery occlusion is still an important cause of lv dysfunction and heart failure . a significant proportion of cases are clinically silent , ie , without symptoms that cause the patients to seek medical care.3,4 important determinants in the development of heart failure are the extent and transmurality of the infarcted wall and development of adverse lv remodeling.58 further , applying 1.5 t cmr transmurality of lv lge has been shown to predict improvement of the myocardial function after revascularization,9 and thereby provide valuable information with regard to preoperative individualized decision making . although some particular artifacts are increased at high - field scanning compared to scanning at 1.5 t , this is outweighed by the benefits of image quality and reduced imaging time.11 acceptable inter - observer reproducibility for solid - state free precession ( ssfp ) and lge images at 3 t has been shown as well.12 on comparing lge imaging at 3 t versus 1.5 t , the snr has been shown to increase by 1.63.9 times , and the contrast - to - noise ratio by 1.93.3 times between an infarcted and a normal myocardium , depending on various sequences that were applied.13 combined with the high spatial resolution of electrocardiogram ( ecg)-triggered cmr acquisitions , this would be expected to give more detailed information about the localization and extent of the nonviable myocardium as well as lv regional wall remodeling ( rwr ) of the corresponding segments . in the present study , the lv myocardium of patients with suspected ischemic heart disease was prospectively examined by high - field 3 t cmr , whereas coronary angiography was used for demonstrating the extent and distribution of cad . the medical history of each patient was then followed for a mean time of 58 months , and the major adverse cardiovascular events ( mace ) were noted . we hypothesize that there is a positive correlation between the presence of nonviable myocardium and cad , and that the extent of nonviable myocardium is associated with the development of lvrwr and decreased lv ejection fraction ( lvef ) . eighty - seven patients ( 59 9 years ; 13 women ) with suspected stable cad undergoing elective coronary angiography ( n = 72 ) or coronary angiography due to unstable acute coronary syndrome including unstable angina pectoris or non - st - elevation myocardial infarction ( n = 15 ) were included . coronary angiography was performed by experienced ( > 15 years ) cardiologists , and a total of 16 coronary artery segments were evaluated for possible stenosis in all patients : ie , 15 segments as per the american heart association standardization criteria16 plus the right atrioventricular branch . the extent of significant cad was scored as no cad , one - vessel disease , two - vessel disease , or three - vessel disease , according to the number of main vessels with significant stenosis . the patients were included over a period of 26 months , and the median follow - up time was 58 ( 4562 ) months . for assessing the distribution of lge , a 17-segment bulls - eye model was applied.18 hence , segments 1 , 2 , 3 , 7 , 8 , 13 , 14 , and 17 corresponded to the myocardium supplied by lad ; segments 5 , 6 , 11 , 12 , and 16 to cx ; and segments 4 , 9 , 10 , and 15 to rca , which then enabled comparison of viability with the angiographic findings of the corresponding coronary arteries . the extent of lge across the wall was noted and allocated into subgroups : subendocardial nonviable myocardium ( comprising < 50% of wall thickness ) ; transmural nonviable myocardium ( comprising 50% of wall thickness ) ; or combined subendocardial and transmural nonviable myocardium . lvrwr was defined as significant thinning of the myocardial wall ( 50% of the wall thickness compared to neighboring segment with viable myocardium).19 this was performed by assessing the ssfp short - axis views of the lv after identifying the end - diastolic phase . a multivariable model was applied , adjusting for lvef ( % ) , number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) . eighty - seven patients ( 59 9 years ; 13 women ) with suspected stable cad undergoing elective coronary angiography ( n = 72 ) or coronary angiography due to unstable acute coronary syndrome including unstable angina pectoris or non - st - elevation myocardial infarction ( n = 15 ) were included . coronary angiography was performed by experienced ( > 15 years ) cardiologists , and a total of 16 coronary artery segments were evaluated for possible stenosis in all patients : ie , 15 segments as per the american heart association standardization criteria16 plus the right atrioventricular branch . the extent of significant cad was scored as no cad , one - vessel disease , two - vessel disease , or three - vessel disease , according to the number of main vessels with significant stenosis . the patients were included over a period of 26 months , and the median follow - up time was 58 ( 4562 ) months . for assessing the distribution of lge , a 17-segment bulls - eye model was applied.18 hence , segments 1 , 2 , 3 , 7 , 8 , 13 , 14 , and 17 corresponded to the myocardium supplied by lad ; segments 5 , 6 , 11 , 12 , and 16 to cx ; and segments 4 , 9 , 10 , and 15 to rca , which then enabled comparison of viability with the angiographic findings of the corresponding coronary arteries . the extent of lge across the wall was noted and allocated into subgroups : subendocardial nonviable myocardium ( comprising < 50% of wall thickness ) ; transmural nonviable myocardium ( comprising 50% of wall thickness ) ; or combined subendocardial and transmural nonviable myocardium . lvrwr was defined as significant thinning of the myocardial wall ( 50% of the wall thickness compared to neighboring segment with viable myocardium).19 this was performed by assessing the ssfp short - axis views of the lv after identifying the end - diastolic phase . a multivariable model was applied , adjusting for lvef ( % ) , number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) . lge in the lv wall demonstrating nonviable myocardium was detected in 35 ( 40% ) patients . all nonviable segments were related to the presence of significant stenosis or occlusion as confirmed by coronary angiography ; moreover , nonviable segments were not detected in angiographically open vessels . known chronic mis ( > 3 months old ) , previous cabg , or pci was overrepresented in the nonviable group , and lvef was significantly decreased ( p < 0.05 ) : 50 16% versus 61 8% . transmurality as well as the segmental localization and distribution of nonviable myocardium was evaluated and compared to the angiographic findings ( figs . of the 35 patients with nonviable myocardium , the number of these segments with regard to the corresponding blood supply territories was comparable : lad n = 18 segments , cx n = 17 , and rca n = 21 . further , the frequency of nonviable segments depicting subendocardial , transmural , or combined subendocardial and transmural nonviable myocardium was not statistically different for the three vessel territories . for the lad territory , the presence of subendocardial , transmural , and combined nonviable myocardium was 5% ( n = 1 ) , 67% ( 12 ) , 28% ( 5 ) ; for the cx territory 12% ( n = 2 ) , 47% ( 8) , 41% ( 7 ) ; and for rca territory 0% ( n = 0 ) , 52% ( 11 ) , 48% ( 10 ) . when allocating the patients with nonviable myocardium into three subgroups according to the number of nonviable segments , ie , low 03 segments , medium 47 segments , and high 8 segments , in addition to the group with only viable myocardium , a significant negative correlation to lvef was noted ( fig . moreover , the nonviable myocardium with evidence of lvrwr , 66% ( n = 23 ) , appeared mainly in the groups with medium and high numbers of nonviable segments ( fig . in nonviable hearts with lvrwr , the average number of nonviable segments was 6.0 3.2 , whereas the number of segments was significantly lower ( p < 0.001 ) , 2.6 1.3 , in hearts without lvrwr . further , in hearts with nonviable myocardium and lvrwr , lvef was significantly reduced ( p < 0.001 ) compared to nonviable hearts without lvrwr , ie , 44 17% versus 62 8% . when comparing patients with stable versus unstable angina , a significant difference in the presence of nonviable segments was found , ie , 1.5 2.6 versus 3.9 4.3 ( p = 0.007 ) , respectively . the incidence of mace was significantly higher ( p < 0.05 ) for those with nonviable myocardial segments than the group with only viable myocardial segments , ie , 19 patients out of 35 ( 54% ) versus 15 out of 52 ( 29% ) . by applying logistic regression analysis , we found that in the multivariable model adjusting for lvef ( % ) , number of nonviable segments , lvrwr ( yes versus no ) , and diagnosis , the number of nonviable segments was associated with mace by an or of 1.33 ( 95% ci , 0.981.80 ; p = 0.063 ) , and also diagnosis was borderline significant with an or of 0.27 ( 95% ci , 0.061.28 ; p = 0.098 ) . in a simple regression model , number of nonviable segments by lge correlated with lvef by an r value of 0.66 , p < 0.001 . furthermore , in a multi - variable stepwise regression model with lvef as dependent variable , including number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) , only the number of nonviable segments by lge was found as a significant predictor for lvef with r = 0.66 , b = 2.8 ( 95% ci : 3.5 to 2.1 ; p < 0.0001 ) . lge in the lv wall demonstrating nonviable myocardium was detected in 35 ( 40% ) patients . all nonviable segments were related to the presence of significant stenosis or occlusion as confirmed by coronary angiography ; moreover , nonviable segments were not detected in angiographically open vessels . known chronic mis ( > 3 months old ) , previous cabg , or pci was overrepresented in the nonviable group , and lvef was significantly decreased ( p < 0.05 ) : 50 16% versus 61 8% . transmurality as well as the segmental localization and distribution of nonviable myocardium was evaluated and compared to the angiographic findings ( figs . of the 35 patients with nonviable myocardium , the number of these segments with regard to the corresponding blood supply territories was comparable : lad n = 18 segments , cx n = 17 , and rca n = 21 . further , the frequency of nonviable segments depicting subendocardial , transmural , or combined subendocardial and transmural nonviable myocardium was not statistically different for the three vessel territories . for the lad territory , the presence of subendocardial , transmural , and combined nonviable myocardium was 5% ( n = 1 ) , 67% ( 12 ) , 28% ( 5 ) ; for the cx territory 12% ( n = 2 ) , 47% ( 8) , 41% ( 7 ) ; and for rca territory 0% ( n = 0 ) , 52% ( 11 ) , 48% ( 10 ) . when allocating the patients with nonviable myocardium into three subgroups according to the number of nonviable segments , ie , low 03 segments , medium 47 segments , and high 8 segments , in addition to the group with only viable myocardium , a significant negative correlation to lvef was noted ( fig . moreover , the nonviable myocardium with evidence of lvrwr , 66% ( n = 23 ) , appeared mainly in the groups with medium and high numbers of nonviable segments ( fig . hence , in nonviable hearts with lvrwr , the average number of nonviable segments was 6.0 3.2 , whereas the number of segments was significantly lower ( p < 0.001 ) , 2.6 1.3 , in hearts without lvrwr . further , in hearts with nonviable myocardium and lvrwr , lvef was significantly reduced ( p < 0.001 ) compared to nonviable hearts without lvrwr , ie , 44 17% versus 62 8% . when comparing patients with stable versus unstable angina , a significant difference in the presence of nonviable segments was found , ie , 1.5 2.6 versus 3.9 4.3 ( p = 0.007 ) , respectively . the incidence of mace was significantly higher ( p < 0.05 ) for those with nonviable myocardial segments than the group with only viable myocardial segments , ie , 19 patients out of 35 ( 54% ) versus 15 out of 52 ( 29% ) . by applying logistic regression analysis , we found that in the multivariable model adjusting for lvef ( % ) , number of nonviable segments , lvrwr ( yes versus no ) , and diagnosis , the number of nonviable segments was associated with mace by an or of 1.33 ( 95% ci , 0.981.80 ; p = 0.063 ) , and also diagnosis was borderline significant with an or of 0.27 ( 95% ci , 0.061.28 ; p = 0.098 ) . in a simple regression model , number of nonviable segments by lge correlated with lvef by an r value of 0.66 , p < 0.001 furthermore , in a multi - variable stepwise regression model with lvef as dependent variable , including number of nonviable segments by lge , lvrwr ( yes versus no ) , and diagnosis ( stable versus unstable coronary syndrome ) , only the number of nonviable segments by lge was found as a significant predictor for lvef with r = 0.66 , b = 2.8 ( 95% ci : 3.5 to 2.1 ; p < 0.0001 ) . in the present study , stable patients with suspected cad and referred for coronary angiography were examined with high - field 3 t cmr . being unaware of the angiographic results , the patients were allocated into a nonviable group and a viable group based on the presence or absence of lge of the lv myocardium . a positive correlation between the amount of scar tissue and increased lv end - diastolic volume has been previously demonstrated.21,28 in the present study , the nonviable segments were directly related to lvrwr . a significant correlation between the number of nonviable segments and the development of lvrwr appeared . most likely , this can be explained by the increased regional wall stress due to the replacement of myocardium with fibrotic tissues characterized by inferior compliance during the diastole and reduced contractile properties at systole . a corresponding theory has been suggested for hypertrophic cardiomyopathies , where increased deposition of fibrotic tissues facilitates an escalation of the condition.27,29 further , in a study on patients treated with primary pci , it was found that hearts exhibiting at least four lv segments with lge was a strong predictor of adverse remodeling.28,30 this correlates well with the present patient cohort , where nonviable hearts with lvrwr had an average of 6.0 nonviable segments , whereas nonviable hearts without lvrwr had an average of 2.3 nonviable segments as defined by the presence of lge . the lvrwr was reflected by a decrease in global lv systolic function , where ef was 44% compared to 62% for the nonviable group not showing evidence of lvrwr . at 1.5 t , it has been shown that in patients with ischemic heart disease and reduced lvef there was a significant association between the extent of lge and increase in mortality and the need of cardiac transplantation.31 as lvef has been widely used a prognostic factor , we analyzed the relationship between lvef and the number of nonviable segments by lge , and found a significant negative correlation . on the other hand , in the multivariable logistic regression model , the number of nonviable segments by lge was found to be a stronger predictor for mace . although increased image resolution and snr is an advantage when using 3 t cmr compared to 1.5 t scanners , the increased magnetic field , however , is also potentially related to more image artifacts . this includes increased susceptibility , field inhomogeneity , and specific absorption rate ( sar ) while using 3 t scanners.32 however , these disadvantages have been considerably overcome , and presently 3 t cmr is emerging as a robust diagnostic technique.11 thus , comparison of lge at 3 t and 1.5 t mr systems has shown similar or superior image quality.3335 in this study , the images were assessable for all patients , and it was demonstrated that nonviable myocardium confirmed by lge was prognostically significant with regard to hard endpoints at a 3 t mr system similar to that noted for a number of 1.5 t cmr studies.7,23,24 the main limitations of this observational study are the small sample size and the restriction of the cohort to one large center . thus , in our study , applying a segment - based comparison of the presence of nonviable myocardium detected by 3 t cmr and significant cad as shown by coronary angiography , a positive correlation was confirmed . the extent of nonviable myocardium correlated with the presence of lvrwr and reduced lv systolic function . hence , this study suggests that 3 t cmr with detection of nonviable myocardium can be used as a noninvasive method for assessing patients with cad , and may contribute to the individualized risk - stratification and treatment strategy of the patient .

owing to demographic aging , dementia has been identified as a major economic and societal issue , and several studies have investigated the economic impact of dementia on health care systems around the world . there is broad agreement that the clinical course of dementia ( ongoing physical and cognitive decline combined with challenging behavior ) strongly promotes nursing home placement . many studies have been performed to identify the crucial risk factors for institutionalization in individuals with dementia . in germany , one acts on the assumption that every year up to 25% of previously community - living dementia patients transfer to nursing homes and that only 20% of the patients eventually die at home . institutional care is reported to be a major cost driver in dementia care especially in more advanced disease stages but there are only a few cross - sectional studies comparing costs of community - based and institutional dementia care . to ensure comparability with our own data referring to 2006 as the base year , we first converted the reported figures to euros by applying the average currency exchange rate of the corresponding year and then inflated these figures to 2006 values by applying the german gross domestic product deflator . the following paragraph focuses on expenditures borne by the health care system and does not account for out - of - pocket payments and informal care . according to our own previous research , per capita expenditures for institutionalized dementia patients are substantially higher than those for community - living ones ( approx . this is in line with the results from the belgian nades study ( institutional care approx . . however , these aggregated figures seemingly fail to answer the question whether costs are equally distributed across the entire observation period . in this regard , the work of sands et al . , which compared health care spending for community - living and nursing home - based medicaid recipients with dementia over a 12-month time frame , is an important advancement . eur 2,600 in month 12 ) but steadily increasing expenditures in the community - living population ( approx . it has to be kept in mind that all the studies cited contrasted two patient strata with different socioeconomic and clinical characteristics , rendering a scientifically sound comparison of both care settings at least to some extent questionable . moreover , the research described does not address the question to what extent a change in health care setting might influence the provision of health care services . corresponding knowledge is essential to detect potential shortfalls and to improve continuity of care provision for individuals with dementia after institutionalization , and is thus highly relevant for health economics and health care services research . to the best of our knowledge , no longitudinal study dealing with these issues by following a defined patient population over a distinct time horizon before and after institutionalization has been published yet . based on longitudinal patient level data from a large german statutory health insurance ( shi ) fund , this paper aims ( a ) to describe the health care service utilization patterns of individuals with dementia from four quarters before until four quarters after transfer to a nursing home ( hereafter institutionalization ) and ( b ) to assess the thereby incurred overall shi expenditures ( i.e. , cost of care from a payer perspective ) . within germany , health and long - term care insurance is a legal obligation , and about 85% of the resident population is insured within statutory funds . these funds are organized by the principles of pay - as - you - go financing and income - dependent but risk - independent contributions . health insurance is designed as a full coverage insurance , which allows access to a broad range of medical services for a small co - payment . nursing care services are not covered by shi but fall under the scope of compulsory long - term care insurance ( ltci ) . in order to make a claim for ltci support , insurants with impaired capabilities in their activities of daily living an independent statutory corporation that is directly supervised by the state 's ministry of social affairs . if the application is accepted , applicants are assigned to one of three care levels ( i.e. adl dependency groups ) . only individuals with a care level assignment receive ltci support in the form of partial reimbursement of nursing care services up to a legally fixed ceiling amount , which depends on the type of services provided and the recipient 's care level . sample selection was based on 2005 - 2007 insurance claims data from aok bavaria , a large regional shi fund , which had been provided for a previous excess cost study . within this sample , 9,147 individuals with dementia had been identified based on 2005 and 2006 inpatient and outpatient diagnosis codes ( icd-10 codes f00 , f01 , f02 , f03 , and g30 ) and anti - dementia drug prescriptions ( atc codes n06da and n06dx01 ) . to be classified as a dementia patient , the documentation of at least one of these dementia indicators within three of four consecutive quarters was required . we identified the residential setting of the 9,147 dementia patients based on 2006 data from the ltci . ltci data only exist for individuals who have made a claim for ltci services , i.e. , for subjects who are considered to be in need of nursing care according to the code of social law . similar to previous research , we classified all individuals without an ltci claim as community living ( n = 3,110 ) . this decision is based on the assumption that individuals without an ltci claim have no need to transfer to a nursing home . for individuals with an ltci claim , institutional care is conditional on nursing home residency , and all nursing home residents but no community - living individuals receive corresponding payments . therefore , the starting date of payments for institutional care equals the date of transfer to a nursing home , and the end date of payments for institutional care if different from the date of death reflects a transfer from a nursing home to the community setting . we classified the remaining 6,037 individuals with an ltci claim as continuously community living in 2006 if institutional care was never documented between january 1 and december 31 ( n = 2,425 ) , as continuously institutionalized in 2006 if institutional care was documented without interruptions from january 1 to december 31 ( n = 2,933 ) , and as transferring between community - based and institutional care in 2006 if payments for institutional care started during 2006 ( n = 679 ) . in 2006 , 18 of the transferring individuals shifted back to community - based care , and 10 individuals showed multiple shifts between community - based and institutional care . these 28 individuals were dropped , and 651 permanently institutionalized individuals were finally included in our analyses . figure 1 visualizes the selection process in a flow chart . within the german shi system , therefore , the quarter of the year was chosen as the reference period for our analyses . to define the quarter of institutionalization ( qi ) for each individual institutional care in 2006 which is reported on a daily basis and determined the respective quarter . for example , for an individual transferring to a nursing home on june 15 , 2006 , qi would have been q2/2006 ; for an individual shifting on october 2 , 2006 , it would have been q4/2006 and so on . based on qi , for each individual , the four quarters before ( i.e. , the period of community - based care ) and after ( i.e. , the period of institutional care ) were identified . given that transfer to a nursing home could take place from q1/2006 earliest to q4/2006 latest , the individual observation periods lasted from q1/2005 to q1/2007 ( for transfers in q1/2006 ) up to q4/2005 to q4/2007 ( for transfers in q4/2006 ) . the main outcome parameter of our analysis was service utilization within the different service domains of shi . these are care of general practitioners and medical specialists , drugs , hospital care , non - physician services , medical aids , rehabilitation , and home health care . within this paper , non - physician services refer to non - medicinal remedies covered by the shi , such as physiotherapy , occupational therapy , or medicinal massages . the term home health care is used to describe the provision of temporary medical nursing care , which contrary to permanent nursing care falls within the scope of shi . the services of general practitioners and medical specialists were already reported as visits per quarter but , for the remaining domains , transformation was necessary . for drugs and medical aids , the exact date of prescription was given . to reach per quarter values , all prescriptions within the distinct time frame were added . home health care , non - physician services , hospital care , and rehabilitation do not refer to a distinct point in time but to a distinct time frame . as home health care is usually prescribed on a monthly basis , we assessed each month of prescription , assigned it to the corresponding quarter , and summed up the prescriptions within a quarter . however , this straightforward approach was not applicable for hospital care , rehabilitation , and non - physician services , as the duration of these services can range over several quarters . regarding these domains , we distributed service utilization to the distinctive phases whenever treatment took place in different quarters ( e.g. , if a hospital stay lasted from june 25 to july 4 , 6 days were assigned to q2/2006 and 4 days to q3/2006 ) . in addition to service utilization , we also analyzed the corresponding expenditure trends as a secondary outcome parameter . to do so , we either summed up costs per unit of utilization ( drugs , medical aids , home health care ) or attributed them proportionally to time of resource use implying constant amounts per day ( hospital , rehabilitation , non - physician services ) . to ensure the comparability of quarter - specific costs , all amounts have been inflated to 2006 values ( year of institutionalization ) by applying the gross domestic product deflator . to account for the longitudinal nature of the data and the accompanying intra - subject correlation , the analyses were based on the generalized estimation equation ( gee ) approach , which represents a marginal model with robust parameter estimates . contrary to mixed models , which would have been an alternative approach to account for intra - subject correlation , marginal models calculate population averages instead of subject - specific trajectories . the applied gee estimates the quarter - specific utilization and cost figures within the study sample accounting for the intra - subject correlation as a nuisance variable . assuming that service utilization and costs of care within a distinct quarter depend on the values observed within the previous quarter , first - order autoregression was chosen as the working correlation . exponentiation of the calculated group - based estimators describes service utilization and costs for each quarter as a percentage of the reference quarter . the distribution of service utilization and costs is zero bounded and positively skewed to the right . to best possibly fit this non - normal distribution , we assumed a negative binomial distribution for service utilization and a gamma distribution for costs . as individuals with zero costs would have been dropped in a gamma model , we assigned all individuals without costs the small positive amount of eur 0.5 to keep them in the analyses . age , gender , comorbidity , and dementia duration were included as cofactors in all analyses . comorbidity was reflected by a modified version of the charlson index , which accounts for all index diseases except for dementia . to reflect the patient - individual morbidity burden at the point of institutionalization , inpatient and outpatient diagnoses in the last two quarters before qi were used to calculate the index . dementia duration was expressed by a dummy variable indicating whether dementia was already prevalent within the year prior to institutionalization or whether dementia was newly developed during the year of institutionalization . we also performed extended models accounting for all possible interactions between age , gender , incidental versus prevalent dementia , and observation quarter . as these interaction terms turned out to be not statistically significant , we decided on the simple model without interaction terms , which allows a more straightforward interpretation of p values . within an additional sensitivity analysis , we excluded individuals who died during the observation period ( n = 91 ) . the results of this analysis differed only marginally from those of the main analysis ; thus , the corresponding results are not included in the paper . all analyses were performed using sas version 9.2 , and a p value of at least 0.01 was defined as being statistically significant . within germany , health and long - term care insurance is a legal obligation , and about 85% of the resident population is insured within statutory funds . these funds are organized by the principles of pay - as - you - go financing and income - dependent but risk - independent contributions . health insurance is designed as a full coverage insurance , which allows access to a broad range of medical services for a small co - payment . nursing care services are not covered by shi but fall under the scope of compulsory long - term care insurance ( ltci ) . in order to make a claim for ltci support , insurants with impaired capabilities in their activities of daily living an independent statutory corporation that is directly supervised by the state 's ministry of social affairs . if the application is accepted , applicants are assigned to one of three care levels ( i.e. adl dependency groups ) . only individuals with a care level assignment receive ltci support in the form of partial reimbursement of nursing care services up to a legally fixed ceiling amount , which depends on the type of services provided and the recipient 's care level . sample selection was based on 2005 - 2007 insurance claims data from aok bavaria , a large regional shi fund , which had been provided for a previous excess cost study . within this sample , 9,147 individuals with dementia had been identified based on 2005 and 2006 inpatient and outpatient diagnosis codes ( icd-10 codes f00 , f01 , f02 , f03 , and g30 ) and anti - dementia drug prescriptions ( atc codes n06da and n06dx01 ) . to be classified as a dementia patient , the documentation of at least one of these dementia indicators within three of four consecutive quarters was required . we identified the residential setting of the 9,147 dementia patients based on 2006 data from the ltci . ltci data only exist for individuals who have made a claim for ltci services , i.e. , for subjects who are considered to be in need of nursing care according to the code of social law . similar to previous research , we classified all individuals without an ltci claim as community living ( n = 3,110 ) . this decision is based on the assumption that individuals without an ltci claim have no need to transfer to a nursing home . for individuals with an ltci claim , the care setting can be assessed based on the provision of distinct ltci services . the ltci service institutional care is conditional on nursing home residency , and all nursing home residents but no community - living individuals receive corresponding payments . therefore , the starting date of payments for institutional care equals the date of transfer to a nursing home , and the end date of payments for institutional care if different from the date of death reflects a transfer from a nursing home to the community setting . we classified the remaining 6,037 individuals with an ltci claim as continuously community living in 2006 if institutional care was never documented between january 1 and december 31 ( n = 2,425 ) , as continuously institutionalized in 2006 if institutional care was documented without interruptions from january 1 to december 31 ( n = 2,933 ) , and as transferring between community - based and institutional care in 2006 if payments for institutional care started during 2006 ( n = 679 ) . in 2006 , 18 of the transferring individuals shifted back to community - based care , and 10 individuals showed multiple shifts between community - based and institutional care . these 28 individuals were dropped , and 651 permanently institutionalized individuals were finally included in our analyses . within the german shi system , physician services are documented and billed on a quarterly basis . therefore , the quarter of the year was chosen as the reference period for our analyses . to define the quarter of institutionalization ( qi ) for each individual institutional care in 2006 which is reported on a daily basis and determined the respective quarter . for example , for an individual transferring to a nursing home on june 15 , 2006 , qi would have been q2/2006 ; for an individual shifting on october 2 , 2006 , it would have been q4/2006 and so on . based on qi , for each individual , the four quarters before ( i.e. , the period of community - based care ) and after ( i.e. , the period of institutional care ) were identified . given that transfer to a nursing home could take place from q1/2006 earliest to q4/2006 latest , the individual observation periods lasted from q1/2005 to q1/2007 ( for transfers in q1/2006 ) up to q4/2005 to q4/2007 ( for transfers in q4/2006 ) . the main outcome parameter of our analysis was service utilization within the different service domains of shi . these are care of general practitioners and medical specialists , drugs , hospital care , non - physician services , medical aids , rehabilitation , and home health care . within this paper , non - physician services refer to non - medicinal remedies covered by the shi , such as physiotherapy , occupational therapy , or medicinal massages . the term home health care is used to describe the provision of temporary medical nursing care , which contrary to permanent nursing care falls within the scope of shi . the services of general practitioners and medical specialists were already reported as visits per quarter but , for the remaining domains , transformation was necessary . for drugs and medical aids , the exact date of prescription was given . to reach per quarter values , all prescriptions within the distinct time frame were added . home health care , non - physician services , hospital care , and rehabilitation do not refer to a distinct point in time but to a distinct time frame . as home health care is usually prescribed on a monthly basis , we assessed each month of prescription , assigned it to the corresponding quarter , and summed up the prescriptions within a quarter . however , this straightforward approach was not applicable for hospital care , rehabilitation , and non - physician services , as the duration of these services can range over several quarters . regarding these domains , we distributed service utilization to the distinctive phases whenever treatment took place in different quarters ( e.g. , if a hospital stay lasted from june 25 to july 4 , 6 days were assigned to q2/2006 and 4 days to q3/2006 ) . in addition to service utilization , we also analyzed the corresponding expenditure trends as a secondary outcome parameter . to do so , we either summed up costs per unit of utilization ( drugs , medical aids , home health care ) or attributed them proportionally to time of resource use implying constant amounts per day ( hospital , rehabilitation , non - physician services ) . to ensure the comparability of quarter - specific costs , all amounts have been inflated to 2006 values ( year of institutionalization ) by applying the gross domestic product deflator . to account for the longitudinal nature of the data and the accompanying intra - subject correlation , the analyses were based on the generalized estimation equation ( gee ) approach , which represents a marginal model with robust parameter estimates . contrary to mixed models , which would have been an alternative approach to account for intra - subject correlation , marginal models calculate population averages instead of subject - specific trajectories . the applied gee estimates the quarter - specific utilization and cost figures within the study sample accounting for the intra - subject correlation as a nuisance variable . assuming that service utilization and costs of care within a distinct quarter depend on the values observed within the previous quarter , first - order autoregression was chosen as the working correlation . exponentiation of the calculated group - based estimators describes service utilization and costs for each quarter as a percentage of the reference quarter . the distribution of service utilization and costs is zero bounded and positively skewed to the right . to best possibly fit this non - normal distribution , we assumed a negative binomial distribution for service utilization and a gamma distribution for costs . as individuals with zero costs would have been dropped in a gamma model , we assigned all individuals without costs the small positive amount of eur 0.5 to keep them in the analyses . age , gender , comorbidity , and dementia duration were included as cofactors in all analyses . comorbidity was reflected by a modified version of the charlson index , which accounts for all index diseases except for dementia . to reflect the patient - individual morbidity burden at the point of institutionalization , inpatient and outpatient diagnoses in the last two quarters before qi were used to calculate the index . dementia duration was expressed by a dummy variable indicating whether dementia was already prevalent within the year prior to institutionalization or whether dementia was newly developed during the year of institutionalization . we also performed extended models accounting for all possible interactions between age , gender , incidental versus prevalent dementia , and observation quarter . as these interaction terms turned out to be not statistically significant , we decided on the simple model without interaction terms , which allows a more straightforward interpretation of p values . within an additional sensitivity analysis , we excluded individuals who died during the observation period ( n = 91 ) . the results of this analysis differed only marginally from those of the main analysis ; thus , the corresponding results are not included in the paper . all analyses were performed using sas version 9.2 , and a p value of at least 0.01 was defined as being statistically significant . the transferees were almost equally distributed across the four quarters of 2006 ( q1 : 24.3% , q2 : 27.5% , q3 : 27.0% , and q4 : 21.2% ) . a total of 497 ( 76.3% ) of the transferring dementia patients were female , and 456 ( 70.1% ) had been diagnosed with dementia prior to the year of transfer . individuals transferring to a nursing home were on average 82.8 years old ( sd 6.9 ) and , given a mean charlson index of 3.9 ( sd 2.9 ) , their comorbidity burden was substantial . table 1 describes the percentage of transferring individuals with service utilization in the distinct shi categories for each observation quarter . by trend , the quota of service users increased over time with a particularly strong increase in qi . home health care was the only exception to this rule , as corresponding services are dispensed with after nursing home transfer . the first pattern was characterized by a steadily increasing user quota from the beginning of the observation period until the phase of institutionalization ( i.e. , qi and , if required , the adjacent quarter ) and stabilization at a higher level afterwards . this pattern applied to general practitioner services , drugs , and non - physician services . however , non - physician services represented an exception in that their user quota literally escalated within qi ( duplication ) , whereas the increase was more continuous in the other three domains . the second pattern showed an increasing user quota from the beginning of the observation period until the phase of institutionalization and a decrease afterwards . this pattern was observed in the use of medical specialists , hospitals , and rehabilitation . the third pattern presented an increasing user quota over the entire observation period with a remarkable level shift from qi onwards ( quadruplication ) . the trend of the mean per capita service utilization across the nine observation quarters as estimated by the gee is shown in table 2 . within all service domains ( except for home health care ) , the estimated volume of service utilization was higher in qi than in the four previous quarters . during the year after institutionalization , the volume of service utilization of general practitioner visits and medical specialist visits decreased gradually to approximately the initial level . regarding hospital and rehabilitation days , there was an abrupt decrease immediately within the first quarter after institutionalization . the number of drug prescriptions , medical aid prescriptions , and non - physician service prescriptions in the institutional setting evened out at a higher level than in the community setting . regarding the last two service domains , this level was remarkably higher than in qi . concerning all service domains , the volume of service use within each quarter prior to institutionalization as well as within each quarter after institutionalization differed significantly from the volume of service use in qi . the only exception was found during the first quarter after institutionalization regarding medical specialist visits and drug prescriptions , which did not differ significantly ( p values of 0.10 and 0.07 , respectively ) . figure 2 visualizes the longitudinal trend in service utilization for the distinct service domains by pointing out the relative difference from qi . it demonstrates that institutionalization had only a temporary effect on acute medical needs represented by physician visits or inpatient treatment , but a long - lasting effect on supportive services such as medical aids and non - physician services . as shown in table 3 , shi expenditures followed an inverse u - shape peaking in qi . indeed , the rise in shi expenditures was already quite pronounced one quarter before institutionalization . figure 3 indicates that escalating expenditures within the quarter prior to institutionalization and qi were almost exclusively explained by hospital expenditures , which accounted for about two thirds of the entire shi expenditures in these two quarters . even from q2_prior to q1_prior , hospital costs almost tripled , whereas the increase did not exceed 20% within the other service domains . expenditures on hospital care , rehabilitation , general practitioners , and medical specialists decreased after institutionalization , but expenditures on drugs and particularly on medical aids and non - physician services remained elevated . they accounted for approximately 5% of shi expenditures during the four quarters before institutionalization but ranged between 15 and 25% within the four quarters after institutionalization . the nursing care expenditures of ltci , which were incurred in addition to the health care expenditures of shi , escalated in the context of institutionalization and remained substantially increased afterwards ( table 3 ) . the transferees were almost equally distributed across the four quarters of 2006 ( q1 : 24.3% , q2 : 27.5% , q3 : 27.0% , and q4 : 21.2% ) . a total of 497 ( 76.3% ) of the transferring dementia patients were female , and 456 ( 70.1% ) had been diagnosed with dementia prior to the year of transfer . individuals transferring to a nursing home were on average 82.8 years old ( sd 6.9 ) and , given a mean charlson index of 3.9 ( sd 2.9 ) , their comorbidity burden was substantial . table 1 describes the percentage of transferring individuals with service utilization in the distinct shi categories for each observation quarter . by trend , the quota of service users increased over time with a particularly strong increase in qi . home health care was the only exception to this rule , as corresponding services are dispensed with after nursing home transfer . the first pattern was characterized by a steadily increasing user quota from the beginning of the observation period until the phase of institutionalization ( i.e. , qi and , if required , the adjacent quarter ) and stabilization at a higher level afterwards . this pattern applied to general practitioner services , drugs , and non - physician services . however , non - physician services represented an exception in that their user quota literally escalated within qi ( duplication ) , whereas the increase was more continuous in the other three domains . the second pattern showed an increasing user quota from the beginning of the observation period until the phase of institutionalization and a decrease afterwards . this pattern was observed in the use of medical specialists , hospitals , and rehabilitation . the third pattern presented an increasing user quota over the entire observation period with a remarkable level shift from qi onwards ( quadruplication ) . the trend of the mean per capita service utilization across the nine observation quarters as estimated by the gee is shown in table 2 . within all service domains ( except for home health care ) , the estimated volume of service utilization was higher in qi than in the four previous quarters . during the year after institutionalization , the volume of service utilization of general practitioner visits and medical specialist visits decreased gradually to approximately the initial level . regarding hospital and rehabilitation days the number of drug prescriptions , medical aid prescriptions , and non - physician service prescriptions in the institutional setting evened out at a higher level than in the community setting . regarding the last two service domains , this level was remarkably higher than in qi . concerning all service domains , the volume of service use within each quarter prior to institutionalization as well as within each quarter after institutionalization differed significantly from the volume of service use in qi . the only exception was found during the first quarter after institutionalization regarding medical specialist visits and drug prescriptions , which did not differ significantly ( p values of 0.10 and 0.07 , respectively ) . figure 2 visualizes the longitudinal trend in service utilization for the distinct service domains by pointing out the relative difference from qi . it demonstrates that institutionalization had only a temporary effect on acute medical needs represented by physician visits or inpatient treatment , but a long - lasting effect on supportive services such as medical aids and non - physician services . table 1 describes the percentage of transferring individuals with service utilization in the distinct shi categories for each observation quarter . by trend , the quota of service users increased over time with a particularly strong increase in qi . home health care was the only exception to this rule , as corresponding services are dispensed with after nursing home transfer . the first pattern was characterized by a steadily increasing user quota from the beginning of the observation period until the phase of institutionalization ( i.e. , qi and , if required , the adjacent quarter ) and stabilization at a higher level afterwards . this pattern applied to general practitioner services , drugs , and non - physician services . however , non - physician services represented an exception in that their user quota literally escalated within qi ( duplication ) , whereas the increase was more continuous in the other three domains . the second pattern showed an increasing user quota from the beginning of the observation period until the phase of institutionalization and a decrease afterwards . this pattern was observed in the use of medical specialists , hospitals , and rehabilitation . the third pattern presented an increasing user quota over the entire observation period with a remarkable level shift from qi onwards ( quadruplication ) . the trend of the mean per capita service utilization across the nine observation quarters as estimated by the gee is shown in table 2 . within all service domains ( except for home health care ) , the estimated volume of service utilization was higher in qi than in the four previous quarters . during the year after institutionalization , the volume of service utilization of general practitioner visits and medical specialist visits decreased gradually to approximately the initial level . regarding hospital and rehabilitation days , there was an abrupt decrease immediately within the first quarter after institutionalization . the number of drug prescriptions , medical aid prescriptions , and non - physician service prescriptions in the institutional setting evened out at a higher level than in the community setting . regarding the last two service domains , this level was remarkably higher than in qi . concerning all service domains , the volume of service use within each quarter prior to institutionalization as well as within each quarter after institutionalization differed significantly from the volume of service use in qi . the only exception was found during the first quarter after institutionalization regarding medical specialist visits and drug prescriptions , which did not differ significantly ( p values of 0.10 and 0.07 , respectively ) . figure 2 visualizes the longitudinal trend in service utilization for the distinct service domains by pointing out the relative difference from qi . it demonstrates that institutionalization had only a temporary effect on acute medical needs represented by physician visits or inpatient treatment , but a long - lasting effect on supportive services such as medical aids and non - physician services . as shown in table 3 , shi expenditures followed an inverse u - shape peaking in qi . indeed , the rise in shi expenditures was already quite pronounced one quarter before institutionalization . figure 3 indicates that escalating expenditures within the quarter prior to institutionalization and qi were almost exclusively explained by hospital expenditures , which accounted for about two thirds of the entire shi expenditures in these two quarters . even from q2_prior to q1_prior , hospital costs almost tripled , whereas the increase did not exceed 20% within the other service domains . expenditures on hospital care , rehabilitation , general practitioners , and medical specialists decreased after institutionalization , but expenditures on drugs and particularly on medical aids and non - physician services remained elevated . they accounted for approximately 5% of shi expenditures during the four quarters before institutionalization but ranged between 15 and 25% within the four quarters after institutionalization . the nursing care expenditures of ltci , which were incurred in addition to the health care expenditures of shi , escalated in the context of institutionalization and remained substantially increased afterwards ( table 3 ) . our analysis of patient - level shi data from 651 dementia patients covering a period from four quarters before to four quarters after nursing home transfer revealed that , within all shi domains , the volume of quarterly per capita utilization escalated in qi compared with the directly adjacent quarter . once institutionalization had taken place , the trend in the level of service utilization did not increase further but stabilized ( drugs : level of qi ; non - physician services and medical aids : increased level ) or even decreased to the initial level ( physician services and inpatient treatment ) . ltci expenditures increased substantially after institutionalization because payments for institutional long - term care per legal definition exceed payments for community - based long - term care . summing up the quarter - specific expenditures resulted in higher health care expenditures during the year prior to institutionalization than during the year after institutionalization ( eur 5,780 vs. 4,478 ) . regarding long - term care expenditures , the situation was reversed ( eur 2,178 vs. 11,699 ) , and they accounted for the most part of the total costs of care . these spending structures within the german shi / ltci system are in line with previous cross - sectional research on expenditures borne by the health care system . however , it has to be kept in mind that a payer perspective disregards out - of - pocket payments and particularly does not account for non - monetary cost components such as unpaid family care . valued informal care in the community setting thus , from a societal perspective , which accounts for monetary and non - monetary cost components , institutional care might even be the less costly option , as has been recently shown by koenig et al . . compared with cross - sectional studies , our disaggregated longitudinal design allows the analysis of utilization and expenditure trends in the context of nursing home transfer . the results suggest that the period of institutionalization has to be considered as an exceptional time frame. regarding outpatient and inpatient treatment , a substantial increase in service utilization is already observed within the quarter prior to institutionalization . obviously , changes in health status which precipitate the decision to institutionalize are already manifest some months before the definite date of transfer . this observation indicates that the health care - seeking behavior of individuals who will be shortly institutionalized is different from the health care - seeking behavior of individuals who will stay in the community setting . indeed , a comparison of cost data between the 651 transferring dementia patients and the 2,425 dementia patients who were continuously community living in 2006 revealed significantly higher health care expenditures within the four quarters prior to institutionalization than within a usual year . institutionalization as a censoring event within cross - sectional analyses on community - living populations and to calculate correspondingly adjusted average per capita costs and utilization figures might be biased towards overestimation . it seems more advisable to consider individuals as a distinct subgroup shortly before institutionalization and to run stratified analyses . moreover , the analyses illustrate that institutionalization is rather associated with an increased demand for continuous care and support ( e.g. , medical aids and drugs ) than with an increased demand for acute medical care ( e.g. , inpatient and outpatient care ) . indeed , outpatient and inpatient care decrease to the initial level after peaking in qi . hence , it can be concluded that the decision to institutionalize is triggered by acute health - deteriorating events such as a hospital stay , but that acute illnesses , which require specialized medical care , do not occur more frequently within the institutional setting . however , dementia is a progressive syndrome , and the patients ' ongoing physical and cognitive decline is not affected by the residential setting per se . the increased demand for medical aids and non - physician services after nursing home transfer matches the hypothesis of deteriorating health in the context of dementia progression . nevertheless , no similar trend within the post - institutionalization period was observed regarding physician services . therefore , the reduced contact frequency with general practitioners and medical specialists in the institutional setting might be an indicator of undersupply in individuals with more advanced dementia . according to a recent health technology assessment ( hta ) analysis on behalf of the german institute of medical documentation and information ( dimdi ) , there is a common opinion among physicians working in germany that visits to nursing homes are not financially interesting , and thus this service is often not offered . to judge our findings comprehensively , the limitations of the applied approach need to be taken into consideration . first , claims data - based analyses are linked to some intrinsic caveats , such as lacking information on disease severity and socioeconomic factors ( household size , income ) . there is broad scientific consensus that these parameters influence health care service utilization , costs of care , and the final decision to transfer to a nursing home . despite these undocumented parameters definitively influence the absolute utilization level , there is no reason to assume that they affect utilization trends themselves , especially within the post - institutionalization period . second , the gee approach required the assignment of small positive values to individuals with zero costs . thus , there is an overestimation of quarter - specific costs , especially in service domains with a generally low user quota ( e.g. , rehabilitation ) . we are aware that this aspect is of particular relevance if cost trends within distinct shi domains are looked at , but we strongly believe that it is not a major issue if the entire shi expenditures are estimated . indeed , the percentage of insurants without any shi costs in a distinct quarter ranged between 6.5 and 0.0% . given this low quota combined with the small amount replaced in case of zero costs ( eur 0.5 ) , we are convinced that we have not introduced substantial bias . third , our analyses did not include a control sample of individuals without dementia transferring to a nursing home . thus , it is not possible to conclude whether the observed profiles are dementia specific or context specific , i.e. , whether they apply to individuals without dementia as well . as institutionalization of individuals without dementia does not happen very often , most studies on risk factors for institutionalization do not compare individuals with and without dementia . such a comparative design was chosen by schoenmakers et al . who reported lack of self - management and problems with care to be the most important motifs for institutionalization in individuals with and without dementia . age , functional impairment , and comorbidity are other shared risk factors for transfer to a nursing home . thus , we assume that individuals with and without dementia show similar health care service utilization profiles in the context of institutionalization , even though there might be a dementia - specific level effect . despite these drawbacks , our study design offers some noteworthy advantages . first of all , claims data provide information on all individuals insured including the oldest of the old , frail individuals , and those in the terminal phase of life . these individuals have a particularly high risk of institutionalization but are often not enrolled in primary data - based studies . moreover , information on service utilization and costs can be obtained directly from the data . basically , this eliminates the issues of non - response and recall bias , which would have occurred , for example , if nursing home residents had been retrospectively asked about their utilization patterns before and after transfer . therefore , we believe that , for our particular research question , claims data yield more reliable results than primary data . additionally , our analyses did not only consider overall costs but also domain - specific trends . this itemized view enables the investigation of common hypotheses regarding interactions between the distinct domains . for example , our analyses did not find a substitution effect between care from medical specialists and general practitioners ( parallel trend ) or a complementary relationship between drug prescriptions and physician visits . finally , changes in the cost structure , which reflect the changing relevance of distinct service domains , are traceable . in particular , it provides evidence that the months around the date of institutionalization represent an exceptional time frame regarding the level of service utilization . this has methodological implications in that the use of setting - related adjusted average per capita figures needs to be scrutinized carefully in further studies . moreover , the analysis demonstrated that institutional dementia care seems not necessarily to be associated with an increased demand for specialized medical care . assuming dementia progression over time , a decrease in specialized medical care services during the post - institutionalization period seems counterintuitive . to comprehensively interpret this observation , further evidence is required to establish to what extent institutionalization affects the quality of care provision .

backgroundcommunity - based and institutional dementia care has been compared in cross - sectional studies , but longitudinal information on the effect of institutionalization on health care service utilization is sparse.methodswe analyzed claims data from 651 dementia patients via generalized estimation equations to assess health care service utilization profiles and corresponding expenditures from four quarters before to four quarters after institutionalization.resultsin all domains , utilization increased in the quarter of institutionalization . afterwards , the use of drugs , medical aids , and non - physician services ( e.g. , occupational therapy and physiotherapy ) remained elevated , but use of in- and outpatient treatment decreased . cost of care showed corresponding profiles.conclusioninstitutional dementia care seems to be associated with an increased demand for supportive services but not necessarily for specialized medical care .

Background Methods Background Information on the German Health Care System Data Source and Sample Selection Observation Period Outcome Parameters Statistics Results Baseline Characteristics Utilization of Health Care Services User Quota within the Distinct Services Mean Volume of Service Utilization Costs within the Distinct Service Domains Discussion Disclosure Statement

owing to demographic aging , dementia has been identified as a major economic and societal issue , and several studies have investigated the economic impact of dementia on health care systems around the world . in germany , one acts on the assumption that every year up to 25% of previously community - living dementia patients transfer to nursing homes and that only 20% of the patients eventually die at home . institutional care is reported to be a major cost driver in dementia care especially in more advanced disease stages but there are only a few cross - sectional studies comparing costs of community - based and institutional dementia care . , which compared health care spending for community - living and nursing home - based medicaid recipients with dementia over a 12-month time frame , is an important advancement . moreover , the research described does not address the question to what extent a change in health care setting might influence the provision of health care services . corresponding knowledge is essential to detect potential shortfalls and to improve continuity of care provision for individuals with dementia after institutionalization , and is thus highly relevant for health economics and health care services research . to the best of our knowledge , no longitudinal study dealing with these issues by following a defined patient population over a distinct time horizon before and after institutionalization has been published yet . based on longitudinal patient level data from a large german statutory health insurance ( shi ) fund , this paper aims ( a ) to describe the health care service utilization patterns of individuals with dementia from four quarters before until four quarters after transfer to a nursing home ( hereafter institutionalization ) and ( b ) to assess the thereby incurred overall shi expenditures ( i.e. , cost of care from a payer perspective ) . only individuals with a care level assignment receive ltci support in the form of partial reimbursement of nursing care services up to a legally fixed ceiling amount , which depends on the type of services provided and the recipient 's care level . sample selection was based on 2005 - 2007 insurance claims data from aok bavaria , a large regional shi fund , which had been provided for a previous excess cost study . within this sample , 9,147 individuals with dementia had been identified based on 2005 and 2006 inpatient and outpatient diagnosis codes ( icd-10 codes f00 , f01 , f02 , f03 , and g30 ) and anti - dementia drug prescriptions ( atc codes n06da and n06dx01 ) . to be classified as a dementia patient , the documentation of at least one of these dementia indicators within three of four consecutive quarters was required . we identified the residential setting of the 9,147 dementia patients based on 2006 data from the ltci . we classified the remaining 6,037 individuals with an ltci claim as continuously community living in 2006 if institutional care was never documented between january 1 and december 31 ( n = 2,425 ) , as continuously institutionalized in 2006 if institutional care was documented without interruptions from january 1 to december 31 ( n = 2,933 ) , and as transferring between community - based and institutional care in 2006 if payments for institutional care started during 2006 ( n = 679 ) . in 2006 , 18 of the transferring individuals shifted back to community - based care , and 10 individuals showed multiple shifts between community - based and institutional care . within the german shi system , therefore , the quarter of the year was chosen as the reference period for our analyses . to define the quarter of institutionalization ( qi ) for each individual institutional care in 2006 which is reported on a daily basis and determined the respective quarter . based on qi , for each individual , the four quarters before ( i.e. , the period of community - based care ) and after ( i.e. these are care of general practitioners and medical specialists , drugs , hospital care , non - physician services , medical aids , rehabilitation , and home health care . within this paper , non - physician services refer to non - medicinal remedies covered by the shi , such as physiotherapy , occupational therapy , or medicinal massages . for drugs and medical aids , the exact date of prescription was given . home health care , non - physician services , hospital care , and rehabilitation do not refer to a distinct point in time but to a distinct time frame . however , this straightforward approach was not applicable for hospital care , rehabilitation , and non - physician services , as the duration of these services can range over several quarters . regarding these domains , we distributed service utilization to the distinctive phases whenever treatment took place in different quarters ( e.g. to do so , we either summed up costs per unit of utilization ( drugs , medical aids , home health care ) or attributed them proportionally to time of resource use implying constant amounts per day ( hospital , rehabilitation , non - physician services ) . to account for the longitudinal nature of the data and the accompanying intra - subject correlation , the analyses were based on the generalized estimation equation ( gee ) approach , which represents a marginal model with robust parameter estimates . assuming that service utilization and costs of care within a distinct quarter depend on the values observed within the previous quarter , first - order autoregression was chosen as the working correlation . exponentiation of the calculated group - based estimators describes service utilization and costs for each quarter as a percentage of the reference quarter . to best possibly fit this non - normal distribution , we assumed a negative binomial distribution for service utilization and a gamma distribution for costs . to reflect the patient - individual morbidity burden at the point of institutionalization , inpatient and outpatient diagnoses in the last two quarters before qi were used to calculate the index . as these interaction terms turned out to be not statistically significant , we decided on the simple model without interaction terms , which allows a more straightforward interpretation of p values . the results of this analysis differed only marginally from those of the main analysis ; thus , the corresponding results are not included in the paper . only individuals with a care level assignment receive ltci support in the form of partial reimbursement of nursing care services up to a legally fixed ceiling amount , which depends on the type of services provided and the recipient 's care level . sample selection was based on 2005 - 2007 insurance claims data from aok bavaria , a large regional shi fund , which had been provided for a previous excess cost study . within this sample , 9,147 individuals with dementia had been identified based on 2005 and 2006 inpatient and outpatient diagnosis codes ( icd-10 codes f00 , f01 , f02 , f03 , and g30 ) and anti - dementia drug prescriptions ( atc codes n06da and n06dx01 ) . to be classified as a dementia patient , the documentation of at least one of these dementia indicators within three of four consecutive quarters was required . for individuals with an ltci claim , the care setting can be assessed based on the provision of distinct ltci services . therefore , the starting date of payments for institutional care equals the date of transfer to a nursing home , and the end date of payments for institutional care if different from the date of death reflects a transfer from a nursing home to the community setting . we classified the remaining 6,037 individuals with an ltci claim as continuously community living in 2006 if institutional care was never documented between january 1 and december 31 ( n = 2,425 ) , as continuously institutionalized in 2006 if institutional care was documented without interruptions from january 1 to december 31 ( n = 2,933 ) , and as transferring between community - based and institutional care in 2006 if payments for institutional care started during 2006 ( n = 679 ) . in 2006 , 18 of the transferring individuals shifted back to community - based care , and 10 individuals showed multiple shifts between community - based and institutional care . therefore , the quarter of the year was chosen as the reference period for our analyses . to define the quarter of institutionalization ( qi ) for each individual institutional care in 2006 which is reported on a daily basis and determined the respective quarter . based on qi , for each individual , the four quarters before ( i.e. , the period of community - based care ) and after ( i.e. these are care of general practitioners and medical specialists , drugs , hospital care , non - physician services , medical aids , rehabilitation , and home health care . within this paper , non - physician services refer to non - medicinal remedies covered by the shi , such as physiotherapy , occupational therapy , or medicinal massages . for drugs and medical aids , the exact date of prescription was given . home health care , non - physician services , hospital care , and rehabilitation do not refer to a distinct point in time but to a distinct time frame . as home health care is usually prescribed on a monthly basis , we assessed each month of prescription , assigned it to the corresponding quarter , and summed up the prescriptions within a quarter . however , this straightforward approach was not applicable for hospital care , rehabilitation , and non - physician services , as the duration of these services can range over several quarters . regarding these domains , we distributed service utilization to the distinctive phases whenever treatment took place in different quarters ( e.g. to do so , we either summed up costs per unit of utilization ( drugs , medical aids , home health care ) or attributed them proportionally to time of resource use implying constant amounts per day ( hospital , rehabilitation , non - physician services ) . to account for the longitudinal nature of the data and the accompanying intra - subject correlation , the analyses were based on the generalized estimation equation ( gee ) approach , which represents a marginal model with robust parameter estimates . assuming that service utilization and costs of care within a distinct quarter depend on the values observed within the previous quarter , first - order autoregression was chosen as the working correlation . exponentiation of the calculated group - based estimators describes service utilization and costs for each quarter as a percentage of the reference quarter . to reflect the patient - individual morbidity burden at the point of institutionalization , inpatient and outpatient diagnoses in the last two quarters before qi were used to calculate the index . as these interaction terms turned out to be not statistically significant , we decided on the simple model without interaction terms , which allows a more straightforward interpretation of p values . the results of this analysis differed only marginally from those of the main analysis ; thus , the corresponding results are not included in the paper . a total of 497 ( 76.3% ) of the transferring dementia patients were female , and 456 ( 70.1% ) had been diagnosed with dementia prior to the year of transfer . this pattern applied to general practitioner services , drugs , and non - physician services . however , non - physician services represented an exception in that their user quota literally escalated within qi ( duplication ) , whereas the increase was more continuous in the other three domains . this pattern was observed in the use of medical specialists , hospitals , and rehabilitation . within all service domains ( except for home health care ) , the estimated volume of service utilization was higher in qi than in the four previous quarters . during the year after institutionalization , the volume of service utilization of general practitioner visits and medical specialist visits decreased gradually to approximately the initial level . the number of drug prescriptions , medical aid prescriptions , and non - physician service prescriptions in the institutional setting evened out at a higher level than in the community setting . concerning all service domains , the volume of service use within each quarter prior to institutionalization as well as within each quarter after institutionalization differed significantly from the volume of service use in qi . it demonstrates that institutionalization had only a temporary effect on acute medical needs represented by physician visits or inpatient treatment , but a long - lasting effect on supportive services such as medical aids and non - physician services . expenditures on hospital care , rehabilitation , general practitioners , and medical specialists decreased after institutionalization , but expenditures on drugs and particularly on medical aids and non - physician services remained elevated . they accounted for approximately 5% of shi expenditures during the four quarters before institutionalization but ranged between 15 and 25% within the four quarters after institutionalization . the nursing care expenditures of ltci , which were incurred in addition to the health care expenditures of shi , escalated in the context of institutionalization and remained substantially increased afterwards ( table 3 ) . the transferees were almost equally distributed across the four quarters of 2006 ( q1 : 24.3% , q2 : 27.5% , q3 : 27.0% , and q4 : 21.2% ) . a total of 497 ( 76.3% ) of the transferring dementia patients were female , and 456 ( 70.1% ) had been diagnosed with dementia prior to the year of transfer . table 1 describes the percentage of transferring individuals with service utilization in the distinct shi categories for each observation quarter . this pattern applied to general practitioner services , drugs , and non - physician services . however , non - physician services represented an exception in that their user quota literally escalated within qi ( duplication ) , whereas the increase was more continuous in the other three domains . this pattern was observed in the use of medical specialists , hospitals , and rehabilitation . within all service domains ( except for home health care ) , the estimated volume of service utilization was higher in qi than in the four previous quarters . during the year after institutionalization , the volume of service utilization of general practitioner visits and medical specialist visits decreased gradually to approximately the initial level . regarding hospital and rehabilitation days the number of drug prescriptions , medical aid prescriptions , and non - physician service prescriptions in the institutional setting evened out at a higher level than in the community setting . concerning all service domains , the volume of service use within each quarter prior to institutionalization as well as within each quarter after institutionalization differed significantly from the volume of service use in qi . it demonstrates that institutionalization had only a temporary effect on acute medical needs represented by physician visits or inpatient treatment , but a long - lasting effect on supportive services such as medical aids and non - physician services . table 1 describes the percentage of transferring individuals with service utilization in the distinct shi categories for each observation quarter . this pattern applied to general practitioner services , drugs , and non - physician services . however , non - physician services represented an exception in that their user quota literally escalated within qi ( duplication ) , whereas the increase was more continuous in the other three domains . this pattern was observed in the use of medical specialists , hospitals , and rehabilitation . within all service domains ( except for home health care ) , the estimated volume of service utilization was higher in qi than in the four previous quarters . during the year after institutionalization , the volume of service utilization of general practitioner visits and medical specialist visits decreased gradually to approximately the initial level . the number of drug prescriptions , medical aid prescriptions , and non - physician service prescriptions in the institutional setting evened out at a higher level than in the community setting . concerning all service domains , the volume of service use within each quarter prior to institutionalization as well as within each quarter after institutionalization differed significantly from the volume of service use in qi . it demonstrates that institutionalization had only a temporary effect on acute medical needs represented by physician visits or inpatient treatment , but a long - lasting effect on supportive services such as medical aids and non - physician services . expenditures on hospital care , rehabilitation , general practitioners , and medical specialists decreased after institutionalization , but expenditures on drugs and particularly on medical aids and non - physician services remained elevated . they accounted for approximately 5% of shi expenditures during the four quarters before institutionalization but ranged between 15 and 25% within the four quarters after institutionalization . the nursing care expenditures of ltci , which were incurred in addition to the health care expenditures of shi , escalated in the context of institutionalization and remained substantially increased afterwards ( table 3 ) . our analysis of patient - level shi data from 651 dementia patients covering a period from four quarters before to four quarters after nursing home transfer revealed that , within all shi domains , the volume of quarterly per capita utilization escalated in qi compared with the directly adjacent quarter . once institutionalization had taken place , the trend in the level of service utilization did not increase further but stabilized ( drugs : level of qi ; non - physician services and medical aids : increased level ) or even decreased to the initial level ( physician services and inpatient treatment ) . summing up the quarter - specific expenditures resulted in higher health care expenditures during the year prior to institutionalization than during the year after institutionalization ( eur 5,780 vs. 4,478 ) . regarding long - term care expenditures , the situation was reversed ( eur 2,178 vs. 11,699 ) , and they accounted for the most part of the total costs of care . these spending structures within the german shi / ltci system are in line with previous cross - sectional research on expenditures borne by the health care system . valued informal care in the community setting thus , from a societal perspective , which accounts for monetary and non - monetary cost components , institutional care might even be the less costly option , as has been recently shown by koenig et al . compared with cross - sectional studies , our disaggregated longitudinal design allows the analysis of utilization and expenditure trends in the context of nursing home transfer . the results suggest that the period of institutionalization has to be considered as an exceptional time frame. regarding outpatient and inpatient treatment , a substantial increase in service utilization is already observed within the quarter prior to institutionalization . indeed , a comparison of cost data between the 651 transferring dementia patients and the 2,425 dementia patients who were continuously community living in 2006 revealed significantly higher health care expenditures within the four quarters prior to institutionalization than within a usual year . moreover , the analyses illustrate that institutionalization is rather associated with an increased demand for continuous care and support ( e.g. , medical aids and drugs ) than with an increased demand for acute medical care ( e.g. hence , it can be concluded that the decision to institutionalize is triggered by acute health - deteriorating events such as a hospital stay , but that acute illnesses , which require specialized medical care , do not occur more frequently within the institutional setting . the increased demand for medical aids and non - physician services after nursing home transfer matches the hypothesis of deteriorating health in the context of dementia progression . to judge our findings comprehensively , the limitations of the applied approach need to be taken into consideration . first , claims data - based analyses are linked to some intrinsic caveats , such as lacking information on disease severity and socioeconomic factors ( household size , income ) . there is broad scientific consensus that these parameters influence health care service utilization , costs of care , and the final decision to transfer to a nursing home . thus , there is an overestimation of quarter - specific costs , especially in service domains with a generally low user quota ( e.g. thus , we assume that individuals with and without dementia show similar health care service utilization profiles in the context of institutionalization , even though there might be a dementia - specific level effect . first of all , claims data provide information on all individuals insured including the oldest of the old , frail individuals , and those in the terminal phase of life . these individuals have a particularly high risk of institutionalization but are often not enrolled in primary data - based studies . finally , changes in the cost structure , which reflect the changing relevance of distinct service domains , are traceable . this has methodological implications in that the use of setting - related adjusted average per capita figures needs to be scrutinized carefully in further studies . moreover , the analysis demonstrated that institutional dementia care seems not necessarily to be associated with an increased demand for specialized medical care . assuming dementia progression over time , a decrease in specialized medical care services during the post - institutionalization period seems counterintuitive .

diseases caused by trypanosomatids constitute a substantial health and socioeconomic problem in several countries , mainly in the americas , sub - saharan africa and tropical and subtropical belt regions . in particular , chagas disease , ( caused by trypanosoma cruzi ) affects 8 to 10 million people in the americas , with an additional 40 million people at risk ( http://www.who.int/tdr ) . t. cruzi has a complex life cycle involving a reduviid insect vector and a mammalian host ( fig . insect vectors become infected when they bite an infected mammal that has trypomastigote forms of the parasite circulating in its bloodstream . trypomastigotes , infective non - dividing forms , are ingested with the blood ; in the insect s digestive tube , they differentiate into dividing and non - infective epimastigote forms . in the terminal portion of the digestive tube , epimastigotes differentiate into metacyclic trypomastigotes , which are eliminated in faeces and deposited on mammals skin while the triatomine bug bites and feeds . trypomastigotes enter the body and invade host cells ; they differentiate into dividing amastigote forms and after proliferating , differentiate into trypomastigotes , passing through a transient epimastigote - like stage . finally , the trypomastigotes lyse host cells and are released into the extracellular medium , where they can invade other cells or the bloodstream , becoming capable of invading other tissues or a non - infected reduviid insect , thus completing the cycle . schematic representation of the trypanosoma cruzi life cycle . replicative , non - infective epimastigote forms ( a ) , predominantly present in the insect vector , give rise to non - replicative , infective metacyclic trypomastigotes ( b ) . metacyclic forms must invade the host cells and differentiate into replicative amastigote forms ( c ) to establish the infection . these forms give rise to a transient stage called intracellular epimastigotes ( d ) , which subsequently differentiate into trypomastigotes ( e ) . the cycle ends when the ingested trypomastigotes differentiate again into epimastigotes ( a ) , which colonize the digestive tube of a new insect . chagas disease presents mainly as two clinical phases in human beings : acute and chronic . the acute phase happens shortly after infection , beginning when the parasite enters the mammalian host . it is either largely asymptomatic or accompanied by non - specific symptoms such as fever and headache . it is characterized by an absence of antibodies and , in most patients , a conspicuous para sitemia starting 1 or 2 weeks after parasite entry . in some cases , specific symptoms such as lymphadenopathy and splenomegaly , myalgia , malaise , muscle pains , sweating , hepatosplenomegaly or heart failure from myocarditis or pericardial effusion the chronic phase , in principle , can last for the patient s entire lifetime , beginning with the decline of parasitemia . the main chronic forms are indeterminate , cardiac ( chronic chagasic cardiomyopathy , or ccc ) and digestive . at lower frequencies , the indeterminate form is characterized by the absence of evident tissue damage and organ dysfunction and can last from several months to the patient s entire life , which is the case for approximately 70% of chronically infected people . this form presents different degrees of severity , ranging from mild symptoms to heart failure ( caused by inflammation and fibrosis ) , frequently followed by sudden death . the main clinical manifestation of ccc is cardiomegaly caused by inflammatory infiltrations , arrhythmias and thromboembolism . the digestive form consists of two syndromes : megaesophagus , leading to dysphagia and regurgitation , and megacolon , leading to severe constipation and faecal retention . in immunocompromised patients , severe compromise of the central nervous system has been also reported . in conclusion , although the majority of t. cruzi infected individuals remain asymptomatic for their entire lives , a percentage of the infected population will develop serious symptoms . despite the fact that chagas disease was first described a century ago , only two therapeutic compounds presently in use have been shown to be useful against human infections by t. cruzi : benznidazole ( bzl ) and nifurtimox ( nf ) ( fig . bzl , a nitroimidazole , was launched in the 1970s ; in most latin american countries , it is the only drug used to treat chagas disease . this treatment is effective for acute phase infections , congenital infections , reactivated infections and early chronic disease . however , its efficacy during the chronic phase is controversial . the key mode of action of bzl seems to be based on interference with the synthesis of macromolecules via covalent binding between nitroreduction intermediates and various cellular components such as dna , lipids and proteins of the parasite . bzl has also been shown to improve phagocytosis , increase trypanosomal death through interferon ( ifn)- production and inhibit t. cruzi nadh - fumarate reductase . the mechanism of action of nf involves the generation of nitroanion radicals by nitroreductases that , in the presence of oxygen , produce reactive intermediates to which t. cruzi is susceptible . a detailed discussion of new drugs with chemotherapeutic perspectives is outside the scope of this review . however , some t. cruzi specific pathways contain proteins / enzymes that are being validated as targets ; several drugs that interfere with these targets are particularly promising . the cysteine proteinases of t. cruzi , which participate in cellular processes such as energy metabolism , differentiation , host cell invasion and evasion of the immune system , have been validated as drug targets . in particular , the use of synthetic inhibitors such as vinyl sulfone - derivatized dipeptides has shown promising results in vivo[9 , 10 ] . in addition , sterol and polyisoprenoid biosynthesis pathways provide promising targets , because ergosterol ( rather than cholesterol ) is the main sterol in t. cruzi membranes . specific inhibitors such as azole derivatives and bisphosphonates [ 13 , 14 ] have been tested in vitro or in vivo . inhibitors of t. cruzi specific enzymes such as trypanothione reductase , arginine kinase and proline racemase have shown promising trypanocidal activities . allopurinol , an inhibitor of purine ( hypoxanthine / guanine)-phosphoribosyl - transferase , has been proposed for treating t. cruzi reactivation infection in patients after heart transplantation . inhibitors of topoisomerases i and ii [ 20 , 21 ] , which are involved in nuclear and kinetoplastid dna replication , were efficient in blocking t. cruzi growth . parasite dna has also been proposed as a target for intercalators and binders with trypanocidal activity [ 2224 ] . t. cruzi s success in maintaining its life cycle is dependent on its capacity to cause chronic infection in the host , thus favouring encounters with insect vectors . to maintain latent infection different cell types and molecules are involved in the response against experimental t. cruzi infection , as summarized in table 1 . generally , the absence of some component(s ) of the immune response leads to greater susceptibility to t. cruzi infection , resulting in higher parasitemia and mortality rates . antigen - presenting cells are among the first cells that become infected by the trypomastigotes when they enter the mammalian host . normally , the cells react by up - regulating il-6 , il-1 , tnf- , il-12 and nitric oxide in an attempt to control the infection . nk cells are among the first line of responders and usually produce high levels of ifn- when stimulated by il-12 . the stimulus of the th1 profile and the cd8 t cells contributes to eliminate the intracellular amastigotes in infected tissues . on the other hand , the parasite antigens stimulate a th2 profile which contributes to the production of specific antibodies . effect of the absence of different molecules of the immune system in the experimental acute infection by trypanosoma cruzi with respect to the innate immune mechanisms triggered by the parasite , it has been found that some pathogen - associated molecular patterns derived from t. cruzi are recognized by specific receptors known as pattern recognition receptors ( prrs ) . the toll - like receptor ( tlr ) family is the best characterized class of mammalian prrs . mice that are unable to signal through most tlrs ( myd88 knockout [ ko ] or myd88/trif double ko ) are highly susceptible to t. cruzi infection , suggesting that resistance to acute t. cruzi infection is dependent on tlr signalling [ 30 , 31 ] . t. cruzi derived glycosylphosphatidylinositol ( gpi ) anchors and glycoinositolphospholipids ( gipls ) were shown to have immunoregulatory properties and were present in significant quantities on the parasitic surface . interestingly , t. cruzi infected tlr-2 ko mice are able to produce pro - inflammatory cytokines , yet parasitemia and mortality rates are not different from wild - type animals . it has also been shown that gipls are recognized via tlr-4 , and tlr-4 ko mice are more susceptible to t. cruzi infection than wild - type mice . t. cruzi dna binds tlr-9 and has been reported to stimulate macrophages and dcs to express interleukin ( il)-12 , tumour necrosis factor ( tnf)- and nitric oxide . in fact , tlr-9 ko mice are highly susceptible to t. cruzi infection , and some results suggest that tlr-2 and tlr-9 cooperate to control t. cruzi replication during acute infection . in summary , these data suggest an important role for tlr signalling pathways in the innate immune response to t. cruzi infection . to unravel the role of cellular immune responses , initial experiments have been performed in which t cells were adoptively transferred from chronically infected to nave mice . significant protection was observed after experimental challenge with t. cruzi . both cd4 and cd8 ( ) t - cell subsets appear to be important for the generation of protective immunity against acute experimental t. cruzi infection [ 26 , 27 , 35 , 36 ] . tarleton and collaborators showed that th1 cd4 t cells are important for controlling t. cruzi infection , while th2 cells contribute to parasite persistence and increased disease severity . attempts to obtain protective vaccines against defined t. cruzi antigens have also provided valuable information about the role of th1-type responses during infection . for example , cruzipain , when used as an antigen in immunization protocols , significantly improved immune responses in animals challenged with a lethal dose of the parasite and precluded tissue damage . cd8 t cells also seem to play a role in t. cruzi infection by killing infected cells through the production of perforin and granzyme b or through the fas / fas ligand pathway . acute infection with the brazil strain of t. cruzi did not enhance the susceptibility of either perforin or granzyme b ko mice . by contrast , previously reported data have shown that mice lacking perforin and granzymes a and b are more susceptible to infection with the tulahuen strain . increased susceptibility to infection with the tulahuen strain was also observed in mice deficient in the fas / fas ligand pathway . t. cruzi antigens that are cd8 t - cell targets have also been studied using chronically infected mice . these studies showed that epitopes from the trans - sialidase family of proteins are immunodominant , and the cd8 t - cell response is focused mainly on very specific epitopes [ 41 , 42 ] . when surface markers of some of these cell populations were analysed , it was found that they were central memory t cells that are maintained even during persistent t. cruzi infection . immunization protocols using plasmid dna coding for the proteins trans - sialidase and amastigote surface protein-2 have also shown that both cellular and humuoral immune responses were induced , and the presence of cd4 th1 and cd8 specific t cells was shown . furthermore , parasitemia and/or mortality reduction was seen in mice infected with the y [ 4446 ] or brazil strains of t. cruzi . the effective participation of t cells in the immune response against t. cruzi is still controversial . these cells were found to be deleterious for the host in a study in which -ko mice had lower mortality rates and fewer areas with skeletal and cardiac inflammatory lesions compared to wild - type mice . on the other hand , an increase in susceptibility was observed in t - cell depleted animals after experimental infection associated with a reduction in ifn- production . the immune response against t. cruzi is also influenced by nk , nk t and regulatory t cells ( t reg ) . the relevance of nk cells in acute t. cruzi infection was demonstrated when normal , nk - depleted mice showed a significant increase in parasitemia and mortality rates [ 50 , 51 ] . nk t cells are activated by glycolipids presented via cd1d molecules and act by limiting parasitemia . the role of regulatory t cells ( cd4 cd25 ) during t. cruzi infection was also shown through depletion . controversial results were obtained with mice infected with different t. cruzi strains : no effect was seen when brazil and tulahuen strains were used , while limited effects were seen with the colombian strain . no role was observed for these cells during chronic infection of mice with the colombian strain . the role of ifn- during t. cruzi infection was demonstrated when ifn- and ifn- receptor ko mice showed higher rates of parasitemia and mortality . infected ifn- ko mice showed increases in cellular infiltrates in the heart and skeletal muscles and reduced survival [ 36 , 56 , 57 ] . inos ko mice showed greater parasitemia in the acute phase and rapid mortality compared to control animals [ 55 , 57 , 58 ] . additionally , nitric oxide seems to have an effect on the generation of the inflammatory heart infiltrate seen during t. cruzi infection by modulating chemokine expression . cardiomyocytes from inos ko mice that were stimulated with ifn- and tnf produced significantly higher levels of the chemokines ccl2 , ccl4 , ccl5 and cxcl2 . other chemokines and chemokine receptors have also been analysed during acute or chronic t. cruzi infections . cxcl9 , cxcl10 and ccl5 are expressed in the heart during both phases of t. cruzi infection [ 60 , 61 ] . their presence correlates with the expression of ifn- and tnf- and the presence of inflammatory cells . however , their ablation did not modulate the severity of heart inflammation . during the acute phase , ccr5 seems to be critical to controlling the migration of cd4 and cd8 t cells to the heart , but it does not seem to play an important role in maintaining an inflammatory response in the heart during chronic infection . il-12 is also extremely important for controlling the infection , as il-12 ko mice show higher rates of parasitemia and mortality compared to controls [ 57 , 64 ] . the role of tnf- is controversial : tnf receptor p55 ko mice [ 25 , 65 ] showed increased parasitemia , while parasitemia and mortality rates in tnfr1 ko mice did not differ from wild - type . il-10 deficiency leads to parasitemia reduction ; however , mortality is accelerated due to a dramatic increase in acute pathology . il-4 ko mice infected with the y strain did not differ from wild - type in terms of parasitemia and mortality . however , when the colombian strain was used , il-4 ko mice showed reduced parasitemia and mortality rates . the importance of antibodies for controlling chronic infection was demonstrated when sera from chronically infected chagasic patients or mice were transferred to nave mice , significantly reducing parasitemia and prolonging survival after challenge with t. cruzi[69 , 70 ] . protective antibodies are also able to agglutinate trypomastigotes in vitro , lyse them in a complement - mediated fashion , facilitate phagocytosis / opsonization and mediate antibody - dependent cellular cytotoxicity [ 73 , 74 ] . during acute t. cruzi infection , b cells also play fundamental roles in both the recruitment of cd4 t cells and cd8 t cells to the spleen and in the generation and maintenance of central memory and effector t cells . ko mice lacking mature b cells ( mt ko ) produced decreased amounts of inflammatory cytokines and fewer central and memory cd4 and cd8 t cells compared to wild - type t. cruzi infected mice . an increase in parasitemia was observed in mumt ko mice , but no difference was seen in terms of survival . b cells are also able to participate in the cross - priming of specific cd8 t cells , inducing systemic and mucosal protective immunity against experimental infection . taken together , these results suggest the important participation of both innate and adaptive immune responses during experimental t. cruzi infection . during the chronic phase of chagas disease , the majority of individuals show potent cellular and humuoral immune responses [ 77 , 78 ] . the relevance of a strong immune response for parasite control has been shown by the fact that chemically immunosuppressed individuals and aids patients can develop symptomatic forms of the disease . several factors may be involved , such as differences in parasite strain , parasite load , infection time , host genetic background and immune response . in animal models , different parasite strains , mouse backgrounds and re - infections can play a role in the development of heart pathology and/or protection [ 8183 ] . regarding human infections , a study with patients acutely infected with t. cruzi has shown that cd4 and cd8 t cells are present in endomyocardial biopsies where myocarditis was also detected in 100% of the cases , reinforcing the role of the immune response in acute pathology . with respect to the chronic phase , a predominance of activated cd8 t cells was found in myocardial biopsy fragments from patients with ccc [ 85 , 86 ] . it is also of interest that peripheral blood mononuclear cells ( pbmcs ) from chronically infected chagasic patients were able to produce ifn- upon stimulation with recombinant t. cruzi derived proteins [ 87 , 88 ] . furthermore , pbmcs from ccc patients produced more ifn- and less il-10 [ 87 , 89 ] . il-10 expression in monocytes from patients with the indeterminate form was higher than in cardiac patients [ 89 , 90 ] . by contrast , analysis of ifn--producing cd8 t cells present in infected patients ( with undetectable , mild or more severe forms of clinical disease ) showed that there was a negative correlation between the capacity of their cells to respond to t. cruzi amastigote antigens and disease severity . a careful analysis of these cells showed that in responding individuals ( individuals with milder heart disease ) , there were early differentiated ( cd27cd28 ) and effector memory ( cd45raccr7 ) cd8 t cells . on the other hand , individuals with more severe forms of the disease presented fully differentiated ( cd27cd28 ) cd8 t cells . the authors suggest that this profile is compatible with the hypothesis that as the disease progresses , there is a gradual clonal exhaustion of the cd8 t - cell population , probably due to continuous antigenic stimulation . another study showed that patients with cardiac forms of chagas disease display a high frequency of circulating cd4 and cd8 t cells lacking the cd28 surface molecule . two cytokines ( il-7 and il-15 ) were suggested as being important for the survival of cd8 t cells in the cardiac infiltrate . on the other hand , cells from patients with chronic infections ( either symptomatic or asymptomatic ) express both inflammatory and anti - inflammatory cytokines [ 93 , 94 ] , suggesting that there is probably immune regulation during the chronic phase . however , preferential expression of tnf- and ifn- ( both inflammatory cytokines ) was observed in cardiac lesions [ 33 , 86 ] and has been associated with progressively severe cardiac disease [ 89 , 95 ] . few reports have focused on the identification the cd8 t - cell targets in chronic chagasic patients [ 41 , 91 , 9698 ] , probably due to the fact that these responses are not very strong . cd8 t - cell responses against t. cruzi peptides derived from the proteins cruzipain , fl-160 , kmp-11 and the trans - sialidase family were detected in infected individuals [ 41 , 97 , 98 ] . peptides derived from the trans - sialidase protein family were able to bind to six of the most common class i hla supertypes , and the stimulated cells showed a lack of polyfunctional cytokine responses , producing only ifn-. evidence has also led to the proposal of an autoimmune hypothesis for the disease , suggesting that the symptoms presented by infected individuals are a consequence of the triggered immune response rather than parasite persistence . however , autoimmunity is not sufficient to explain the multifocal nature of myocarditis and the preferential location of fibrosis in certain regions , such as the apical or posterior left ventricular wall in the cardiac form of chagas disease . moreover , as mentioned above , frequent positive xenodiagnosis during the chronic phase of the disease and episodes of reactivation in immunocompromised patients has provided evidence that the parasite is present even under active control of the host immunological system . in fact , recent studies have described a positive correlation between myocardial parasite persistence and high - grade myocarditis [ 101 , 102 ] . these studies reinforce the notion that a combination of immune response and parasite persistence determines the development of chagas disease pathology . however , the specific mechanisms that trigger the symptoms seen during the chronic phase are still elusive . as previously mentioned , the resistance developed during experimental chagas disease depends on innate immune responses as well as on a prevalent th1 response at the beginning of infection . in addition , the antibodies produced by a th2 response can contribute to the control of in vivo parasite replication ( schematized in fig . several works have shown that chemotherapy acts by unbalancing the equilibrium between the host immune response and the parasite in favour of the host [ 66 , 103 ] . however , it is important to stress that the results obtained with both drugs used for treatment , bzl and nf , differ strongly according to the disease phase , the time extension and the drug dose , as well as the patient s age and geographical origin . in vitro phagocytosis and intracellular destruction of trypomastigotes by mouse peritoneal macrophages collected from animals treated with nf and bzl the effects of in vivo treatment with bzl on parasite - macrophage interaction have been studied using the y strain of t. cruzi . drug - resistant and drug - susceptible parasites from this strain were used , and it was observed that bzl enhanced phagocytosis , parasite destruction and cytokine release by macrophages when a drug - sensitive strain was used . bzl treatment was also evaluated in ifn- , il-12 , tnf receptor and inos ko mice . although bzl treatment was able to cure 100% of wild - type mice , it was not as effective when various ko mice were used . the induction of a stable parasite - specific cd8 t - cell population with the characteristics of central memory was observed in chronically infected mice treated with bzl . these cells also expand more rapidly and provide greater protection after challenge compared to cells from non - treated chronically infected mice . it was also observed that splenomegaly persisted in spite of amelioration of clinical and parasitological signs in infected and bzl - treated mice . this was due to a preferential expansion of effector and memory cd8 t cells but not of recently activated cd4 and cd8 t cells , suggesting that bzl directly affects immunoregulation in t. cruzi infected mice . the effect of bzl treatment was also studied in two ccc mouse models , showing contradictory results . one model showed that treatment was able to reduce the severity of cardiac autoimmune disease , while in the other , early treatment with bzl did not alter the intensity of ccc . pbmcs from cured patients treated with bzl during the acute phase showed stronger proliferative responses and ifn- production compared to non - cured patients . it was also demonstrated that most individuals with an indeterminate clinical form show a dominant regulatory cytokine profile , whereas individuals with ccc display a dominant inflammatory cytokine pattern . interestingly , an inversion of the cytokine profile ( from an inflammatory to a regulatory profile or vice versa ) was found after in vivo treatment of indeterminate and cardiac individuals with bzl . cytokine expression in t. cruzi infected children treated with bzl shifted toward a type 1 modulated immune profile , and ifn- was mainly produced by nk cells and cd8 t lymphocytes . although a pro - inflammatory immune response is commonly related to chagas disease pathogenesis , it is also important for treatment effectiveness . a longitudinal study performed to evaluate immunological status following bzl treatment during early indeterminate chagas disease demonstrated that bzl treatment induced substantial t- and b - cell activation . as discussed above , several lines of evidence show that bzl , in addition to having trypanocidal activity , acts as an immunomodulator . more specifically , the fact that bzl is known to affect mammalian host cells raised the hypothesis that it might be affecting macrophage metabolism . revelli and collaborators showed that bzl down - regulates nitric oxide and pro - inflammatory cytokine synthesis ( il-6 , il-1 ) by lipopolysaccharide ( lps)-stimulated raw 264.7 murine macrophages and leads to an inhibition of inos gene expression through the inhibition of nf-b activation . moreover , in a rat model of acute t. cruzi infection , systemic treatment with bzl led to a marked reduction of nitric oxide derived metabolites , suggesting that the beneficial properties of bzl may depend on both trypanocidal action and immunomodulating effects . these properties of bzl have been further demonstrated by its ability to increase survival and decrease serum levels of il-6 and tnf- in c57bl/6 mice challenged systemically with lps . in an infectious - based situation of systemic inflammatory response ( cecal ligation and puncture , clp ) , mice treated with bzl had an increased survival rate and a significant reduction of tnf- levels and bacteremia 24 hrs after clp . such beneficial properties may broaden the potential use of bzl in hyperacute cases where inflammatory responses become harmful . there are limited data concerning the effects of nf on immune responses , mainly because treatment with this drug was discontinued in the 1980s . at the experimental level , no gross changes in cell - mediated immunity were recorded in nf - treated mice . however , an impaired ppd skin reaction was seen in guinea pigs when this compound was administered . from a clinical standpoint , chronically infected human beings who were treated for two months with nf had a detectable peripheral leukocyte migration inhibition test to t. cruzi antigens . animals that were parasitologically sterilized by nf treatment and re - infected with a small number of parasites showed parasitemia and mortality rates similar to control animals ( infected , non - treated ) . however , when re - infection was performed with a large number of parasites , parasitemia and mortality were increased compared to controls . immunological studies have shown that nf treatment reduces the levels of antibodies engaged in parasite destruction , reducing either complement - dependent lysis or antibody - dependent cytotoxicity . no difference was observed when treated and non - treated mice were compared in terms of t - cell mediated immunity . therefore , it seems that nf treatment leads to a loss of resistance to re - infection with a high number of parasites . during the last 40 years , therapy for chagas disease has been based solely on two drugs : nf and bzl . the search for new trypanocidal drugs as well as other strategies to improve immune responses during infection ( i.e. therapeutic vaccines ) are currently under way [ 123 , 124 ] . treatment of the symptomatic complications of chagas disease , mainly related to heart failure , is also effective in improving quality of life . heart transplantation and cellular therapy with stem cells are therapeutic options for patients with advanced ccc , but both require optimization of the etiological treatment . as more data become available regarding the relevance of parasite persistence in the development of chronic infection , the search for new treatments becomes extremely critical . future challenges for developing new drugs for chagas disease reside in finding compounds that show both trypanocidal and immunomodulatory activities . despite the vast literature regarding the anti - t . cruzi activity of myriad compounds , only a small fraction of studies explore the characteristics mentioned above . cruzi activity could be evaluated as co - adjuvant drugs , interacting synergistically with nf or bzl . this strategy would increase the spectrum of available combinations and possibly contribute to reducing the dose and thus avoiding the toxic effects of the drugs currently in use . in summary , the drugs that are available to treat diseases caused by trypanosomatids are somewhat effective . however , for the reasons discussed above , new drugs that are able to act alone or in concert with current treatments as well as with strategies such as immunotherapy and vaccines are urgently needed . these are relevant goals because they could aid in reducing undesired secondary effects , thereby optimizing the quality of life of patients and diminishing treatment evasion . in this way , funding of all steps related to the development of new therapies , from validation of new targets to evaluation of new drugs ( from the bench top to clinical trials ) , is a necessity .

abstractthe final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order . among the pathogenic trypanosomatids , some species are of particular interest due to their medical importance . these species include the agent responsible for chagas disease , trypanosoma cruzi . approximately 8 to 10 million people are infected in the americas , and approximately 40 million are at risk . in the present review , we discuss in detail the immune mechanisms elicited during infection by t. cruzi and the effects of chemotherapy in controlling parasite proliferation and on the host immune system .

Introduction Chagas disease Chemotherapy Immune response in experimental Immune response in human beings infected with Immune response in the treatment of chagasic infection The need for new therapeutic alternatives for Chagas disease Conclusions

diseases caused by trypanosomatids constitute a substantial health and socioeconomic problem in several countries , mainly in the americas , sub - saharan africa and tropical and subtropical belt regions . in particular , chagas disease , ( caused by trypanosoma cruzi ) affects 8 to 10 million people in the americas , with an additional 40 million people at risk ( http://www.who.int/tdr ) . t. cruzi has a complex life cycle involving a reduviid insect vector and a mammalian host ( fig . insect vectors become infected when they bite an infected mammal that has trypomastigote forms of the parasite circulating in its bloodstream . in the terminal portion of the digestive tube , epimastigotes differentiate into metacyclic trypomastigotes , which are eliminated in faeces and deposited on mammals skin while the triatomine bug bites and feeds . schematic representation of the trypanosoma cruzi life cycle . replicative , non - infective epimastigote forms ( a ) , predominantly present in the insect vector , give rise to non - replicative , infective metacyclic trypomastigotes ( b ) . metacyclic forms must invade the host cells and differentiate into replicative amastigote forms ( c ) to establish the infection . chagas disease presents mainly as two clinical phases in human beings : acute and chronic . it is characterized by an absence of antibodies and , in most patients , a conspicuous para sitemia starting 1 or 2 weeks after parasite entry . at lower frequencies , the indeterminate form is characterized by the absence of evident tissue damage and organ dysfunction and can last from several months to the patient s entire life , which is the case for approximately 70% of chronically infected people . this form presents different degrees of severity , ranging from mild symptoms to heart failure ( caused by inflammation and fibrosis ) , frequently followed by sudden death . the main clinical manifestation of ccc is cardiomegaly caused by inflammatory infiltrations , arrhythmias and thromboembolism . the digestive form consists of two syndromes : megaesophagus , leading to dysphagia and regurgitation , and megacolon , leading to severe constipation and faecal retention . in immunocompromised patients , severe compromise of the central nervous system has been also reported . in conclusion , although the majority of t. cruzi infected individuals remain asymptomatic for their entire lives , a percentage of the infected population will develop serious symptoms . despite the fact that chagas disease was first described a century ago , only two therapeutic compounds presently in use have been shown to be useful against human infections by t. cruzi : benznidazole ( bzl ) and nifurtimox ( nf ) ( fig . bzl , a nitroimidazole , was launched in the 1970s ; in most latin american countries , it is the only drug used to treat chagas disease . the key mode of action of bzl seems to be based on interference with the synthesis of macromolecules via covalent binding between nitroreduction intermediates and various cellular components such as dna , lipids and proteins of the parasite . bzl has also been shown to improve phagocytosis , increase trypanosomal death through interferon ( ifn)- production and inhibit t. cruzi nadh - fumarate reductase . the mechanism of action of nf involves the generation of nitroanion radicals by nitroreductases that , in the presence of oxygen , produce reactive intermediates to which t. cruzi is susceptible . however , some t. cruzi specific pathways contain proteins / enzymes that are being validated as targets ; several drugs that interfere with these targets are particularly promising . the cysteine proteinases of t. cruzi , which participate in cellular processes such as energy metabolism , differentiation , host cell invasion and evasion of the immune system , have been validated as drug targets . in addition , sterol and polyisoprenoid biosynthesis pathways provide promising targets , because ergosterol ( rather than cholesterol ) is the main sterol in t. cruzi membranes . allopurinol , an inhibitor of purine ( hypoxanthine / guanine)-phosphoribosyl - transferase , has been proposed for treating t. cruzi reactivation infection in patients after heart transplantation . inhibitors of topoisomerases i and ii [ 20 , 21 ] , which are involved in nuclear and kinetoplastid dna replication , were efficient in blocking t. cruzi growth . t. cruzi s success in maintaining its life cycle is dependent on its capacity to cause chronic infection in the host , thus favouring encounters with insect vectors . to maintain latent infection different cell types and molecules are involved in the response against experimental t. cruzi infection , as summarized in table 1 . generally , the absence of some component(s ) of the immune response leads to greater susceptibility to t. cruzi infection , resulting in higher parasitemia and mortality rates . antigen - presenting cells are among the first cells that become infected by the trypomastigotes when they enter the mammalian host . nk cells are among the first line of responders and usually produce high levels of ifn- when stimulated by il-12 . the stimulus of the th1 profile and the cd8 t cells contributes to eliminate the intracellular amastigotes in infected tissues . on the other hand , the parasite antigens stimulate a th2 profile which contributes to the production of specific antibodies . effect of the absence of different molecules of the immune system in the experimental acute infection by trypanosoma cruzi with respect to the innate immune mechanisms triggered by the parasite , it has been found that some pathogen - associated molecular patterns derived from t. cruzi are recognized by specific receptors known as pattern recognition receptors ( prrs ) . mice that are unable to signal through most tlrs ( myd88 knockout [ ko ] or myd88/trif double ko ) are highly susceptible to t. cruzi infection , suggesting that resistance to acute t. cruzi infection is dependent on tlr signalling [ 30 , 31 ] . t. cruzi derived glycosylphosphatidylinositol ( gpi ) anchors and glycoinositolphospholipids ( gipls ) were shown to have immunoregulatory properties and were present in significant quantities on the parasitic surface . interestingly , t. cruzi infected tlr-2 ko mice are able to produce pro - inflammatory cytokines , yet parasitemia and mortality rates are not different from wild - type animals . it has also been shown that gipls are recognized via tlr-4 , and tlr-4 ko mice are more susceptible to t. cruzi infection than wild - type mice . t. cruzi dna binds tlr-9 and has been reported to stimulate macrophages and dcs to express interleukin ( il)-12 , tumour necrosis factor ( tnf)- and nitric oxide . in fact , tlr-9 ko mice are highly susceptible to t. cruzi infection , and some results suggest that tlr-2 and tlr-9 cooperate to control t. cruzi replication during acute infection . in summary , these data suggest an important role for tlr signalling pathways in the innate immune response to t. cruzi infection . significant protection was observed after experimental challenge with t. cruzi . both cd4 and cd8 ( ) t - cell subsets appear to be important for the generation of protective immunity against acute experimental t. cruzi infection [ 26 , 27 , 35 , 36 ] . tarleton and collaborators showed that th1 cd4 t cells are important for controlling t. cruzi infection , while th2 cells contribute to parasite persistence and increased disease severity . attempts to obtain protective vaccines against defined t. cruzi antigens have also provided valuable information about the role of th1-type responses during infection . for example , cruzipain , when used as an antigen in immunization protocols , significantly improved immune responses in animals challenged with a lethal dose of the parasite and precluded tissue damage . cd8 t cells also seem to play a role in t. cruzi infection by killing infected cells through the production of perforin and granzyme b or through the fas / fas ligand pathway . t. cruzi antigens that are cd8 t - cell targets have also been studied using chronically infected mice . these studies showed that epitopes from the trans - sialidase family of proteins are immunodominant , and the cd8 t - cell response is focused mainly on very specific epitopes [ 41 , 42 ] . when surface markers of some of these cell populations were analysed , it was found that they were central memory t cells that are maintained even during persistent t. cruzi infection . immunization protocols using plasmid dna coding for the proteins trans - sialidase and amastigote surface protein-2 have also shown that both cellular and humuoral immune responses were induced , and the presence of cd4 th1 and cd8 specific t cells was shown . furthermore , parasitemia and/or mortality reduction was seen in mice infected with the y [ 4446 ] or brazil strains of t. cruzi . the effective participation of t cells in the immune response against t. cruzi is still controversial . these cells were found to be deleterious for the host in a study in which -ko mice had lower mortality rates and fewer areas with skeletal and cardiac inflammatory lesions compared to wild - type mice . on the other hand , an increase in susceptibility was observed in t - cell depleted animals after experimental infection associated with a reduction in ifn- production . the immune response against t. cruzi is also influenced by nk , nk t and regulatory t cells ( t reg ) . the role of regulatory t cells ( cd4 cd25 ) during t. cruzi infection was also shown through depletion . controversial results were obtained with mice infected with different t. cruzi strains : no effect was seen when brazil and tulahuen strains were used , while limited effects were seen with the colombian strain . the role of ifn- during t. cruzi infection was demonstrated when ifn- and ifn- receptor ko mice showed higher rates of parasitemia and mortality . infected ifn- ko mice showed increases in cellular infiltrates in the heart and skeletal muscles and reduced survival [ 36 , 56 , 57 ] . inos ko mice showed greater parasitemia in the acute phase and rapid mortality compared to control animals [ 55 , 57 , 58 ] . additionally , nitric oxide seems to have an effect on the generation of the inflammatory heart infiltrate seen during t. cruzi infection by modulating chemokine expression . other chemokines and chemokine receptors have also been analysed during acute or chronic t. cruzi infections . cxcl9 , cxcl10 and ccl5 are expressed in the heart during both phases of t. cruzi infection [ 60 , 61 ] . their presence correlates with the expression of ifn- and tnf- and the presence of inflammatory cells . during the acute phase , ccr5 seems to be critical to controlling the migration of cd4 and cd8 t cells to the heart , but it does not seem to play an important role in maintaining an inflammatory response in the heart during chronic infection . il-10 deficiency leads to parasitemia reduction ; however , mortality is accelerated due to a dramatic increase in acute pathology . during acute t. cruzi infection , b cells also play fundamental roles in both the recruitment of cd4 t cells and cd8 t cells to the spleen and in the generation and maintenance of central memory and effector t cells . ko mice lacking mature b cells ( mt ko ) produced decreased amounts of inflammatory cytokines and fewer central and memory cd4 and cd8 t cells compared to wild - type t. cruzi infected mice . b cells are also able to participate in the cross - priming of specific cd8 t cells , inducing systemic and mucosal protective immunity against experimental infection . taken together , these results suggest the important participation of both innate and adaptive immune responses during experimental t. cruzi infection . during the chronic phase of chagas disease , the majority of individuals show potent cellular and humuoral immune responses [ 77 , 78 ] . the relevance of a strong immune response for parasite control has been shown by the fact that chemically immunosuppressed individuals and aids patients can develop symptomatic forms of the disease . regarding human infections , a study with patients acutely infected with t. cruzi has shown that cd4 and cd8 t cells are present in endomyocardial biopsies where myocarditis was also detected in 100% of the cases , reinforcing the role of the immune response in acute pathology . with respect to the chronic phase , a predominance of activated cd8 t cells was found in myocardial biopsy fragments from patients with ccc [ 85 , 86 ] . it is also of interest that peripheral blood mononuclear cells ( pbmcs ) from chronically infected chagasic patients were able to produce ifn- upon stimulation with recombinant t. cruzi derived proteins [ 87 , 88 ] . by contrast , analysis of ifn--producing cd8 t cells present in infected patients ( with undetectable , mild or more severe forms of clinical disease ) showed that there was a negative correlation between the capacity of their cells to respond to t. cruzi amastigote antigens and disease severity . on the other hand , individuals with more severe forms of the disease presented fully differentiated ( cd27cd28 ) cd8 t cells . the authors suggest that this profile is compatible with the hypothesis that as the disease progresses , there is a gradual clonal exhaustion of the cd8 t - cell population , probably due to continuous antigenic stimulation . two cytokines ( il-7 and il-15 ) were suggested as being important for the survival of cd8 t cells in the cardiac infiltrate . on the other hand , cells from patients with chronic infections ( either symptomatic or asymptomatic ) express both inflammatory and anti - inflammatory cytokines [ 93 , 94 ] , suggesting that there is probably immune regulation during the chronic phase . few reports have focused on the identification the cd8 t - cell targets in chronic chagasic patients [ 41 , 91 , 9698 ] , probably due to the fact that these responses are not very strong . cd8 t - cell responses against t. cruzi peptides derived from the proteins cruzipain , fl-160 , kmp-11 and the trans - sialidase family were detected in infected individuals [ 41 , 97 , 98 ] . peptides derived from the trans - sialidase protein family were able to bind to six of the most common class i hla supertypes , and the stimulated cells showed a lack of polyfunctional cytokine responses , producing only ifn-. evidence has also led to the proposal of an autoimmune hypothesis for the disease , suggesting that the symptoms presented by infected individuals are a consequence of the triggered immune response rather than parasite persistence . however , autoimmunity is not sufficient to explain the multifocal nature of myocarditis and the preferential location of fibrosis in certain regions , such as the apical or posterior left ventricular wall in the cardiac form of chagas disease . moreover , as mentioned above , frequent positive xenodiagnosis during the chronic phase of the disease and episodes of reactivation in immunocompromised patients has provided evidence that the parasite is present even under active control of the host immunological system . these studies reinforce the notion that a combination of immune response and parasite persistence determines the development of chagas disease pathology . as previously mentioned , the resistance developed during experimental chagas disease depends on innate immune responses as well as on a prevalent th1 response at the beginning of infection . in addition , the antibodies produced by a th2 response can contribute to the control of in vivo parasite replication ( schematized in fig . several works have shown that chemotherapy acts by unbalancing the equilibrium between the host immune response and the parasite in favour of the host [ 66 , 103 ] . however , it is important to stress that the results obtained with both drugs used for treatment , bzl and nf , differ strongly according to the disease phase , the time extension and the drug dose , as well as the patient s age and geographical origin . in vitro phagocytosis and intracellular destruction of trypomastigotes by mouse peritoneal macrophages collected from animals treated with nf and bzl the effects of in vivo treatment with bzl on parasite - macrophage interaction have been studied using the y strain of t. cruzi . drug - resistant and drug - susceptible parasites from this strain were used , and it was observed that bzl enhanced phagocytosis , parasite destruction and cytokine release by macrophages when a drug - sensitive strain was used . this was due to a preferential expansion of effector and memory cd8 t cells but not of recently activated cd4 and cd8 t cells , suggesting that bzl directly affects immunoregulation in t. cruzi infected mice . one model showed that treatment was able to reduce the severity of cardiac autoimmune disease , while in the other , early treatment with bzl did not alter the intensity of ccc . interestingly , an inversion of the cytokine profile ( from an inflammatory to a regulatory profile or vice versa ) was found after in vivo treatment of indeterminate and cardiac individuals with bzl . cytokine expression in t. cruzi infected children treated with bzl shifted toward a type 1 modulated immune profile , and ifn- was mainly produced by nk cells and cd8 t lymphocytes . although a pro - inflammatory immune response is commonly related to chagas disease pathogenesis , it is also important for treatment effectiveness . moreover , in a rat model of acute t. cruzi infection , systemic treatment with bzl led to a marked reduction of nitric oxide derived metabolites , suggesting that the beneficial properties of bzl may depend on both trypanocidal action and immunomodulating effects . there are limited data concerning the effects of nf on immune responses , mainly because treatment with this drug was discontinued in the 1980s . from a clinical standpoint , chronically infected human beings who were treated for two months with nf had a detectable peripheral leukocyte migration inhibition test to t. cruzi antigens . during the last 40 years , therapy for chagas disease has been based solely on two drugs : nf and bzl . the search for new trypanocidal drugs as well as other strategies to improve immune responses during infection ( i.e. treatment of the symptomatic complications of chagas disease , mainly related to heart failure , is also effective in improving quality of life . heart transplantation and cellular therapy with stem cells are therapeutic options for patients with advanced ccc , but both require optimization of the etiological treatment . as more data become available regarding the relevance of parasite persistence in the development of chronic infection , the search for new treatments becomes extremely critical . future challenges for developing new drugs for chagas disease reside in finding compounds that show both trypanocidal and immunomodulatory activities . this strategy would increase the spectrum of available combinations and possibly contribute to reducing the dose and thus avoiding the toxic effects of the drugs currently in use . in summary , the drugs that are available to treat diseases caused by trypanosomatids are somewhat effective .

pedigree - based linkage analyses have been quite successful in identifying the genes for hundreds of simple mendelian diseases ( like huntington 's disease ) , and for a few complex diseases ( like early - onset alzheimer 's and early - onset breast cancer ) . although a few groups have focused on a small number of large , extended pedigrees , due to the difficulty of obtaining large multiplex families , genome - wide scans using dense maps of polymorphic markers in small pedigrees have become the standard strategy for finding bipolar genes through linkage . to circumvent , problems inherent , in complex diseases , nonparametric methods have recently been utilized , where mode of inheritance , allele frequency , or penetrance parameters ( currently unknown for bipolar disorder ) are not needed to assess linkage between phenotype and genotype . in what may be a preview of things to come , investigators from several countries recently pooled their genotypic information from 11 different genome - wide linkage scans , ( with a total sample of 5179 individuals from 1067 families ) , and found successful , genome - wide significant evidence of linkage to chromosomes 6q and 8q . table 1 summarizes key findings from a number of linkage analyses performed over the last 20 years , indicating the chromosomal regions , phenotypes focused on , and the lod scores for each region . as we increase our sample sizes ( mainly through collaborative efforts from multiple sites ) , improve the phenotypic definition of bipolar disorder ( possibly through endophenotype discoveries ) and discover improved meta - analysis tools , it is hoped that linkage analyses will assist , the field in better understanding where the most critical loci for bipolar disorder ( predisposition genes of moderate effect ) arc located in the human genome . in the current , excitement over genome - wide association ( gwa ) analyses ( see section below ) , it , would certainly be unwise to overlook the benefits of family - based linkage analyses to contribute to the identification of genes for complex diseases . until recently , it , was not practical to consider gwa studies to try to detect genes for bipolar disorder . to screen the whole genome and detect , genes that are associated with bipolar disorder requires that the gene variant responsible for the phenotype ( ie , bipolar disorder ) is in tight , linkage disequilibrium with the variant , ( typically either a microsatellite or a single nucleotide polymorphism , snp ) being studied . linkage disequilibrium is a technical term that indicates that , two genetic loci are so close that , specific alleles for the loci segregate together more often than would be expected by chance . at , the genome level , areas of linkage disequilibrium , at least in outbred populations , are very small , thus requiring that hundreds of thousands of snps be genotyped per person . although such studies are now becoming possible ( see the genome - wide association studies section below ) , many investigators have focused on particular genes , to determine whether they might , be associated with bipolar disorder . this candidate gene approach usually requires an a priori hypothesis that a. gene , due to its location near a linkage peak and/or because of the function of its gene product , might play a role in bipolar disorder . systematic analyses of genes in peak regions found from linkage studies have been rare ( ie , where all genes under the linkage peak are carefully screened ) . however , analyses of genes in these peak regions ( positional candidates ) have led to positive associations for a number of genes on chromosomes including 5 , 12 , 13 , 18 , and 22 . a large number of genes have been studied because of a hypothesized role based on neurophysiology , including genes that play a role in circadian rhythms , the dopaminergic pathway ( drd1 , drd4 , dat1 ) , the serotinergic pathway ( httlpr , htr2a ) , neural development and neurotrophism ( bdnf , ncam 1 ) . in addition , as genes have been discovered for schizophrenia , investigators have also analyzed whether these genes might , be associated with bipolar disorder , with several studies now suggesting that variations in the neuregulin 1 gene and the g72/30 gene are associated with bipolar disorder or manic psychosis . replication of genetic association studies has been difficult , in part because the sample sizes necessary to detect , genes are of small effect size . other difficulties in candidate gene studies are similar to problems faced in all association studies : poorly matched cases and controls can lead to false - positive or -negative results , definitions of bipolar disorder vary across studies , and genes may have different , effects based on background population issues ( genetic background and environmental background ) . of all the specific candidate genes shown in one study or another to be associated with bipolar disorder , at this point , none of these findings have been robust enough or tested in large enough samples to definitively implicate them in the genesis of bipolar disorder . recently , with the advent of genetic chips that can analyze over 500 000 snps , and the knowledge - base provided by analysis of the human genome , it , has become possible to construct gw studies in outbred populations . in this approach , a case - control or trio approach ( affected subjects , plus their parents ) is utilized , typically requiring thousands of subjects , and 500 000 or more snps arc analyzed in order to determine specific genes or regions associated with a disorder . the approach has recently provided promising results in studies of type ii diabetes , cancer , and other medical conditions which can be classified as common and complex diseases , and this has led to efforts in the united states , the united kingdom , and elsewhere , to pursue gwa studies on a large scale . the potential advantage of whole - genome association studies is that such studies may be able to pick out . have major effect , on a. disease , and ( if the sample size is big enough ) potentially overcome complications when disorders are multigcnic . on the other hand , sample sizes needed for analyses may be difficult , to reach without major investments , the cost , of the technology is not . trivial , rare alleles with major effects may be overlooked , stratification issues and multiple testing issues become even more critical than in linkage studies , selection of individual cases may dilute the study of genetic forms of bipolar disorder , and replication will remain a difficult issue , leading some to temper the expectations we might expect from gwa analyses . gwa studies in bipolar disorder were initially pursued in the costa rican population , with microsatellitcs placed relatively sparsely across the genome . although these studies yielded potentially interesting linkage disequilibrium between bipolar disorder and specific chromosomal regions , the sparseness of the map did not . gwa studies of bipolar disorder , using dense snp maps , have been reported thus far . baum et al used a. two - stage strategy , beginning with 461 bipolar cases and 563 controls and following up significant findings in a sample of 772 bipolar cases and 876 controls , and found evidence for novel genes potentially associated with bipolar disorder , including a gene for diacylglycerol kinase , which plays a key role in the lithium sensitive phosphatidyl inositol pathway . a study by the wellcome trust case control consortium utilized 2000 bipolar cases and 3000 controls , and reported on a number of snps showing evidence of association , some at , specific loci that had not . formerly been implicated in studies of bipolar disorder . for both of these recent gwa studies , additional genes or regions comparisons across studies , replication studies for specific genes in new samples , combined analyses and even larger case - control studies will be necessary to adequately separate the wheat from the chaff . an additional gwa study of bipolar disorder is currently under way in the united states , as part of a private - public joint venture known as the gain collaborative group . the true cost , versus benefit , of such massive ventures , compared with the potentially more modest , costs of continuing and combining linkage studies and following these up with focused fine mapping , has yet to be determined . it is known that neuropsychiatrie disorders and their phenotypes do not , follow classic mendelian genetics , but rather a complex genetic pattern where multiple genes are involved and environment also modifies the course of illness . it , is the interaction of all these aspects that lead to the phenotypic appearance of these complex disorders . these difficulties , as well as the relatively slow process in identifying genes for complex disorders , has led many investigators to begin to focus on identifying genes for endophenotypes . the term endophenotype has been defined as an internal , intermediate phenotype that may fill the gap in the causal chain between genes and distal diseases . an endophenotypc can be an inherited neurophysiological , neuropsychological , cognitive , neuroanatomical , biochemical , or endocrinological trait . the current diagnostic and classification of psychiatric disorders is not based on pathophysiology or etiology , but . is based on nosological tradition , expert , consensus , psychometric reliability and clinical utility . endophenotypes , if accurately defined , could represent more basic biological phenomena than the more complex related phenotype . theoretically , it . might , then be easier to identify genetic variants associated with an endophenotype than it . would be to identify variants associated with a. more complex phenotype . often quantitative and occur in affecteds and unaffecteds , endophenotypes also allow more persons per family to participate and contribute linkage information . quantitative linkage and association methods can also be utilized . in order for an endophenotype to be useful in the identification of genetic markers for a disorder it must , has to be associated with the illness in the population ; ( ii ) it . has to be heritable ; ( iii ) it should be primarily state -independent , ( manifests in an individual whether or not , the illness is active ) ; ( iv ) it should segregate with illness within families ; and finally ( v ) the endophenotype found in affected family members should be found in nonaffected family members at a higher rate than in the general population . another aspect which should be taken into consideration when identifying an endophenotype is the feasibility and reliability of its measurement . following are a number of preliminary studies which suggest , possible endophenotypes for bipolar disorder that derive from neuroanatomy and neuropsychology . importantly , we do not , know at this point , whether any of these meet all of the criteria necessary to be fully considered as an endophenotype for bipolar disorder . future studies need to be done , especially in terms of measuring hcritability and segregation with disease , for these and other potential endophenotypes . when looking at , biological structures of the brain , there are studies that suggest specific regions of the brain as endophenotypes for bipolar disorder . macdonald et al indicated that , a genetic risk for bipolar disorder was specifically associated with gray - matter deficits in the right anterior cingulate gyrus and ventral striatum . two studies revealed that the risk of white - matter abnormalities is more than threefold higher in patients with bipolar disorder than in healthy controls . a meta - analysis of magnetic resonance imaging ( mri ) brain measurements done in multiple studies reviewed by mcdonald et al showed right lateral ventricular volume was increased in bipolar subjects . was found to be present , early in the disorder and was more pronounced with recurring episodes of bipolar . poor performance on verbal memory tests was consistently found as a characteristic of bipolar disorder . impaired planning ( speed of information processing ) lithium is a treatment for bipolar disorder , and has been shown to modify the phase and period of circadian rhythms in a variety of species involving the glycogen synthase kinase 3 ( gsk-3 ) inhibitor . there is preliminary evidence of an association between a polymorphism in the gsk-3- promoter gene and bipolar disorder , suggesting that genetic factors involved in the regulation of the human circadian clock might represent , another endophenotype for bipolar disorder . for more thorough reviews of the role of endophenotypes for bipolar disorder , until recently , it , was not practical to consider gwa studies to try to detect genes for bipolar disorder . to screen the whole genome and detect , genes that are associated with bipolar disorder requires that the gene variant responsible for the phenotype ( ie , bipolar disorder ) is in tight , linkage disequilibrium with the variant , ( typically either a microsatellite or a single nucleotide polymorphism , snp ) being studied . linkage disequilibrium is a technical term that indicates that , two genetic loci are so close that , specific alleles for the loci segregate together more often than would be expected by chance . at , the genome level , areas of linkage disequilibrium , at least in outbred populations , are very small , thus requiring that hundreds of thousands of snps be genotyped per person . although such studies are now becoming possible ( see the genome - wide association studies section below ) , many investigators have focused on particular genes , to determine whether they might , be associated with bipolar disorder . this candidate gene approach usually requires an a priori hypothesis that a. gene , due to its location near a linkage peak and/or because of the function of its gene product , might play a role in bipolar disorder . systematic analyses of genes in peak regions found from linkage studies have been rare ( ie , where all genes under the linkage peak are carefully screened ) . however , analyses of genes in these peak regions ( positional candidates ) have led to positive associations for a number of genes on chromosomes including 5 , 12 , 13 , 18 , and 22 . a large number of genes have been studied because of a hypothesized role based on neurophysiology , including genes that play a role in circadian rhythms , the dopaminergic pathway ( drd1 , drd4 , dat1 ) , the serotinergic pathway ( httlpr , htr2a ) , neural development and neurotrophism ( bdnf , ncam 1 ) . in addition , as genes have been discovered for schizophrenia , investigators have also analyzed whether these genes might , be associated with bipolar disorder , with several studies now suggesting that variations in the neuregulin 1 gene and the g72/30 gene are associated with bipolar disorder or manic psychosis . replication of genetic association studies has been difficult , in part because the sample sizes necessary to detect , genes are of small effect size . other difficulties in candidate gene studies are similar to problems faced in all association studies : poorly matched cases and controls can lead to false - positive or -negative results , definitions of bipolar disorder vary across studies , and genes may have different , effects based on background population issues ( genetic background and environmental background ) . of all the specific candidate genes shown in one study or another to be associated with bipolar disorder , at this point , none of these findings have been robust enough or tested in large enough samples to definitively implicate them in the genesis of bipolar disorder . recently , with the advent of genetic chips that can analyze over 500 000 snps , and the knowledge - base provided by analysis of the human genome , it , has become possible to construct gw studies in outbred populations . in this approach , a case - control or trio approach ( affected subjects , plus their parents ) is utilized , typically requiring thousands of subjects , and 500 000 or more snps arc analyzed in order to determine specific genes or regions associated with a disorder . the approach has recently provided promising results in studies of type ii diabetes , cancer , and other medical conditions which can be classified as common and complex diseases , and this has led to efforts in the united states , the united kingdom , and elsewhere , to pursue gwa studies on a large scale . the potential advantage of whole - genome association studies is that such studies may be able to pick out . have major effect , on a. disease , and ( if the sample size is big enough ) potentially overcome complications when disorders are multigcnic . on the other hand , sample sizes needed for analyses may be difficult , to reach without major investments , the cost , of the technology is not . trivial , rare alleles with major effects may be overlooked , stratification issues and multiple testing issues become even more critical than in linkage studies , selection of individual cases may dilute the study of genetic forms of bipolar disorder , and replication will remain a difficult issue , leading some to temper the expectations we might expect from gwa analyses . gwa studies in bipolar disorder were initially pursued in the costa rican population , with microsatellitcs placed relatively sparsely across the genome . although these studies yielded potentially interesting linkage disequilibrium between bipolar disorder and specific chromosomal regions , the sparseness of the map did not . gwa studies of bipolar disorder , using dense snp maps , have been reported thus far . baum et al used a. two - stage strategy , beginning with 461 bipolar cases and 563 controls and following up significant findings in a sample of 772 bipolar cases and 876 controls , and found evidence for novel genes potentially associated with bipolar disorder , including a gene for diacylglycerol kinase , which plays a key role in the lithium sensitive phosphatidyl inositol pathway . a study by the wellcome trust case control consortium utilized 2000 bipolar cases and 3000 controls , and reported on a number of snps showing evidence of association , some at , specific loci that had not . formerly been implicated in studies of bipolar disorder . for both of these recent gwa studies , additional genes or regions comparisons across studies , replication studies for specific genes in new samples , combined analyses and even larger case - control studies will be necessary to adequately separate the wheat from the chaff . an additional gwa study of bipolar disorder is currently under way in the united states , as part of a private - public joint venture known as the gain collaborative group . the true cost , versus benefit , of such massive ventures , compared with the potentially more modest , costs of continuing and combining linkage studies and following these up with focused fine mapping , has yet to be determined . it is known that neuropsychiatrie disorders and their phenotypes do not , follow classic mendelian genetics , but rather a complex genetic pattern where multiple genes are involved and environment also modifies the course of illness . it , is the interaction of all these aspects that lead to the phenotypic appearance of these complex disorders . these difficulties , as well as the relatively slow process in identifying genes for complex disorders , has led many investigators to begin to focus on identifying genes for endophenotypes . the term endophenotype has been defined as an internal , intermediate phenotype that may fill the gap in the causal chain between genes and distal diseases . an endophenotypc can be an inherited neurophysiological , neuropsychological , cognitive , neuroanatomical , biochemical , or endocrinological trait . the current diagnostic and classification of psychiatric disorders is not based on pathophysiology or etiology , but . is based on nosological tradition , expert , consensus , psychometric reliability and clinical utility . endophenotypes , if accurately defined , could represent more basic biological phenomena than the more complex related phenotype . theoretically , it might , then be easier to identify genetic variants associated with an endophenotype than it . would be to identify variants associated with a. more complex phenotype . often quantitative and occur in affecteds and unaffecteds , endophenotypes also allow more persons per family to participate and contribute linkage information . quantitative linkage and association methods can also be utilized . in order for an endophenotype to be useful in the identification of genetic markers for a disorder it must , has to be associated with the illness in the population ; ( ii ) it . has to be heritable ; ( iii ) it should be primarily state -independent , ( manifests in an individual whether or not , the illness is active ) ; ( iv ) it should segregate with illness within families ; and finally ( v ) the endophenotype found in affected family members should be found in nonaffected family members at a higher rate than in the general population . another aspect which should be taken into consideration when identifying an endophenotype is the feasibility and reliability of its measurement . following are a number of preliminary studies which suggest , possible endophenotypes for bipolar disorder that derive from neuroanatomy and neuropsychology . importantly , we do not , know at this point , whether any of these meet all of the criteria necessary to be fully considered as an endophenotype for bipolar disorder . future studies need to be done , especially in terms of measuring hcritability and segregation with disease , for these and other potential endophenotypes . when looking at , biological structures of the brain , there are studies that suggest specific regions of the brain as endophenotypes for bipolar disorder . macdonald et al indicated that , a genetic risk for bipolar disorder was specifically associated with gray - matter deficits in the right anterior cingulate gyrus and ventral striatum . two studies revealed that the risk of white - matter abnormalities is more than threefold higher in patients with bipolar disorder than in healthy controls . a meta - analysis of magnetic resonance imaging ( mri ) brain measurements done in multiple studies reviewed by mcdonald et al showed right lateral ventricular volume was increased in bipolar subjects . was found to be present , early in the disorder and was more pronounced with recurring episodes of bipolar . poor performance on verbal memory tests was consistently found as a characteristic of bipolar disorder . impaired planning ( speed of information processing ) lithium is a treatment for bipolar disorder , and has been shown to modify the phase and period of circadian rhythms in a variety of species involving the glycogen synthase kinase 3 ( gsk-3 ) inhibitor . there is preliminary evidence of an association between a polymorphism in the gsk-3- promoter gene and bipolar disorder , suggesting that genetic factors involved in the regulation of the human circadian clock might represent , another endophenotype for bipolar disorder . for more thorough reviews of the role of endophenotypes for bipolar disorder , see refs 122 and 123 . when looking at , biological structures of the brain , there are studies that suggest specific regions of the brain as endophenotypes for bipolar disorder . macdonald et al indicated that , a genetic risk for bipolar disorder was specifically associated with gray - matter deficits in the right anterior cingulate gyrus and ventral striatum . two studies revealed that the risk of white - matter abnormalities is more than threefold higher in patients with bipolar disorder than in healthy controls . a meta - analysis of magnetic resonance imaging ( mri ) brain measurements done in multiple studies reviewed by mcdonald et al showed right lateral ventricular volume was increased in bipolar subjects . was found to be present , early in the disorder and was more pronounced with recurring episodes of bipolar . poor performance on verbal memory tests was consistently found as a characteristic of bipolar disorder . impaired planning ( speed of information processing ) lithium is a treatment for bipolar disorder , and has been shown to modify the phase and period of circadian rhythms in a variety of species involving the glycogen synthase kinase 3 ( gsk-3 ) inhibitor . there is preliminary evidence of an association between a polymorphism in the gsk-3- promoter gene and bipolar disorder , suggesting that genetic factors involved in the regulation of the human circadian clock might represent , another endophenotype for bipolar disorder . for more thorough reviews of the role of endophenotypes for bipolar disorder , the last two decades have been a time of vigorous activity in the field of bipolar disorder genetic studies . although success in the ultimate goals of clearly identifying which genes play a role in this disorder has been modest , this is not unusual , given the complexity of the disorder and the challenge of identifying genes for any disorder that is not . moving from the pioneering work in the 20th century to define the genetic basis of bipolar disorder , through carefully designed family and twin studies , a number of teams throughout the world have focused their energies on gathering large numbers of multiplex families , in order to carry out genome - wide linkage studies to identify bipolar gene loci . these studies have used fairly modest numbers of families , compared with the recommended number for complex diseases , and , perhaps as could be expected , the linkage scores have been modest in all studies published , ranging at best , up to lod scores in the range of 3 to 4 . although meta - analyses have been performed , few studies have combined large numbers of families to interrogate specific loci , with the largest systematically gathered samples coming primarily from the nimh genetics consortium and the uk wellcome trust . consortium . joint , analyses combining data from multiple groups are only just now beginning to occur . smaller sets of families , from special populations known as population isolates have also yielded a. number of linkage regions with modest . systematic fine mapping of these regions may yield specific genes of interest , for bipolar disorder , as was seen in similar linkage studies of schizophrenia . candidate genes studies have also yielded a number of potentially associated genes deserving of further study in combined , large samples . new technologies now make gwa studies possible , and such studies will soon add a. number of additional genes to the pool of potentially associated genes for bipolar disorder . endophenotype studies will most likely also add a number of novel genes to consider in terms of how they might , indirectly contribute to bipolar disorder of mood destabilization . technologies that allow detection of copy number variants and chromosomal variations , as well as analyses of methylation patterns ( epigenetics ) , genomic expression , and proteomic analyses will add further gene candidates which can be targeted for study at the genomic level . as each new piece of data comes in from these studies , a major challenge for the field will be to sort out and keep track of the various findings . the use of bioinformati.es to review convergent evidence from multiple types of studies will become a critical component of research planning and interpretation of results . iterative research , in which variants are discovered for a bipolar phenotype , and then those subjects who carry the variant are studied in more detail ( deep phenotyping ) may help to more clearly link gene variants to bipolar phenotypes . ultimately large collaborative studies , which clearly delineate specific phenotypes ( categorical and quantitative ) and take population genetics carefully into account , will be needed to , one by one , determine the exact correlation between gene variants and risk for the disease ( or trait ) of interest . scientists and clinicians who may have hoped that one or a few genes would eventually be identified that , would explain the majority of risk for bipolar disorder must , face the reality that there are likely to be many genes of relatively small effect , involved in bipolar disorder , and the genetic dissection of this disorder will be a subtle and complex process . genetic testing for bipolar disorder will likely ultimately require careful weighing of the presence or absence of many gene variants , when counseling is being done at the population level . as specific genes are clearly identified to play a role in bipolar disorder , it remains quite possible that , within specific families or clusters , genes of moderate effect will be discovered , but , we must face the fact that , thus far , no clear bipolar disease causing variant , has been discovered in any family studied . in the next , decade , a feasible goal might , be to clearly implicate at , least a handful of genes ( through well - powered replication studies or meta - analyses ) , from which the biochemical pathways underlying the disease can be more thoroughly studied at the level of cell biology and physiology . such approaches may yield clear pharmacologic targets which can intervene in disease processes that have their origin in genetic risk variants , at . times by acting on an enzyme or protein that is part , of the biochemical pathway rather than on the gene or gene product , itself . it . is likely that over the next , decade , the field of bipolar genetics will shift , from the current emphasis on identifying the genes which play a. role in this disease , to understanding the pathophysiology of the disease from a new perspective ( ie , by study of the pathways tied to the genes which play a role in the disease ) . along with this work , last begin to be understood to be a complex behavioral phenotype , with many components and subtypes . for a. disorder that involves some of the fundamental behaviors and experiences of relevance to the human race , including regulation of activity levels , the ability to feel euphoria and dysphoria , to control social impulses , to create , to have racing thoughts , and to over- or undervalue one 's capacities , it is perhaps not surprising that the molecular underpinnings of the bipolar condition will prove to be complex and subtle , and span a. multiplicity of gene and protein networks . indeed , the gene variants which contribute to bipolar disorder may have evolved because of their specific value in helping individuals or groups adapt to socially and physically challenging and changing environments . to understand the genetics of bipolar disorder may in the end , not be any less of a. task than to understand the genetics of human psychology and behavior .

bipolar disorder , especially the most severe type ( type i ) , has a strong genetic component . family studies suggest that a small number of genes of modest effect are involved in this disorder . family - based studies have identified a number of chromosomal regions linked to bipolar disorder , and progress is currently being made in identifying positional candidate genes within those regions , number of candidate genes have also shown evidence of association with bipolar disorder , and genome - wide association studies are now under way , using dense genetic maps . replication studies in larger or combined datasets are needed to definitively assign a role for specific genes in this disorder . this review covers our current knowledge of the genetics of bipolar disorder , and provides a commentary on current approaches used to identify the genes involved in this complex behavioral disorder .

Linkage studies Association studies Candidate genes Genome-wide association studies Endophenotypes Potential neuroanatomical endophenotypes Potential neuropsychological endophenotypes Summary and future directions in genetic studies of bipolar disorder

pedigree - based linkage analyses have been quite successful in identifying the genes for hundreds of simple mendelian diseases ( like huntington 's disease ) , and for a few complex diseases ( like early - onset alzheimer 's and early - onset breast cancer ) . although a few groups have focused on a small number of large , extended pedigrees , due to the difficulty of obtaining large multiplex families , genome - wide scans using dense maps of polymorphic markers in small pedigrees have become the standard strategy for finding bipolar genes through linkage . to circumvent , problems inherent , in complex diseases , nonparametric methods have recently been utilized , where mode of inheritance , allele frequency , or penetrance parameters ( currently unknown for bipolar disorder ) are not needed to assess linkage between phenotype and genotype . in what may be a preview of things to come , investigators from several countries recently pooled their genotypic information from 11 different genome - wide linkage scans , ( with a total sample of 5179 individuals from 1067 families ) , and found successful , genome - wide significant evidence of linkage to chromosomes 6q and 8q . table 1 summarizes key findings from a number of linkage analyses performed over the last 20 years , indicating the chromosomal regions , phenotypes focused on , and the lod scores for each region . as we increase our sample sizes ( mainly through collaborative efforts from multiple sites ) , improve the phenotypic definition of bipolar disorder ( possibly through endophenotype discoveries ) and discover improved meta - analysis tools , it is hoped that linkage analyses will assist , the field in better understanding where the most critical loci for bipolar disorder ( predisposition genes of moderate effect ) arc located in the human genome . in the current , excitement over genome - wide association ( gwa ) analyses ( see section below ) , it , would certainly be unwise to overlook the benefits of family - based linkage analyses to contribute to the identification of genes for complex diseases . to screen the whole genome and detect , genes that are associated with bipolar disorder requires that the gene variant responsible for the phenotype ( ie , bipolar disorder ) is in tight , linkage disequilibrium with the variant , ( typically either a microsatellite or a single nucleotide polymorphism , snp ) being studied . although such studies are now becoming possible ( see the genome - wide association studies section below ) , many investigators have focused on particular genes , to determine whether they might , be associated with bipolar disorder . this candidate gene approach usually requires an a priori hypothesis that a. gene , due to its location near a linkage peak and/or because of the function of its gene product , might play a role in bipolar disorder . systematic analyses of genes in peak regions found from linkage studies have been rare ( ie , where all genes under the linkage peak are carefully screened ) . however , analyses of genes in these peak regions ( positional candidates ) have led to positive associations for a number of genes on chromosomes including 5 , 12 , 13 , 18 , and 22 . a large number of genes have been studied because of a hypothesized role based on neurophysiology , including genes that play a role in circadian rhythms , the dopaminergic pathway ( drd1 , drd4 , dat1 ) , the serotinergic pathway ( httlpr , htr2a ) , neural development and neurotrophism ( bdnf , ncam 1 ) . in addition , as genes have been discovered for schizophrenia , investigators have also analyzed whether these genes might , be associated with bipolar disorder , with several studies now suggesting that variations in the neuregulin 1 gene and the g72/30 gene are associated with bipolar disorder or manic psychosis . other difficulties in candidate gene studies are similar to problems faced in all association studies : poorly matched cases and controls can lead to false - positive or -negative results , definitions of bipolar disorder vary across studies , and genes may have different , effects based on background population issues ( genetic background and environmental background ) . of all the specific candidate genes shown in one study or another to be associated with bipolar disorder , at this point , none of these findings have been robust enough or tested in large enough samples to definitively implicate them in the genesis of bipolar disorder . recently , with the advent of genetic chips that can analyze over 500 000 snps , and the knowledge - base provided by analysis of the human genome , it , has become possible to construct gw studies in outbred populations . in this approach , a case - control or trio approach ( affected subjects , plus their parents ) is utilized , typically requiring thousands of subjects , and 500 000 or more snps arc analyzed in order to determine specific genes or regions associated with a disorder . the approach has recently provided promising results in studies of type ii diabetes , cancer , and other medical conditions which can be classified as common and complex diseases , and this has led to efforts in the united states , the united kingdom , and elsewhere , to pursue gwa studies on a large scale . trivial , rare alleles with major effects may be overlooked , stratification issues and multiple testing issues become even more critical than in linkage studies , selection of individual cases may dilute the study of genetic forms of bipolar disorder , and replication will remain a difficult issue , leading some to temper the expectations we might expect from gwa analyses . gwa studies in bipolar disorder were initially pursued in the costa rican population , with microsatellitcs placed relatively sparsely across the genome . although these studies yielded potentially interesting linkage disequilibrium between bipolar disorder and specific chromosomal regions , the sparseness of the map did not . gwa studies of bipolar disorder , using dense snp maps , have been reported thus far . baum et al used a. two - stage strategy , beginning with 461 bipolar cases and 563 controls and following up significant findings in a sample of 772 bipolar cases and 876 controls , and found evidence for novel genes potentially associated with bipolar disorder , including a gene for diacylglycerol kinase , which plays a key role in the lithium sensitive phosphatidyl inositol pathway . a study by the wellcome trust case control consortium utilized 2000 bipolar cases and 3000 controls , and reported on a number of snps showing evidence of association , some at , specific loci that had not . formerly been implicated in studies of bipolar disorder . for both of these recent gwa studies , additional genes or regions comparisons across studies , replication studies for specific genes in new samples , combined analyses and even larger case - control studies will be necessary to adequately separate the wheat from the chaff . an additional gwa study of bipolar disorder is currently under way in the united states , as part of a private - public joint venture known as the gain collaborative group . these difficulties , as well as the relatively slow process in identifying genes for complex disorders , has led many investigators to begin to focus on identifying genes for endophenotypes . following are a number of preliminary studies which suggest , possible endophenotypes for bipolar disorder that derive from neuroanatomy and neuropsychology . importantly , we do not , know at this point , whether any of these meet all of the criteria necessary to be fully considered as an endophenotype for bipolar disorder . when looking at , biological structures of the brain , there are studies that suggest specific regions of the brain as endophenotypes for bipolar disorder . macdonald et al indicated that , a genetic risk for bipolar disorder was specifically associated with gray - matter deficits in the right anterior cingulate gyrus and ventral striatum . two studies revealed that the risk of white - matter abnormalities is more than threefold higher in patients with bipolar disorder than in healthy controls . poor performance on verbal memory tests was consistently found as a characteristic of bipolar disorder . impaired planning ( speed of information processing ) lithium is a treatment for bipolar disorder , and has been shown to modify the phase and period of circadian rhythms in a variety of species involving the glycogen synthase kinase 3 ( gsk-3 ) inhibitor . there is preliminary evidence of an association between a polymorphism in the gsk-3- promoter gene and bipolar disorder , suggesting that genetic factors involved in the regulation of the human circadian clock might represent , another endophenotype for bipolar disorder . for more thorough reviews of the role of endophenotypes for bipolar disorder , until recently , it , was not practical to consider gwa studies to try to detect genes for bipolar disorder . to screen the whole genome and detect , genes that are associated with bipolar disorder requires that the gene variant responsible for the phenotype ( ie , bipolar disorder ) is in tight , linkage disequilibrium with the variant , ( typically either a microsatellite or a single nucleotide polymorphism , snp ) being studied . although such studies are now becoming possible ( see the genome - wide association studies section below ) , many investigators have focused on particular genes , to determine whether they might , be associated with bipolar disorder . this candidate gene approach usually requires an a priori hypothesis that a. gene , due to its location near a linkage peak and/or because of the function of its gene product , might play a role in bipolar disorder . systematic analyses of genes in peak regions found from linkage studies have been rare ( ie , where all genes under the linkage peak are carefully screened ) . however , analyses of genes in these peak regions ( positional candidates ) have led to positive associations for a number of genes on chromosomes including 5 , 12 , 13 , 18 , and 22 . a large number of genes have been studied because of a hypothesized role based on neurophysiology , including genes that play a role in circadian rhythms , the dopaminergic pathway ( drd1 , drd4 , dat1 ) , the serotinergic pathway ( httlpr , htr2a ) , neural development and neurotrophism ( bdnf , ncam 1 ) . in addition , as genes have been discovered for schizophrenia , investigators have also analyzed whether these genes might , be associated with bipolar disorder , with several studies now suggesting that variations in the neuregulin 1 gene and the g72/30 gene are associated with bipolar disorder or manic psychosis . other difficulties in candidate gene studies are similar to problems faced in all association studies : poorly matched cases and controls can lead to false - positive or -negative results , definitions of bipolar disorder vary across studies , and genes may have different , effects based on background population issues ( genetic background and environmental background ) . of all the specific candidate genes shown in one study or another to be associated with bipolar disorder , at this point , none of these findings have been robust enough or tested in large enough samples to definitively implicate them in the genesis of bipolar disorder . recently , with the advent of genetic chips that can analyze over 500 000 snps , and the knowledge - base provided by analysis of the human genome , it , has become possible to construct gw studies in outbred populations . in this approach , a case - control or trio approach ( affected subjects , plus their parents ) is utilized , typically requiring thousands of subjects , and 500 000 or more snps arc analyzed in order to determine specific genes or regions associated with a disorder . on the other hand , sample sizes needed for analyses may be difficult , to reach without major investments , the cost , of the technology is not . trivial , rare alleles with major effects may be overlooked , stratification issues and multiple testing issues become even more critical than in linkage studies , selection of individual cases may dilute the study of genetic forms of bipolar disorder , and replication will remain a difficult issue , leading some to temper the expectations we might expect from gwa analyses . gwa studies in bipolar disorder were initially pursued in the costa rican population , with microsatellitcs placed relatively sparsely across the genome . although these studies yielded potentially interesting linkage disequilibrium between bipolar disorder and specific chromosomal regions , the sparseness of the map did not . gwa studies of bipolar disorder , using dense snp maps , have been reported thus far . baum et al used a. two - stage strategy , beginning with 461 bipolar cases and 563 controls and following up significant findings in a sample of 772 bipolar cases and 876 controls , and found evidence for novel genes potentially associated with bipolar disorder , including a gene for diacylglycerol kinase , which plays a key role in the lithium sensitive phosphatidyl inositol pathway . a study by the wellcome trust case control consortium utilized 2000 bipolar cases and 3000 controls , and reported on a number of snps showing evidence of association , some at , specific loci that had not . formerly been implicated in studies of bipolar disorder . for both of these recent gwa studies , additional genes or regions comparisons across studies , replication studies for specific genes in new samples , combined analyses and even larger case - control studies will be necessary to adequately separate the wheat from the chaff . an additional gwa study of bipolar disorder is currently under way in the united states , as part of a private - public joint venture known as the gain collaborative group . these difficulties , as well as the relatively slow process in identifying genes for complex disorders , has led many investigators to begin to focus on identifying genes for endophenotypes . following are a number of preliminary studies which suggest , possible endophenotypes for bipolar disorder that derive from neuroanatomy and neuropsychology . importantly , we do not , know at this point , whether any of these meet all of the criteria necessary to be fully considered as an endophenotype for bipolar disorder . future studies need to be done , especially in terms of measuring hcritability and segregation with disease , for these and other potential endophenotypes . when looking at , biological structures of the brain , there are studies that suggest specific regions of the brain as endophenotypes for bipolar disorder . two studies revealed that the risk of white - matter abnormalities is more than threefold higher in patients with bipolar disorder than in healthy controls . poor performance on verbal memory tests was consistently found as a characteristic of bipolar disorder . impaired planning ( speed of information processing ) lithium is a treatment for bipolar disorder , and has been shown to modify the phase and period of circadian rhythms in a variety of species involving the glycogen synthase kinase 3 ( gsk-3 ) inhibitor . there is preliminary evidence of an association between a polymorphism in the gsk-3- promoter gene and bipolar disorder , suggesting that genetic factors involved in the regulation of the human circadian clock might represent , another endophenotype for bipolar disorder . for more thorough reviews of the role of endophenotypes for bipolar disorder , see refs 122 and 123 . when looking at , biological structures of the brain , there are studies that suggest specific regions of the brain as endophenotypes for bipolar disorder . two studies revealed that the risk of white - matter abnormalities is more than threefold higher in patients with bipolar disorder than in healthy controls . poor performance on verbal memory tests was consistently found as a characteristic of bipolar disorder . impaired planning ( speed of information processing ) lithium is a treatment for bipolar disorder , and has been shown to modify the phase and period of circadian rhythms in a variety of species involving the glycogen synthase kinase 3 ( gsk-3 ) inhibitor . there is preliminary evidence of an association between a polymorphism in the gsk-3- promoter gene and bipolar disorder , suggesting that genetic factors involved in the regulation of the human circadian clock might represent , another endophenotype for bipolar disorder . for more thorough reviews of the role of endophenotypes for bipolar disorder , the last two decades have been a time of vigorous activity in the field of bipolar disorder genetic studies . although success in the ultimate goals of clearly identifying which genes play a role in this disorder has been modest , this is not unusual , given the complexity of the disorder and the challenge of identifying genes for any disorder that is not . moving from the pioneering work in the 20th century to define the genetic basis of bipolar disorder , through carefully designed family and twin studies , a number of teams throughout the world have focused their energies on gathering large numbers of multiplex families , in order to carry out genome - wide linkage studies to identify bipolar gene loci . these studies have used fairly modest numbers of families , compared with the recommended number for complex diseases , and , perhaps as could be expected , the linkage scores have been modest in all studies published , ranging at best , up to lod scores in the range of 3 to 4 . smaller sets of families , from special populations known as population isolates have also yielded a. number of linkage regions with modest . systematic fine mapping of these regions may yield specific genes of interest , for bipolar disorder , as was seen in similar linkage studies of schizophrenia . candidate genes studies have also yielded a number of potentially associated genes deserving of further study in combined , large samples . new technologies now make gwa studies possible , and such studies will soon add a. number of additional genes to the pool of potentially associated genes for bipolar disorder . endophenotype studies will most likely also add a number of novel genes to consider in terms of how they might , indirectly contribute to bipolar disorder of mood destabilization . technologies that allow detection of copy number variants and chromosomal variations , as well as analyses of methylation patterns ( epigenetics ) , genomic expression , and proteomic analyses will add further gene candidates which can be targeted for study at the genomic level . iterative research , in which variants are discovered for a bipolar phenotype , and then those subjects who carry the variant are studied in more detail ( deep phenotyping ) may help to more clearly link gene variants to bipolar phenotypes . ultimately large collaborative studies , which clearly delineate specific phenotypes ( categorical and quantitative ) and take population genetics carefully into account , will be needed to , one by one , determine the exact correlation between gene variants and risk for the disease ( or trait ) of interest . scientists and clinicians who may have hoped that one or a few genes would eventually be identified that , would explain the majority of risk for bipolar disorder must , face the reality that there are likely to be many genes of relatively small effect , involved in bipolar disorder , and the genetic dissection of this disorder will be a subtle and complex process . genetic testing for bipolar disorder will likely ultimately require careful weighing of the presence or absence of many gene variants , when counseling is being done at the population level . as specific genes are clearly identified to play a role in bipolar disorder , it remains quite possible that , within specific families or clusters , genes of moderate effect will be discovered , but , we must face the fact that , thus far , no clear bipolar disease causing variant , has been discovered in any family studied . in the next , decade , a feasible goal might , be to clearly implicate at , least a handful of genes ( through well - powered replication studies or meta - analyses ) , from which the biochemical pathways underlying the disease can be more thoroughly studied at the level of cell biology and physiology . is likely that over the next , decade , the field of bipolar genetics will shift , from the current emphasis on identifying the genes which play a. role in this disease , to understanding the pathophysiology of the disease from a new perspective ( ie , by study of the pathways tied to the genes which play a role in the disease ) . for a. disorder that involves some of the fundamental behaviors and experiences of relevance to the human race , including regulation of activity levels , the ability to feel euphoria and dysphoria , to control social impulses , to create , to have racing thoughts , and to over- or undervalue one 's capacities , it is perhaps not surprising that the molecular underpinnings of the bipolar condition will prove to be complex and subtle , and span a. multiplicity of gene and protein networks . indeed , the gene variants which contribute to bipolar disorder may have evolved because of their specific value in helping individuals or groups adapt to socially and physically challenging and changing environments . to understand the genetics of bipolar disorder may in the end , not be any less of a. task than to understand the genetics of human psychology and behavior .

to elucidate metabolic effects of hif activation , we profiled the metabolome of cd8 t - lymphocytes with low ( vhl ) , or high ( vhldlck referred to as vhl ) hif signalling , and hif-1-vhl double knockouts ( hif1vhldlck referred to as hif1vhl ) to control for a specific contribution of hif-114 . vhlhif1 cluster with vhl , indicating that hif-1 mediates significant metabolic changes following vhl deletion . glycolysis is critical for sustaining effector function13,17 and these data indicate that vhl suppresses glycolysis via inhibition of hif-1 , ( extended data fig . vhl loss suppresses late and increases early tricarboxylic acid ( tca ) cycle intermediates ( extended data fig . furthermore , increases in 2hg depend on hif-1 when vhl is deleted ( extended data fig . this was validated using quantitative mass spectrometry in vhl and vhl hif1 cd8 t - lymphocytes ( fig . 1d ) , as well as in vhl - null cell lines , that express either hif-1 ( rcc4 ) or hif-2 ( 786-o ) , reconstituted with vhl ( fig . 1e and extended data fig . deletion of vhl in murine embryonic fibroblasts from vhl mice increases 2hg levels ( fig . hence , the vhl - hif axis regulates 2hg levels and constitutive hif-1 signalling underlies this effect in vhl - null cd8 t - lymphocytes . r-2hg is produced by isocitrate dehydrogenase 1/2 mutations in different cancers1820 ; accumulation of s-2hg , the other enantiomer of 2hg , occurs in the context of hypoxia21,22 and mitochondrial dysfunction23,24 . 2hg before activation and at sea level oxygen is undetectable , whereas levels at the same oxygenation are elevated 2 - 4 days after tcr stimulation ( fig . 2a).when activated cd8 t - lymphocytes are exposed to 1% oxygen , the intracellular concentration of 2hg reaches millimolar levels ( fig . given such high levels of 2hg , we sequenced25 idh1/2 , to preclude the unlikely possibility that culturing primary murine cd8 t - lymphocytes in hypoxia gives rise to mutations known to cause r-2hg production in humans18 ; we found no evidence for such mutations ( extended data fig . 3a - e ) . resolving the enantiomers of 2hg indicates that s-2hg constitutes the majority of the 2hg pool ( fig . primary human cd8 t - lymphocytes also accumulate s-2hg in hypoxia ( fig . 2e , f ) , indicating that this phenomenon is not limited to murine lymphocytes . we carried out deletion of loxp - flanked hif1 or epas1 ( referred to as hif2 ) genes in cd8 t - lymphocytes , with cre recombinase expressed under the distal lck promoter ( dlck)26 ( extended data fig . 2c ) . 2hg accumulation is abolished in hif1 dlck ( referred to as hif1 ) , but not hif2 dlck ( referred to as hif2 ) cells under hypoxia ( fig . 2i ) ; furthermore , the levels of s-2hg are significantly decreased in spleens of hif1 dlck mice ( fig . 2i ) , there is also a slight decrease in the levels of r-2hg ( fig . 2hg is present in urine of healthy individuals and is elevated in patients with 2hg acidurias27 . 2j ) indicating that hif-1 in the t - lymphocyte ( cd4 and cd8 ) compartment makes a contribution to s-2hg production in vivo . activated hif1 cd8 t - lymphocytes in 21% oxygen have lower 2hg at extended time points ( extended data fig . 2 g ) , indicating a contribution of hif-1 in non - hypoxic conditions also . we next sought to determine the metabolic route by which hif-1 promotes s-2hg production in cd8 t - lymphocytes . transcriptionally , hypoxic cd8 t - lymphocytes show induction of glycolysis and suppression of the tca cycle ( extended data fig . recent reports implicate lactate and malate dehydrogenases ( ldha and mdh1/2 ) , as enzymatic sources of s-2hg in hypoxia21,22 . in hif1 cd8 t - lymphocytes , the hypoxic expression of these enzymes suggests that mdh1 and mdh2 are unlikely to mediate the hypoxia - induced accumulation of s-2hg ( extended data fig . 2j ) . confirming this , knockdown of mdh1 or mdh2 does not decrease s-2hg in hypoxia ( extended data fig . 2k - m ) ; knockdown of mdh1 leads to marginal increases in s-2hg ( extended data fig . 2k ) , whilst also increasing r-2hg levels in hypoxic cd8 t - lymphocytes ( extended data fig . 2n).over - expression of ldha in hypoxic hif1 cd8 t - lymphocytes ( extended data fig . consistent with this , ldha expression in hypoxic cd8 t - lymphocytes is hif-1-dependent ( extended data fig . we next performed c - labelling experiments and in agreement with previous reports21,23 , u - c - glucose and u - c - glutamine tracing indicates that glutamine is the major source of 2hg , ( fig . the m+5 isotopologue dominates , suggesting direct conversion of glutamine - derived 2-oxoglutarate to 2hg23,21 . furthermore , the glutamate pool increases in hypoxic ( extended data fig . 2p ) and vhl cd8 t - lymphocytes ( extended data fig . 1a ) and depends on hif-1 ( extended data fig . 2q ) but not hif-2 ( extended data fig . inhibition of pyruvate dehydrogenase ( pdh ) by pyruvate dehydrogenase kinases ( pdk ) , promotes glutaminolysis2830 . we reasoned that pdk supports s-2hg production by diverting glutamine - derived 2-oxoglutarate to ldha , and consistent with this , dichloroacetate ( dca ) abrogates hypoxia - induced 2hg accumulation ( extended data fig . pdk1 expression is impaired in hypoxic hif1 cd8 t - lymphocytes ( extended data fig . 2s ) , and re - expression of pdk1 in this context increases s-2hg in hypoxia ( fig . inhibition of pdk activity also impedes hypoxia - induced increases in the glutamate pool ( extended data fig . hence , hif-1 drives s-2hg production in hypoxic cd8 t - lymphocytes via the pdk - pdh axis and ldha induction ( extended data fig . t - lymphocytes by treatment with cell permeable s-2hg , suggesting that s-2hg augments hif signalling in normoxia and hypoxia . additionally , there is increased phosphorylation of pdh - e1 ( extended data fig . 5a and b ) , elevated glucose uptake , lactate secretion ( extended data fig . since hif-1 supports effector functions in cd8 t - lymphocytes13,15 , we reasoned that s-2hg promotes effector differentiation via hif-1. however , unexpectedly , there is suppression of effector cytokine production ( extended data fig . 5 g ) and there is a clear increase in apoptosis at doses > 300 m ( extended data fig . 5h and i ) . 3a ) , with increased viability in the absence of il-2 supplementation ( extended data fig . these effects are robustly mediated at the transcriptional level after prolonged treatment with s-2hg ( fig 3b . 5 m ) and this is reversible upon withdrawal of treatment ( extended data fig . 5n).the effect does not occur when treating cells cultured in vehicle for 7 days ( extended data fig . 5n ) , demonstrating that s-2hg treatment of newly activated cells maintains this phenotypic marker . importantly , cd62l downregulation does not occur when hif-1 is absent15 , masking the effect of s-2hg on cd62l following hif-1 deletion ( extended data fig . 3c ) , cd44 , 41bb , eomes and less pd-1 in a hif-1-independent manner ( extended data fig . 5q ) . to determine the role of endogenously produced s-2hg , over - expression of l2hgdh ( extended data fig . 5r ) , a dehydrogenase that oxidizes s-2hg , was performed . over - expression of l2hgdh l2hgdh over - expression leads to an increase in the proportion of klrg1 cells , which are decreased in the presence of exogenous s-2hg ( fig . conversely , successful shrna - mediated knockdown of l2hgdh by hairpin # 3 ( extended data fig . 3 g ) , especially in 1% oxygen , promoting maintenance of cd62l ( fig . 3h ) . suppression of l2hgdh blocks loss of cd62l in response to low oxygen ( fig . these data demonstrate that l2hgdh activity regulates the expression of key phenotypic makers of cd8 t - lymphocytes , by controlling endogenous s-2hg levels . transcriptionally , s-2hg treatment increases expression of eomes , ccr6 , bcl-6 , cd62l and tcf-7 , with repression of prdm1 , after 7 days ( extended data fig . this transcriptional program is similar to gene expression changes in memory cd8 t - lymphocytes , suggesting that s-2hg treatment of cd8 t - lymphocytes ex vivo may enhance long term persistence and survival in the context of adoptive cell transfer33 . we thus co - transferred cfse - labelled vehicle and s-2hg treated cd45.1.1 or cd45.1.2 ot - i cd8 t - lymphocytes into lymphodepleted mice ( extended data fig . adoptively transferred cd45.1 ot - i cd8 t - lymphocytes , pre - treated with s-2hg , show dramatically enhanced persistence 30 days after transfer ( fig . 4d ) , expressing elevated cd44 , cd127 and bcl-2 levels relative to nave cells ( fig . consistent with this , ot - i cd8 t - lymphocytes , pre - treated with s-2hg are more proficient at controlling tumour growth in vivo in both lymphodepleted ( fig . 4 g ) and lymphoreplete ( fig . these data demonstrate that s-2hg treatment ex vivo maintains cells in a state with increased proliferative and survival capacity , when transferred in vivo , that is otherwise decreased by effector differentiation . mtor is a modifier of cd8 t - lymphocyte differentiation ; however , we do not observe mtor inhibition36,38 at doses of s-2hg needed for this to occur ( extended data fig . s - hg treatment ex vivo may be selecting cells that express higher levels of anti - apoptotic genes . two critical anti - apoptotic genes implicated in cd8 t - lymphocyte survival are bcl-2 and bcl - xl39,40 . moreover , over - expression of bcl-2 or bcl - xl ( extended data fig . 8e ) does not influence the expression of cd62l , cd44 or cd127 in the presence or absence of s-2hg ( extended data fig . 8f - i ) , indicating that s-2hg is exerting these phenotypic changes independently of bcl-2 or bcl - xl . inhibition of 2-oxoglutarate - dependent dioxygenases that demethylate histones ( jumonji c containing proteins ) or oxidise 5-methylcytosine in dna ( ten - eleven translocation ( tet ) proteins ) may mediate s-2hg s effect 32,4143 . 9a ) ; in particular , di- and tri - methylation on h3k27 are reciprocally altered , indicating inhibition of h3k27me3 demethylation ( fig . the h3k27me2/3 demethylase , kdm6a ( utx ) , is an important regulator of thymocyte differentiation44 ; changes in h3k27me3 levels correlate with genes associated with regulation of cd8 t - lymphocyte differentiation11 . global levels of h3k27me3 in cd8 t - lymphocytes are reduced following activation , but remain high with s-2hg treatment in a hif-1-independent manner ( fig . 9d ) and inhibition of utx reproduces the effect of s-2hg treatment on cd62l expression ( fig 5d and extended data fig . levels of h3k27me3 are highest in central memory ( cd62lcd44 ) and nave ( cd62lcd44 ) cd8 t - lymphocytes , relative to effectors ( cd62lcd44 ) ( fig . 5e ) . to determine if histone methylation changes occur at the transcription start site ( tss ) of cd62l with s - hg treatment , we performed chip - pcr for h3k27me3 , h3k4me3 and rna pol ii , on nave and activated cd8 t - lymphocytes cultured with or without s-2hg ( fig . however , nave and s-2hg treated cd8 t - lymphocytes have higher enrichment for h3k4me3 at the tss that is reduced in vehicle - treated cells . additionally , s-2hg treated cd8 t - lymphocytes have markedly higher rna pol ii biding than both nave and vehicle treated cells . thus s-2hg is promoting cd62l transcription directly with enrichment of h3k4me3 at the tss and indirectly via preservation of h3k27me3 elsewhere in the genome . total levels of 5-methylcytosine ( 5mc ) in genomic dna are largely unchanged by tcr triggering ( fig . . however , total levels of 5-hydroxymethylcytosine ( 5hmc ) decrease following tcr triggering ( fig . 5hmc removal in genomic dna can occur via tet - mediated oxidation ( active ) as well as dna replication ( passive)45,46 . s-2hg treatment induces small changes in total 5hmc and 5mc in a time - dependent manner ( fig . 6b , c ) ; at day 3 , there are marginally higher levels of 5hmc ( statistically non - significant ) with no changes in 5mc ( fig . this is likely due to decreased proliferation in the presence of s-2hg ( extended data fig 5 g ) . following sustained treatment , at days 7 and 9 , cells have less 5hmc and more 5mc ( fig . 5mc changes did not reach statistical significance at any time point ; changes in 5hmc were statistically significant at day 7 only . nonetheless , the changes at days 7 and 9 are consistent with inhibition of tet proteins in the presence of sustained s-2hg treatment . knockdown of tet2 recapitulates the effect of s-2hg on cd62l , indicating that tet2 also contributes to cd8 t - lymphocyte effector differentiation ( fig . 10a ) , implicating dna demethylation as an added epigenetic modifier of cd8 t - cell differentiation12 . we performed both 5hmc and 5mc dip - pcr around the tss of cd62l ( fig . nave cells have the highest and lowest enrichment for 5hmc and 5mc respectively , while s-2hg treated cells display the opposite pattern . our data support a model in which both utx and tet2 contribute to effector differentiation of cd8 t - lymphocytes in vitro . the activity of these epigenetic modifiers is altered by s-2hg in a fashion that inhibits effector differentiation . tcr - triggering induced loss of 5hmc in gdna , whereas 5mc levels were relatively stable ( fig . 6a ) and s-2hg treatment produced reciprocal changes in 5mc and 5hmc levels at later time points ( fig . we investigated 5mc and 5hmc presence at and around the tss of cd62l ( fig . s-2hg treatment causes reciprocal 5mc increases and 5hmc decreases in this region consistent with inhibition of tet proteins and transcriptional repression47 , yet there is robust expression of cd62l with s-2hg treatment ( fig . 6d and extended data figure 10a ) , indicating that s-2hg - induced dna methylation changes elsewhere in the genome may indirectly promote cd62l expression . s-2hg - treated and nave cd8 t - lymphocytes have high enrichment for the active h3k4me3 mark49 at the tss that is lost in vehicle - treated cells . at least in s-2hg treated cells , this is accompanied by binding of rna - pol ii . despite global increases in h3k27me3 with s-2hg ( fig . 5a - c ) , the lack of h3k27me3 at the tss of cd62l is not surprising , as this mark is associated with repression49 . 9e and f ) , indicating that h3k27me3 deposition at other genomic sites can indirectly promote cd62l expression . due to the relationship between histone methylation and other marks such as histone acetylation , modulation of the latter may have similar effects to those seen with s-2hg treatment50 . adoptively transferred cells treated with s-2hg ex vivo have an increased capacity to proliferate and persist in vivo , with enhanced anti - tumour efficacy ( fig . 4 ) , demonstrating a new strategy to improve persistence of adoptive cell therapies for cancer . the data presented uncover a metabolic - epigenetic axis that controls aspects of t - cell fate . factors regulating endogenous s-2hg levels , such as hif signalling , tcr triggering , l2hdgh activity and potentially others , can alter the differentiation of cd8 t - lymphocytes and thus shape the immune response . a/ illustration of central carbon metabolism in cd8 lymphocytes , including glycolysis and the tricarboxylic acid cycle , depicting relative levels of detected metabolites between vhl ( n=5 ) , vhldlck ( n=5 ) and hif1 vhldlck ( n=3 ) cd8 t - lymphocyte groups . b/ glucose consumption and lactate production in vhl and vhldlck cd8 t - lymphocytes 7 days after activation with cd3 and cd28 antibodies . c/ immunoblot analysis for hif-1 and laminb1 , using nuclear extracts prepared from vhl and vhl cd8 t - lymphocytes cultured in 21% oxygen . e/ immunoblot analysis for hif-1 , hif-2 and -tubulin , on whole cell extracts prepared from rcc4 and 786-o renal cancer cell lines , with and without expression of functional vhl . f/ deletion efficiency of vhl in vhl mefs following infection with adeno - cre virus , n=3 individual preparations . two - tailed t - test ( b ) , one - way anova for multiple comparisons ( a ) . error bars denote s.d . and each dot represents an individual mouse in a and b. * p<0.05 , * * p<0.01 , * * * p<0.001 . for immunoblot source images , see supplementary fig . 1 . b/ h - nmr analysis for 2hg from cd8 t - lymphocytes cultured as in fig c/ deletion efficiency of hif1 or hif2 in cd8 t - lymphocytes , isolated from hif1dlck or hif2dlck mice . d/ total 2hg levels , normalized to viable cell count or protein content , in hif1 and hif1dlck cd8 t - lymphocytes cultured as in fig . e/ total 2hg levels , normalized to viable cells or protein content , in hif2 and hif2dlck cd8 t - lymphocytes cultured as in fig . f/ illustration outlining the workflow for metabolite extraction , deletion efficiency and viability experiments in hif1 , hif1dlck , hif2 and hif2dlck cd8 t - lymphocytes . also shown g/ total amount of 2hg in hif1 ( n=6 ) and hif1dlck ( n=7 ) cd8 t - lymphocytes , at indicated times following activation . h/ heat map indicating qpcr measurement of expression of enzymes involved in central carbon metabolism in cd8 t - lymphocytes cultured as in fig . 2b i/ lc - ms / ms quantification of total intracellular succinate , fumarate and malate levels in cd8 t - lymphocytes isolated from c57bl/6j mice and cultured as in fig . j/ heat map indicating qpcr measurement , in hif1 ( n=4 ) and hif1dlck ( n=3 ) cd8 t - lymphocytes growing in 1% oxygen , of expression of enzymes implicated in the hypoxic production of s-2hg . k/ qpcr validation of shrna - knockdowns in cd8 t - lymphocytes isolated from c57bl/6j mice . l/ lc - ms / ms quantification of s- and r-2hg in cd8 t - lymphocytes isolated from c57bl/6j mice , with shrna - mediated knockdown of mdh1 ; n=4 pools of 4 mice per pool . m/ lc - ms / ms quantification of s- and r-2hg in cd8 t - lymphocytes isolated from c57bl/6j mice , with shrna - mediated knockdown of mdh2 ; n=4 pools of 4 mice per pool . n/ lc - ms / ms quantification of r-2hg in cd8 t - lymphocytes isolated from c57bl/6j mice , with shrna - mediated knockdown of ldha ; n=4 pools of 4 mice per pool . o/ validation of pdk1-flag and ldha - flag expression in hif1dlck cd8 t - lymphocytes by immunoblot analysis for flag . p/ lc - ms / ms quantification of total intracellular glutamate levels in cd8 t - lymphocytes cultured as in fig . q - r/ lc - ms / ms quantification of total intracellular glutamate levels in hif1 , hif1dlck , hif2 and hif2dlck cd8 t - lymphocytes cultured as in fig . s/ immunoblot of cytosolic fractions for phospho - pdh e1 ( s232 ) and total pdh - e1 in cd8 t - lymphocytes cultured in 1% oxygen in the presence of the indicated concentration of dca for 48h . t/ total intracellular concentration of 2hg in cd8 t - lymphocytes from c57bl/6j mice cultured as in fig . 2b and treated with 5 mm dca for the latter 48h of culture ; n=4 mice . u/ total intracellular amount of 2hg , nomralized to viable cell count or protein content in cd8 t - lymphocytes from c57bl/6j mice cultured as in fig . 2b and treated with 5 mm dca for the latter 48h of culture ; n=4 mice . v/ total intracellular amount of glutamate in cd8 t - lymphocytes from c57bl/6j mice cultured as in fig . 2b and treated with 5 mm dca for the latter 48h of culture ; n=4 mice . two - way anova for grouped data ( d , e , f , q , r , t , u , v ) . paired t - test for matched comparisons ( i , p ) , one - way anova for multiple matched comparisons ( g , l , m , n ) . error bars denote s.d . and each dot represents an individual mouse in g , i and p. ns= non - significant , * p<0.05 , * * p<0.01 , * * * p<0.0001 . experiments were performed with indicated numbers of mice from multiple occasions . for immunoblot source images , a/ illustration outlining the workflow for mutational analysis of idh1 and idh2 b/ sanger sequencing chromatograms validating the presence of wild type idh1 as compared to the c57bl/6j ncbi reference sequence . c/ alignment of mouse and human idh1 protein indicating conservation of active site arginine residues . d/ sanger sequencing chromatograms validating the presence of wild type idh2 as compared to the c57bl/6j ncbi reference sequence . e/ alignment of mouse and human idh2 protein indicating conservation of active site arginine residues . proposed mechanism by which hif-1 controls s-2hg production in cd8t - lymphocytes and c - labelling strategy using u - c - glucose ( m+6 ) and u - c - glutamine ( m+5 ) to label endogenous 2hg . b/ isotopologue distribution of 2hg ( as a percentage of the total pool ) in cd8 t - lymphocytes , after labelling with u - c - glucose for 1 , 5 and 24 h in both 21% and 1% oxygen conditions ; n=3 mice per time point . c/ isotopologue distribution of 2hg ( as a percentage of the total pool ) in cd8 t - lymphocytes , after labelling with u - c - glutamine for 1 , 5 and 24 h in both 21% and 1% oxygen conditions ; n=3 mice per time point . a - b/ immunoblot analysis of nuclear and cytosolic fractions , prepared from cd8 t - lymphocytes cultured in ( a ) 21% and ( b ) 1% oxygen , for hif-1 , hdac1 , phospho - pdh e1 ( s232 ) and total pdh - e1. cells were activated for 48 h with cd3+cd28 antibodies and then expanded for a further 4 days in the presence of il-2 followed by treatment with the indicated concentration of s-2hg for 16 hours . c/ glucose consumption and lactate production of c57bl/6j , hif1 ( n=12 ) and hif1dlck ( n=4 ) cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 16 hours as in extended data fig . 5a . d/ vegf - a production of wild type c57bl/6j , hif1 ( n=16 ) and hif1dlck ( n=4 ) cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 16 hours as in extended data fig . 5a . e/ representative flow cytometry plots of ifn- vs tnf- in siinfekl re - stimulated ot - i cd8 t - lymphocytes , as a function of increasing doses of s-2hg - octyl ester for 7 days . total splenocytes were activated for 48 h with 1000 nm siinfekl and then expanded for a further 4 days in the presence of il-2 followed by treatment with 500 m s-2hg - octyl ester for 24 h. ot - i cd8 t - lymphocytes were incubated with target and control cells for 4 hours ; n=3 mice per condition . g/ cfse dilution assay , with associated statistics ( n=4 mice per condition ) at day 3 of cd8 t - lymphocytes activated with cd3+cd28 antibodies and cultured with or without 500 m s-2hg - octyl ester from day 0 . viability and annexin v assay of cd8 t - lymphocytes treated with increasing s-2hg doses for 4 days , n=4 mice . i/ viability of cd8 t - lymphocytes cultured with 300 m s-2hg - octyl ester for the indicated number of days , n=4 mice . j/ amount of ifn- protein in the media of wild type c57bl/6j , hif1 ( n=8 ) and hif1 dlck ( n=4 ) cd8 t - lymphocytes treated for 24 h with or without 500 m s-2hg - octyl ester . k / viability of hif1 ( n=8 ) and hif1 dlck ( n=4 ) ot - i cd8 t - lymphocytes activated with 1000 nm siinfekl peptide and cultured for 7 days with or without 500 m s-2hg - octyl ester in the absence of il-2 supplementation from day 0 . l/ expression of ifng mrna in cd8 t - lymphocytes treated for either 24 h or 7 days with or without 500 m s-2hg - octyl ester . cd44 and cd62l surface expression on ot - i cd8 t - lymphocytes treated with increasing doses of s-2hg for 7 days . left panel : % cd62l cd8 t - lymphocytes , treated for 7 days with 500 m s-2hg - octyl ester , followed by washout or maintenance of the compound and follow up every 3 day , for 9 more days ; n=4 mice . right panel : % cd62l cd8 t - lymphocytes , treated for 7 days with vehicle , followed by addition of 500 m s-2hg - octyl ester or vehicle and follow up every 3 day , for 9 more days ; n=4 mice . gated on live , cd8 cells . o/ cd44 and cd62l surface expression on hif1 and hif1 dlck cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 1 , 7 and 10 days following treatment . p/ cd44 and cd62l surface expression on hif2 and hif2 dlck cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 1 , 7 and 10 days following treatment . q/ flow cytometric characterisation of indicated phenotypic markers on hif1 ( n=4 ) and hif1 dlck ( n=4 ) cd8 t - lymphocytes treated for 7 days with 500 m s-2hg - octyl ester . r/ validation of l2hgdh - flag expression in cd8 t - lymphocytes from c57bl/6j mice by immunoblot analysis for flag . s/ qpcr validation of l2hgdh knockdowns in cd8 t - lymphocytes isolated from c57bl/6j mice . t - lymphocytes in response to shscramble or shl2hgdh # 3 in 21% or 1% oxygen is shown on the right . u / qpcr quantification of prdm1 , sell , eomes , tcf7 , bcl6 and ccr6 expression in cd8 t - lymphocytes treated for 1 or 7 days with or without 500 m s-2hg - octyl ester . paired t - test for matched comparisons ( g ) and two - way anova for grouped data ( c , d , j , k , l , q ) . each dot in c , d , g , j , k , l , q , t represents an individual mouse . ns = non - significant , * p<0.05 , * * p<0.01 , * * * p<0.001 . experiments were performed with indicated numbers of mice from multiple occasions . for immunoblot source images , c/ representative flow cytometry plots of recalling cd45.1 cd8 t - lymphocytes in indicated organs on day 7 post vaccination ( day 37 post transfer ) . immunoblot analysis on cytosolic extracts for mtor signalling in cd8 t - lymphocytes treated with the indicated doses of s-2hg for 24 h. for immunoblot source images , see supplementary fig . 1 . a / qpcr quantification of bcl2 and bclxl mrna levels in response to 500 m s-2hg - octyl ester treatment for either 1 or 7 days . n=4 mice b / immunnoblot analysis for bcl-2 and bcl - xl protein in response to increasing doses of s-2hg - octyl ester for 9 days . c / qpcr quantification of bcl2 and bclxl mrna levels in response to 300 m s-2hg - octyl ester treatment for either 1 , 7 or 9 days . n=4 mice d / representative flow cytometry histograms of bcl-2 and bcl - xl abundance in cd8 + t - lymphocytes treated with 300 m s-2hg - octyl ester for 9 days . quantification and associated statistics are shown in the graph on the right . e / immunoblot analysis confirming the expression of bcl - xl - flag and bcl-2-flag in ot - i in cd8 t - lymphocytes . f - h / cd62l ( f ) , cd127 ( g ) and cd44 ( h ) surface expression in ot - i cd8 t - lymphocytes transduced with retrovirus expressing either bcl-2-flag or bcl - xl - flag and treated with the indicated concentration of s-2hg - octyl ester for 7 days . n=2 mice i/ representative flow cytometry histograms of cd62l , cd127 and cd44 surface expression in ot - i cd8 t - lymphocytes transduced with retrovirus expressing either bcl-2-flag or bcl - xl - flag and treated with the indicated concentration of s-2hg - octyl ester for 7 days . the associated statistics of these flow cytometry data are shown in f , g and h. * * p<0.01 , ns= non - significant . paired t - test for matched comparisons ( d ) and two - way anova for grouped data ( a , b ) . one - way anova of matched samples for multiple comparisons ( c , f , g , h ) . ns = non - significant , * * p<0.01 , gmfi = geometric mean fluorescence intensity . experiments were performed with indicated numbers of mice from at least two occasions . for immunoblot source images , a/ immunoblot analysis on nuclear extracts for histone h3 methylation marks in activated cd8 t - lymphocytes treated with the indicated doses of s-2hg for 7 days . b / representative flow cytometry histograms of h3k27me3 staining as a function of increasing s-2hg - octyl ester concentration . h3k27me3 staining in cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester and stained with or without fluorophore conjugated c36b11 antibody . d/ qpcr measurement for expression of utx in unstimulated and stimulated cd8 t - lymphocytes ; n=4 mice . e/ represetative flow cytomery plots of cd44 vs cd62l expression , with associated statistics , on activated cd8 t - lymphocytes after 4 days of treatment with 500 m s-2hg - octyl ester or 1 m gskj4 . gated on live , cd8 cells . f/ representative flow cytometry plots of cd44 vs cd62l expression on cd8 t - lymphocytes with shrna - mediated knockdown of utx , 7 days after trasnduction . g/ igg control chip - qpcr for h3k4me3 , h3k27me and rna pol ii at and around the tss for cd62l , in freshly isolated nave or activated cd8 + t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 7 days . experiments were performed with indicated numbers of mice from at least two occasions . for immunoblot source images , a/ representative flow cytometry plots of cd44 vs cd6l expression on cd8 t - lymphocytes with shrna - mediated knockdown of tet2 , 7 days after trasnduction . b/ igg control dip - qpcr for 5mc and 5hmc at and around the tss for cd62l , in freshly isolated nave or activated cd8 + t - lymphocytes treated with or without 500 m s-2hg for 7 days . supplemental information is linked to the online version of the paper at www.nature.com/nature methods can be found in the supplementary information which is linked to the online version of the paper at www.nature.com/nature

r-2-hydroxyglutarate accumulates to millimolar levels in cancers with gain - of - function isocitrate dehydrogenase 1/2 mutations . these levels of r-2-hydroxyglutarate affect 2-oxoglutarate - dependent dioxygenases . both r- and s-2-hydroxyglutarate , the other enantiomer of this metabolite , are detectible in healthy individuals , yet their physiological function remains elusive . here we show that cd8 + t - lymphocytes accumulate 2-hydroxyglutarate in response to t - cell receptor triggering . this increases to millimolar levels in physiological oxygen conditions , via a hypoxia inducible factor 1 alpha - dependent mechanism . s-2-hydroxyglutarate predominates over r-2-hydroxyglutarate in activated t cells , and we demonstrate alterations in markers of cd8 + t - lymphocyte differentiation in response to this metabolite . modulation of histone and dna demethylation as well as hypoxia inducible factor 1 alpha stability mediate these effects . s-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation , persistence and anti - tumour capacity of adoptively transferred cd8 + t - lymphocytes . thus s-2-hydroxyglutarate acts as an immunometabolite that links environmental context , via a metabolic - epigenetic axis , to immune fate and function .

The VHL-HIF-1 axis regulates 2HG production HIF-1 regulates S-2HG production S-2HG alters CD8 S-2HG alters methylation in CD8 Discussion Extended Data Supplementary Material

to elucidate metabolic effects of hif activation , we profiled the metabolome of cd8 t - lymphocytes with low ( vhl ) , or high ( vhldlck referred to as vhl ) hif signalling , and hif-1-vhl double knockouts ( hif1vhldlck referred to as hif1vhl ) to control for a specific contribution of hif-114 . this was validated using quantitative mass spectrometry in vhl and vhl hif1 cd8 t - lymphocytes ( fig . 1d ) , as well as in vhl - null cell lines , that express either hif-1 ( rcc4 ) or hif-2 ( 786-o ) , reconstituted with vhl ( fig . hence , the vhl - hif axis regulates 2hg levels and constitutive hif-1 signalling underlies this effect in vhl - null cd8 t - lymphocytes . r-2hg is produced by isocitrate dehydrogenase 1/2 mutations in different cancers1820 ; accumulation of s-2hg , the other enantiomer of 2hg , occurs in the context of hypoxia21,22 and mitochondrial dysfunction23,24 . 2a).when activated cd8 t - lymphocytes are exposed to 1% oxygen , the intracellular concentration of 2hg reaches millimolar levels ( fig . given such high levels of 2hg , we sequenced25 idh1/2 , to preclude the unlikely possibility that culturing primary murine cd8 t - lymphocytes in hypoxia gives rise to mutations known to cause r-2hg production in humans18 ; we found no evidence for such mutations ( extended data fig . primary human cd8 t - lymphocytes also accumulate s-2hg in hypoxia ( fig . 2i ) ; furthermore , the levels of s-2hg are significantly decreased in spleens of hif1 dlck mice ( fig . 2j ) indicating that hif-1 in the t - lymphocyte ( cd4 and cd8 ) compartment makes a contribution to s-2hg production in vivo . activated hif1 cd8 t - lymphocytes in 21% oxygen have lower 2hg at extended time points ( extended data fig . we next sought to determine the metabolic route by which hif-1 promotes s-2hg production in cd8 t - lymphocytes . transcriptionally , hypoxic cd8 t - lymphocytes show induction of glycolysis and suppression of the tca cycle ( extended data fig . in hif1 cd8 t - lymphocytes , the hypoxic expression of these enzymes suggests that mdh1 and mdh2 are unlikely to mediate the hypoxia - induced accumulation of s-2hg ( extended data fig . 2k ) , whilst also increasing r-2hg levels in hypoxic cd8 t - lymphocytes ( extended data fig . 2n).over - expression of ldha in hypoxic hif1 cd8 t - lymphocytes ( extended data fig . 2p ) and vhl cd8 t - lymphocytes ( extended data fig . t - lymphocytes by treatment with cell permeable s-2hg , suggesting that s-2hg augments hif signalling in normoxia and hypoxia . these data demonstrate that l2hgdh activity regulates the expression of key phenotypic makers of cd8 t - lymphocytes , by controlling endogenous s-2hg levels . this transcriptional program is similar to gene expression changes in memory cd8 t - lymphocytes , suggesting that s-2hg treatment of cd8 t - lymphocytes ex vivo may enhance long term persistence and survival in the context of adoptive cell transfer33 . adoptively transferred cd45.1 ot - i cd8 t - lymphocytes , pre - treated with s-2hg , show dramatically enhanced persistence 30 days after transfer ( fig . consistent with this , ot - i cd8 t - lymphocytes , pre - treated with s-2hg are more proficient at controlling tumour growth in vivo in both lymphodepleted ( fig . mtor is a modifier of cd8 t - lymphocyte differentiation ; however , we do not observe mtor inhibition36,38 at doses of s-2hg needed for this to occur ( extended data fig . s - hg treatment ex vivo may be selecting cells that express higher levels of anti - apoptotic genes . two critical anti - apoptotic genes implicated in cd8 t - lymphocyte survival are bcl-2 and bcl - xl39,40 . inhibition of 2-oxoglutarate - dependent dioxygenases that demethylate histones ( jumonji c containing proteins ) or oxidise 5-methylcytosine in dna ( ten - eleven translocation ( tet ) proteins ) may mediate s-2hg s effect 32,4143 . the h3k27me2/3 demethylase , kdm6a ( utx ) , is an important regulator of thymocyte differentiation44 ; changes in h3k27me3 levels correlate with genes associated with regulation of cd8 t - lymphocyte differentiation11 . global levels of h3k27me3 in cd8 t - lymphocytes are reduced following activation , but remain high with s-2hg treatment in a hif-1-independent manner ( fig . levels of h3k27me3 are highest in central memory ( cd62lcd44 ) and nave ( cd62lcd44 ) cd8 t - lymphocytes , relative to effectors ( cd62lcd44 ) ( fig . to determine if histone methylation changes occur at the transcription start site ( tss ) of cd62l with s - hg treatment , we performed chip - pcr for h3k27me3 , h3k4me3 and rna pol ii , on nave and activated cd8 t - lymphocytes cultured with or without s-2hg ( fig . however , nave and s-2hg treated cd8 t - lymphocytes have higher enrichment for h3k4me3 at the tss that is reduced in vehicle - treated cells . additionally , s-2hg treated cd8 t - lymphocytes have markedly higher rna pol ii biding than both nave and vehicle treated cells . 5hmc removal in genomic dna can occur via tet - mediated oxidation ( active ) as well as dna replication ( passive)45,46 . knockdown of tet2 recapitulates the effect of s-2hg on cd62l , indicating that tet2 also contributes to cd8 t - lymphocyte effector differentiation ( fig . 10a ) , implicating dna demethylation as an added epigenetic modifier of cd8 t - cell differentiation12 . our data support a model in which both utx and tet2 contribute to effector differentiation of cd8 t - lymphocytes in vitro . s-2hg - treated and nave cd8 t - lymphocytes have high enrichment for the active h3k4me3 mark49 at the tss that is lost in vehicle - treated cells . adoptively transferred cells treated with s-2hg ex vivo have an increased capacity to proliferate and persist in vivo , with enhanced anti - tumour efficacy ( fig . the data presented uncover a metabolic - epigenetic axis that controls aspects of t - cell fate . factors regulating endogenous s-2hg levels , such as hif signalling , tcr triggering , l2hdgh activity and potentially others , can alter the differentiation of cd8 t - lymphocytes and thus shape the immune response . a/ illustration of central carbon metabolism in cd8 lymphocytes , including glycolysis and the tricarboxylic acid cycle , depicting relative levels of detected metabolites between vhl ( n=5 ) , vhldlck ( n=5 ) and hif1 vhldlck ( n=3 ) cd8 t - lymphocyte groups . b/ glucose consumption and lactate production in vhl and vhldlck cd8 t - lymphocytes 7 days after activation with cd3 and cd28 antibodies . b/ h - nmr analysis for 2hg from cd8 t - lymphocytes cultured as in fig c/ deletion efficiency of hif1 or hif2 in cd8 t - lymphocytes , isolated from hif1dlck or hif2dlck mice . d/ total 2hg levels , normalized to viable cell count or protein content , in hif1 and hif1dlck cd8 t - lymphocytes cultured as in fig . e/ total 2hg levels , normalized to viable cells or protein content , in hif2 and hif2dlck cd8 t - lymphocytes cultured as in fig . also shown g/ total amount of 2hg in hif1 ( n=6 ) and hif1dlck ( n=7 ) cd8 t - lymphocytes , at indicated times following activation . 2b i/ lc - ms / ms quantification of total intracellular succinate , fumarate and malate levels in cd8 t - lymphocytes isolated from c57bl/6j mice and cultured as in fig . j/ heat map indicating qpcr measurement , in hif1 ( n=4 ) and hif1dlck ( n=3 ) cd8 t - lymphocytes growing in 1% oxygen , of expression of enzymes implicated in the hypoxic production of s-2hg . k/ qpcr validation of shrna - knockdowns in cd8 t - lymphocytes isolated from c57bl/6j mice . l/ lc - ms / ms quantification of s- and r-2hg in cd8 t - lymphocytes isolated from c57bl/6j mice , with shrna - mediated knockdown of mdh1 ; n=4 pools of 4 mice per pool . m/ lc - ms / ms quantification of s- and r-2hg in cd8 t - lymphocytes isolated from c57bl/6j mice , with shrna - mediated knockdown of mdh2 ; n=4 pools of 4 mice per pool . n/ lc - ms / ms quantification of r-2hg in cd8 t - lymphocytes isolated from c57bl/6j mice , with shrna - mediated knockdown of ldha ; n=4 pools of 4 mice per pool . o/ validation of pdk1-flag and ldha - flag expression in hif1dlck cd8 t - lymphocytes by immunoblot analysis for flag . p/ lc - ms / ms quantification of total intracellular glutamate levels in cd8 t - lymphocytes cultured as in fig . q - r/ lc - ms / ms quantification of total intracellular glutamate levels in hif1 , hif1dlck , hif2 and hif2dlck cd8 t - lymphocytes cultured as in fig . t/ total intracellular concentration of 2hg in cd8 t - lymphocytes from c57bl/6j mice cultured as in fig . u/ total intracellular amount of 2hg , nomralized to viable cell count or protein content in cd8 t - lymphocytes from c57bl/6j mice cultured as in fig . b/ isotopologue distribution of 2hg ( as a percentage of the total pool ) in cd8 t - lymphocytes , after labelling with u - c - glucose for 1 , 5 and 24 h in both 21% and 1% oxygen conditions ; n=3 mice per time point . c/ isotopologue distribution of 2hg ( as a percentage of the total pool ) in cd8 t - lymphocytes , after labelling with u - c - glutamine for 1 , 5 and 24 h in both 21% and 1% oxygen conditions ; n=3 mice per time point . c/ glucose consumption and lactate production of c57bl/6j , hif1 ( n=12 ) and hif1dlck ( n=4 ) cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 16 hours as in extended data fig . e/ representative flow cytometry plots of ifn- vs tnf- in siinfekl re - stimulated ot - i cd8 t - lymphocytes , as a function of increasing doses of s-2hg - octyl ester for 7 days . total splenocytes were activated for 48 h with 1000 nm siinfekl and then expanded for a further 4 days in the presence of il-2 followed by treatment with 500 m s-2hg - octyl ester for 24 h. ot - i cd8 t - lymphocytes were incubated with target and control cells for 4 hours ; n=3 mice per condition . g/ cfse dilution assay , with associated statistics ( n=4 mice per condition ) at day 3 of cd8 t - lymphocytes activated with cd3+cd28 antibodies and cultured with or without 500 m s-2hg - octyl ester from day 0 . viability and annexin v assay of cd8 t - lymphocytes treated with increasing s-2hg doses for 4 days , n=4 mice . i/ viability of cd8 t - lymphocytes cultured with 300 m s-2hg - octyl ester for the indicated number of days , n=4 mice . l/ expression of ifng mrna in cd8 t - lymphocytes treated for either 24 h or 7 days with or without 500 m s-2hg - octyl ester . cd44 and cd62l surface expression on ot - i cd8 t - lymphocytes treated with increasing doses of s-2hg for 7 days . left panel : % cd62l cd8 t - lymphocytes , treated for 7 days with 500 m s-2hg - octyl ester , followed by washout or maintenance of the compound and follow up every 3 day , for 9 more days ; n=4 mice . right panel : % cd62l cd8 t - lymphocytes , treated for 7 days with vehicle , followed by addition of 500 m s-2hg - octyl ester or vehicle and follow up every 3 day , for 9 more days ; n=4 mice . o/ cd44 and cd62l surface expression on hif1 and hif1 dlck cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 1 , 7 and 10 days following treatment . q/ flow cytometric characterisation of indicated phenotypic markers on hif1 ( n=4 ) and hif1 dlck ( n=4 ) cd8 t - lymphocytes treated for 7 days with 500 m s-2hg - octyl ester . s/ qpcr validation of l2hgdh knockdowns in cd8 t - lymphocytes isolated from c57bl/6j mice . t - lymphocytes in response to shscramble or shl2hgdh # 3 in 21% or 1% oxygen is shown on the right . u / qpcr quantification of prdm1 , sell , eomes , tcf7 , bcl6 and ccr6 expression in cd8 t - lymphocytes treated for 1 or 7 days with or without 500 m s-2hg - octyl ester . for immunoblot source images , c/ representative flow cytometry plots of recalling cd45.1 cd8 t - lymphocytes in indicated organs on day 7 post vaccination ( day 37 post transfer ) . immunoblot analysis on cytosolic extracts for mtor signalling in cd8 t - lymphocytes treated with the indicated doses of s-2hg for 24 h. for immunoblot source images , see supplementary fig . a / qpcr quantification of bcl2 and bclxl mrna levels in response to 500 m s-2hg - octyl ester treatment for either 1 or 7 days . c / qpcr quantification of bcl2 and bclxl mrna levels in response to 300 m s-2hg - octyl ester treatment for either 1 , 7 or 9 days . n=4 mice d / representative flow cytometry histograms of bcl-2 and bcl - xl abundance in cd8 + t - lymphocytes treated with 300 m s-2hg - octyl ester for 9 days . e / immunoblot analysis confirming the expression of bcl - xl - flag and bcl-2-flag in ot - i in cd8 t - lymphocytes . f - h / cd62l ( f ) , cd127 ( g ) and cd44 ( h ) surface expression in ot - i cd8 t - lymphocytes transduced with retrovirus expressing either bcl-2-flag or bcl - xl - flag and treated with the indicated concentration of s-2hg - octyl ester for 7 days . n=2 mice i/ representative flow cytometry histograms of cd62l , cd127 and cd44 surface expression in ot - i cd8 t - lymphocytes transduced with retrovirus expressing either bcl-2-flag or bcl - xl - flag and treated with the indicated concentration of s-2hg - octyl ester for 7 days . for immunoblot source images , a/ immunoblot analysis on nuclear extracts for histone h3 methylation marks in activated cd8 t - lymphocytes treated with the indicated doses of s-2hg for 7 days . h3k27me3 staining in cd8 t - lymphocytes treated with or without 500 m s-2hg - octyl ester and stained with or without fluorophore conjugated c36b11 antibody . d/ qpcr measurement for expression of utx in unstimulated and stimulated cd8 t - lymphocytes ; n=4 mice . e/ represetative flow cytomery plots of cd44 vs cd62l expression , with associated statistics , on activated cd8 t - lymphocytes after 4 days of treatment with 500 m s-2hg - octyl ester or 1 m gskj4 . f/ representative flow cytometry plots of cd44 vs cd62l expression on cd8 t - lymphocytes with shrna - mediated knockdown of utx , 7 days after trasnduction . g/ igg control chip - qpcr for h3k4me3 , h3k27me and rna pol ii at and around the tss for cd62l , in freshly isolated nave or activated cd8 + t - lymphocytes treated with or without 500 m s-2hg - octyl ester for 7 days . for immunoblot source images , a/ representative flow cytometry plots of cd44 vs cd6l expression on cd8 t - lymphocytes with shrna - mediated knockdown of tet2 , 7 days after trasnduction . b/ igg control dip - qpcr for 5mc and 5hmc at and around the tss for cd62l , in freshly isolated nave or activated cd8 + t - lymphocytes treated with or without 500 m s-2hg for 7 days .

high - throughput screening has become a cornerstone in the drug discovery process , whereby active compounds are rapidly identified and used as starting points for drug development . although robust and reliable assays are crucial for the success of a high - throughput screening campaign , the most valuable asset in any screening center is the collection of compounds . the library not only represents a large capital investment but also may consist of scarce chemicals that have been synthesized in - house , purified from natural sources , or obtained from collaborators . to efficiently support screening projects because of the advancement in technology , facilities are exploiting the benefits of miniaturization , which include reducing the use of reagents , compounds , and other consumables . chemical collections are normally supplied in a 96-well format , which holds relatively large volumes . therefore , it is necessary to compress the samples to higher density formats ( eg , 384-well plates ) by transferring aliquots into suitable vessels in a process known as compound reformatting . automation can expedite this process , ensure consistency , and offer reliability . herein , we report our approach to reformat more than half a million compounds acquired from commercial sources . our strategy enables an efficient and robust workflow that simultaneously incorporates the replating , compression , and replication processes . the automated capper and decapper ( acd ) , central seal press ( csp ) , manual cap press , automated plate piercer , accumulator , mid size store ( mss ) , tube - based stbr96 , and stbr384 racks were acquired from remp ag ( switzerland , now part of nexus biosystems ) . the vprep conventional pipetting station , plateloc sealer , and 96lt 200 l pipette tips were obtained from velocity 11 ( now part of agilent technologies , santa clara , ca ) . polypropylene plates in a 384-well format ( pp384 ) were purchased from corning incorporated ( corning , ny ) . lpr240 carousel - style plate hotels and str240 automated incubators were purchased from liconic instruments ( woburn , ma ) . the automation system and its software ( cellario version 2.4 ) were developed by highres biosolutions ( woburn , ma ) . the compound reformatting process involves several operations that can be divided as follows : stage 1 ( replating ) and stage 2 ( compression and replication ) ( fig . 1 ) . the first stage entails generating replicates from each vendor plate and transferring them to a long - term storage format with an identical layout . the compound solutions can then be stored in individually capped tubes arranged in bar - coded racks in a 96-well format ( stbr96 tube racks ) . to achieve this , an acd capped / decapped the tubes of the stbr96 racks and then aliquots ( 200 l ) were transferred from the vendor plates by using a vprep liquid handling station that uses air - displacement technology and a 96lt pipettor head . to accelerate the workflow and minimize cost , the same pipette tips used in stage 1 were re - used in stage 2 to dispense their corresponding compounds ( 10 l ) into pp384 plates ( for primary screening ) and stbr384 tube racks ( for cherry - picking ) . in the stbr384 tube racks , after the transfer , a csp sealed each individual tube in the stbr384 racks with pierceable aluminum sheets . this compression stage condensed 4 vendor plates into a single 384-well plate with a checkered pattern ( fig . ( 2 ) shows the various instrument setups , particularly how we established a communication network between the devices and the scheduler s ( ie , cellario s ) control environment . each individual device / instrument was tested and optimized during the validation of the automation protocol ; trial - and - error approaches were often applied . the trial - and - error process involves the manual adjustment and calibration of the robots , the various automation components and instruments to ensure the proper labware movement on the deck . it is important to register each compound with a unique identifier and to plan for its tracking by customizing its bar code . to enable this , an alphanumeric check character was included to ensure the uniqueness of the bar code so that no repeated bar code was used in the sequence . preparing the system and samples before each automation run required a substantial amount of time ( fig . 3 ) . the stbr96 racks had to be inspected off - deck ( see below ) , and the samples / labware were scanned offline to be registered into an alternate database , which was then compared with the log file generated during the automation process . at the beginning of the run , the scheduler generated a unique run order number based on selected protocols that were previously created . the process was then simulated by making a gantt chart describing the steps and estimating the timing of each task . the vendor plates , the stbr96 racks , and the tip boxes were placed in 3 separate liconic incubators . a fourth liconic incubator was used exclusively to store the stbr384 racks and pp384 plates . incubators 3 and 4 performed the additional step of loading back the stbr96 , stbr384 , and pp384 copies . because liconic incubators can hold 11 stackers each , different densities of liconic stackers were used to hold different consumables : 22-level stackers for stbr384 tube racks and pp384 plates , 17-level stackers for stbr96 racks , and 8-level stackers for deep - well plates and pipette tip boxes . to initiate the reformatting process , the empty stbr96 racks were first moved to the acd where the tubes were decapped and scanned to detect bar codes before being transferred to the vprep pipetting station . after the liquid - pipetting cycle , the empty vendor plates and used tips were disposed . meanwhile , the stbr384 racks and the pp384 were sealed by the csp and the plateloc , respectively . finally , the vessels containing the reformatted compounds were sent back to their respective liconic incubators . the reformatting process was performed on a higres biosolution automated robotic deck featuring 2 stubli robotic arms , each with custom , end - effector , collision - sensing tools and a pair of pneumatic grippers that shuffles the labware components between stations . a dehumidifier kept the humidity less than 20% , minimizing water absorption of the dmso - dissolved compounds . a dry nitrogen line also controlled humidity and oxygen levels in the chamber , preventing degradation of the chemicals by oxidation . throughout the reformatting process cellario software was used as a dynamic time scheduler , which assigns operational steps , controls timing during the continuous batched - reformatting process , and uses barcodes to track each labware s location . protocols created using cellario optimize the trafficking on the deck on the basis of particular timings , delay and priority logics , instrument availability , and input event sequences . the automation log data and compound - mapping files of vendor plates and those of newly reformatted plates can be subsequently extracted . compound tracking throughout the process was accomplished by tracking well numbers , custom bar codes on each plate , and the unique identifiers of each compound in the collection . the replating and compression stages from a single run of a vendor plate generated 1 remp96 copy for long - term storage , 2 pp384 copies for use directly in primary screens , and 3 remp384 copies for cherry - picking and follow - up experiments . all resulting copies were maintained in a fully automated and humidity - controlled 20c storage facility ( remp mss ) . the facility houses a one - aisle robot equipped with rack grippers , a handling platform , and a punching device , allowing plate delivery and retrieval . this robot can also hand in selected tubes from stbr96 and stbr384 racks . to minimize compound degradation , the reformatted plates are stored at -20 c under low humidity in the mss store , which is designed for long - term storage . in addition , the stbr96 and stbr384 rack systems allow for individual compound cherry - picking , minimizing the chances of compound degradation due to multiple freeze - thaw cycles . even though we utilize one of the most favorable conditions for long - term storage of compounds , degradation is still possible due to the labile nature of certain compounds . compounds that are known to be very reactive were filtered out and not included in our collection . the automation protocols created using cellario were first validated by performing dry runs ( i.e. runs without compounds or reagents ) . because the software has real - time monitoring capabilities , the labware location indicated by cellario was confirmed by visually tracking the location of the physical items on the deck . additionally , bar code and time records were extracted from the database to corroborate the location of the compounds within a given plate . fluorescence measurements using fluorescein isothiocyanate ( fitc ) were used to calculate liquid - dispensing variations among pipette tips and volume deviations among wells . additionally , the total volume of liquid delivered was monitored by measuring the weight of the transferred solutions . problems in the acd s capping and decapping steps could lead to system failure , resulting in cessation of all operations . to ensure that the caps were smoothly decapped and correctly recapped , each stbr96 and stbr384 plate was visually inspected for damage , and a dedicated stand - alone acd was used to manually perform the capping / decapping operations before loading the tube racks into the automation system many of the devices on the automation system require an air supply with steady pressure to ensure consistency during operations . we used dry air generated by an in - house air compressor and stored in reserve tanks to ensure that proper air pressure was supplied to each device . all devices were equipped with gages and valves for pressure control . to ensure continuous power during a reformatting process , an uninterruptible power supply battery for the automation system was wired in place to handle unexpected short - term power loss and sudden voltage fluctuations . the automated capper and decapper ( acd ) , central seal press ( csp ) , manual cap press , automated plate piercer , accumulator , mid size store ( mss ) , tube - based stbr96 , and stbr384 racks were acquired from remp ag ( switzerland , now part of nexus biosystems ) . the vprep conventional pipetting station , plateloc sealer , and 96lt 200 l pipette tips were obtained from velocity 11 ( now part of agilent technologies , santa clara , ca ) . polypropylene plates in a 384-well format ( pp384 ) were purchased from corning incorporated ( corning , ny ) . lpr240 carousel - style plate hotels and str240 automated incubators were purchased from liconic instruments ( woburn , ma ) . the automation system and its software ( cellario version 2.4 ) were developed by highres biosolutions ( woburn , ma ) . the compound reformatting process involves several operations that can be divided as follows : stage 1 ( replating ) and stage 2 ( compression and replication ) ( fig . 1 ) . the first stage entails generating replicates from each vendor plate and transferring them to a long - term storage format with an identical layout . the compound solutions can then be stored in individually capped tubes arranged in bar - coded racks in a 96-well format ( stbr96 tube racks ) . to achieve this , an acd capped / decapped the tubes of the stbr96 racks and then aliquots ( 200 l ) were transferred from the vendor plates by using a vprep liquid handling station that uses air - displacement technology and a 96lt pipettor head . to accelerate the workflow and minimize cost , the same pipette tips used in stage 1 were re - used in stage 2 to dispense their corresponding compounds ( 10 l ) into pp384 plates ( for primary screening ) and stbr384 tube racks ( for cherry - picking ) . in the stbr384 tube racks , after the transfer , a csp sealed each individual tube in the stbr384 racks with pierceable aluminum sheets . this compression stage condensed 4 vendor plates into a single 384-well plate with a checkered pattern ( fig . ( 2 ) shows the various instrument setups , particularly how we established a communication network between the devices and the scheduler s ( ie , cellario s ) control environment . each individual device / instrument was tested and optimized during the validation of the automation protocol ; trial - and - error approaches were often applied . the trial - and - error process involves the manual adjustment and calibration of the robots , the various automation components and instruments to ensure the proper labware movement on the deck . it is important to register each compound with a unique identifier and to plan for its tracking by customizing its bar code . to enable this , an alphanumeric check character was included to ensure the uniqueness of the bar code so that no repeated bar code was used in the sequence . preparing the system and samples before each automation run required a substantial amount of time ( fig . 3 ) . the stbr96 racks had to be inspected off - deck ( see below ) , and the samples / labware were scanned offline to be registered into an alternate database , which was then compared with the log file generated during the automation process . at the beginning of the run , the scheduler generated a unique run order number based on selected protocols that were previously created . the process was then simulated by making a gantt chart describing the steps and estimating the timing of each task . the vendor plates , the stbr96 racks , and the tip boxes were placed in 3 separate liconic incubators . a fourth liconic incubator was used exclusively to store the stbr384 racks and pp384 plates . incubators 3 and 4 performed the additional step of loading back the stbr96 , stbr384 , and pp384 copies . because liconic incubators can hold 11 stackers each , different densities of liconic stackers were used to hold different consumables : 22-level stackers for stbr384 tube racks and pp384 plates , 17-level stackers for stbr96 racks , and 8-level stackers for deep - well plates and pipette tip boxes . to initiate the reformatting process , the empty stbr96 racks were first moved to the acd where the tubes were decapped and scanned to detect bar codes before being transferred to the vprep pipetting station . after the liquid - pipetting cycle , the empty vendor plates and used tips were disposed . the stbr384 racks and the pp384 were sealed by the csp and the plateloc , respectively . finally , the vessels containing the reformatted compounds were sent back to their respective liconic incubators . the reformatting process was performed on a higres biosolution automated robotic deck featuring 2 stubli robotic arms , each with custom , end - effector , collision - sensing tools and a pair of pneumatic grippers that shuffles the labware components between stations . a dehumidifier kept the humidity less than 20% , minimizing water absorption of the dmso - dissolved compounds . a dry nitrogen line also controlled humidity and oxygen levels in the chamber , preventing degradation of the chemicals by oxidation . throughout the reformatting process cellario software was used as a dynamic time scheduler , which assigns operational steps , controls timing during the continuous batched - reformatting process , and uses barcodes to track each labware s location . protocols created using cellario optimize the trafficking on the deck on the basis of particular timings , delay and priority logics , instrument availability , and input event sequences . the automation log data and compound - mapping files of vendor plates and those of newly reformatted plates can be subsequently extracted . compound tracking throughout the process was accomplished by tracking well numbers , custom bar codes on each plate , and the unique identifiers of each compound in the collection . the replating and compression stages from a single run of a vendor plate generated 1 remp96 copy for long - term storage , 2 pp384 copies for use directly in primary screens , and 3 remp384 copies for cherry - picking and follow - up experiments . all resulting copies were maintained in a fully automated and humidity - controlled 20c storage facility ( remp mss ) . the facility houses a one - aisle robot equipped with rack grippers , a handling platform , and a punching device , allowing plate delivery and retrieval . this robot can also hand in selected tubes from stbr96 and stbr384 racks . to minimize compound degradation , the reformatted plates are stored at -20 c under low humidity in the mss store , which is designed for long - term storage . in addition , the stbr96 and stbr384 rack systems allow for individual compound cherry - picking , minimizing the chances of compound degradation due to multiple freeze - thaw cycles . even though we utilize one of the most favorable conditions for long - term storage of compounds , degradation is still possible due to the labile nature of certain compounds . compounds that are known to be very reactive were filtered out and not included in our collection . the automation protocols created using cellario were first validated by performing dry runs ( i.e. runs without compounds or reagents ) . because the software has real - time monitoring capabilities , the labware location indicated by cellario was confirmed by visually tracking the location of the physical items on the deck . additionally , bar code and time records were extracted from the database to corroborate the location of the compounds within a given plate . fluorescence measurements using fluorescein isothiocyanate ( fitc ) were used to calculate liquid - dispensing variations among pipette tips and volume deviations among wells . additionally , the total volume of liquid delivered was monitored by measuring the weight of the transferred solutions . problems in the acd s capping and decapping steps could lead to system failure , resulting in cessation of all operations . to ensure that the caps were smoothly decapped and correctly recapped , each stbr96 and stbr384 plate was visually inspected for damage , and a dedicated stand - alone acd was used to manually perform the capping / decapping operations before loading the tube racks into the automation system many of the devices on the automation system require an air supply with steady pressure to ensure consistency during operations . we used dry air generated by an in - house air compressor and stored in reserve tanks to ensure that proper air pressure was supplied to each device . all devices were equipped with gages and valves for pressure control . to ensure continuous power during a reformatting process , an uninterruptible power supply battery for the automation system was wired in place to handle unexpected short - term power loss and sudden voltage fluctuations . in a span of 42 weeks , we were able to reformat approximately 5.2 10 compounds ( fig . the first few weeks were allocated to performing pilot reformatting before increasing the throughput , reaching a maximal output of 55 040 compounds per week by week 16 . for a reformatting run involving 88 vendor plates , the entire reformatting process took 9 h. cellario s dynamic scheduling capabilities reduced the process by 6 h compared to a static method , which would have taken 15 h for completion ( fig . the specific operations follow a linear succession , and the system is on hold until a specific task is concluded . in contrast , in a dynamic environment , the workflow progresses based on resource allocation ( eg , when labware is being handled by a specific device , the robotic arms shuffle other items to optimize time ) . the time saved by using a dynamic approach instead of a static one increases as the number of plates increases from 24 to 88 ( fig . however , knowing that a system failure could lead to loss of a significant number of compounds , it is advisable to have somebody monitor the entire process to enable prompt troubleshooting and error recovery . therefore , the throughput is limited to some extent by the availability of such a designated monitor . out of a total of 8251 vendor plates , 23 plates were lost or discarded due to automation error , resulting in an overall failure rate of 0.28% . the movement of the stubli robotic arms accounted for 46 % of the time needed to complete the entire protocol ( fig . time in incubators 1 and 2 accounted for only 4% of the total time , and time in incubators 3 and 4 accounted for 8% . notably , capping / decapping of the stbr96 by the acd , sealing of stbr384 by the csp , and sealing of the pp384 screening plates by the plateloc occupied only a small fraction of the total time . the relative time for each task remained virtually constant when the number of vendor plates was increased from 24 to 88 because there is a proportional increase in every task as the number of plates increases ( fig . one stbr96 copy , 2 pp384 copies , and 3 stbr384 copies were generated from each vendor plate . although only 1 copy of stbr96 was created , this step took 27 % of the total process time ( fig . because four 96-well vendor plates were compressed into a single 384-well plate or rack , 96-well labware were shuffled by the robotic arms to the vprep station 4 times for every 384-well vessel , which remained on designated vprep shelves until the 4-cycle process was completed . in our 384-well , screening - ready formats , columns 1 , 2 , 13 , and 14 correspond with empty wells of the original vendor plates and are reserved for control or reference samples needed for subsequent biologic assays . preserving compound integrity is vital for the quality and reliability of screening campaigns and follow - up confirmations [ 5 , 7 ] . therefore , our approach was to store each sample in individual tubes by using remp tube technology consumables : only those compounds of interest are taken out of the 20c store facility . in comparison to the use of traditional microtiter plates , the tube system reduces multiple freeze - thaw cycles of irrelevant compounds and decreases the chance of cross - contamination . therefore , compounds for long - term storage and cherry - picking are stocked in stbr96 and stbr384 , respectively . this standard reformatting protocol was also developed to generate compound copies to be used directly for screening in a 384-well format ( pp384 ) . the screening copies can be ordered from the mss and returned when the screening is complete , with freeze - and - thaw cycles automatically recorded by the system . condensing compounds from 96-well plates to higher density vessels conserves reagents , compounds , and labware while reducing screening time . therefore , the effort invested in compressing compounds can be advantageous in the long term , particularly in the case of a major project involving millions of compounds . concomitantly executing the replating , compression , and replication stages second , operating costs can be reduced by reusing the pipette tips when the same compounds are transferred during different stages . additionally , having all the compounds readily available in a consistent layout facilitates the screening process , data analysis , and interpretation . in general however , this would be at the expense of increasing the chances of dropping plates or racks from the grippers , which would squander precious chemicals . additionally , the vprep pipetting station had a limited number of shelves , constraining the amount of labware that it could handle . therefore , integrating a second pipetting station could reduce the burden of the robotic arms and increase the throughput . however , this approach would introduce the risk of triggering system deadlock or a traffic jam . the 2 robots that were programmed to move 5 different types of labware among 8 locations consumed nearly half of the total process time . the routes between these different devices , as well as robot acceleration and deceleration can be further optimized . in addition , changes in the layout of the stations on the deck can increase process speed . to increase the throughput , we used a dynamic software environment and concurrently executed the replating , compression , and replication processes . system maintenance , compound registration into the database , and labware restocking were some of the factors that prevented a constant throughput . most academic institutions perform the reformatting process in a semi - automation and static mode , generating only 384pp plates for long - term and immediate usage . this approach is more economically feasible , but it increases the likelihood for human error , cross - contamination , compound degradation , while increasing process time . some of the screening centers in the private sector employ strategies that focus on the long - term stability of the compounds by storing them solely in individual tubes as solutions or dry powder , which result in significant costs and the lack of screening copies for immediate use . our strategy incorporates elements from the above - mentioned methods to generate concomitantly multiple copies for short and long - term use , while minimizing cost and process time . the compound repository of st . jude children s research hospital is composed of over half a million compounds , with a growing number from both external and internal sources . reformatted compound plates resulting from the procedure described in this report have been used in 9 full - deck ( ie , entire library ) and 12 partial - deck screening campaigns . this method can be applied in institutions with limited resources if the dynamic process used simulates a priority to optimize the timing of events ; therefore , our experience completing this major task is of use to emerging screening facilities , particularly those in academic institutions .

large - scale screening of small organic compounds has become a standard and essential practice in the early discovery of chemical entities with potential therapeutic use . to effectively support high - throughput screening campaigns , compound collections have to be in suitable formats , which requires a process known as compound reformatting . here we report our approach to reformat the newly - established chemical repository of a large - scale screening facility at st . jude children s research hospital , which comprises more than half a million compounds , mostly from commercial sources . we highlight the timeline for a reformatting process , the importance of standardizing the operational procedures , and the advantages and disadvantages of using automation . the end result of our reformatting process is the concurrent generation of copies for long - term storage , screening , and cherry - picking ; all of which facilitate compound management and high - throughput screening .

INTRODUCTION MATERIALS AND METHODOLOGY Instrumentation and Consumables Reformatting Process Quality Control and Performance RESULTS DISCUSSION CONFLICT OF INTEREST

high - throughput screening has become a cornerstone in the drug discovery process , whereby active compounds are rapidly identified and used as starting points for drug development . although robust and reliable assays are crucial for the success of a high - throughput screening campaign , the most valuable asset in any screening center is the collection of compounds . the library not only represents a large capital investment but also may consist of scarce chemicals that have been synthesized in - house , purified from natural sources , or obtained from collaborators . to efficiently support screening projects because of the advancement in technology , facilities are exploiting the benefits of miniaturization , which include reducing the use of reagents , compounds , and other consumables . chemical collections are normally supplied in a 96-well format , which holds relatively large volumes . therefore , it is necessary to compress the samples to higher density formats ( eg , 384-well plates ) by transferring aliquots into suitable vessels in a process known as compound reformatting . automation can expedite this process , ensure consistency , and offer reliability . herein , we report our approach to reformat more than half a million compounds acquired from commercial sources . our strategy enables an efficient and robust workflow that simultaneously incorporates the replating , compression , and replication processes . the automated capper and decapper ( acd ) , central seal press ( csp ) , manual cap press , automated plate piercer , accumulator , mid size store ( mss ) , tube - based stbr96 , and stbr384 racks were acquired from remp ag ( switzerland , now part of nexus biosystems ) . the vprep conventional pipetting station , plateloc sealer , and 96lt 200 l pipette tips were obtained from velocity 11 ( now part of agilent technologies , santa clara , ca ) . polypropylene plates in a 384-well format ( pp384 ) were purchased from corning incorporated ( corning , ny ) . the compound reformatting process involves several operations that can be divided as follows : stage 1 ( replating ) and stage 2 ( compression and replication ) ( fig . the first stage entails generating replicates from each vendor plate and transferring them to a long - term storage format with an identical layout . to accelerate the workflow and minimize cost , the same pipette tips used in stage 1 were re - used in stage 2 to dispense their corresponding compounds ( 10 l ) into pp384 plates ( for primary screening ) and stbr384 tube racks ( for cherry - picking ) . in the stbr384 tube racks , after the transfer , a csp sealed each individual tube in the stbr384 racks with pierceable aluminum sheets . ( 2 ) shows the various instrument setups , particularly how we established a communication network between the devices and the scheduler s ( ie , cellario s ) control environment . the trial - and - error process involves the manual adjustment and calibration of the robots , the various automation components and instruments to ensure the proper labware movement on the deck . it is important to register each compound with a unique identifier and to plan for its tracking by customizing its bar code . to enable this , an alphanumeric check character was included to ensure the uniqueness of the bar code so that no repeated bar code was used in the sequence . the stbr96 racks had to be inspected off - deck ( see below ) , and the samples / labware were scanned offline to be registered into an alternate database , which was then compared with the log file generated during the automation process . at the beginning of the run , the scheduler generated a unique run order number based on selected protocols that were previously created . the vendor plates , the stbr96 racks , and the tip boxes were placed in 3 separate liconic incubators . a fourth liconic incubator was used exclusively to store the stbr384 racks and pp384 plates . incubators 3 and 4 performed the additional step of loading back the stbr96 , stbr384 , and pp384 copies . because liconic incubators can hold 11 stackers each , different densities of liconic stackers were used to hold different consumables : 22-level stackers for stbr384 tube racks and pp384 plates , 17-level stackers for stbr96 racks , and 8-level stackers for deep - well plates and pipette tip boxes . to initiate the reformatting process , the empty stbr96 racks were first moved to the acd where the tubes were decapped and scanned to detect bar codes before being transferred to the vprep pipetting station . after the liquid - pipetting cycle , the empty vendor plates and used tips were disposed . meanwhile , the stbr384 racks and the pp384 were sealed by the csp and the plateloc , respectively . finally , the vessels containing the reformatted compounds were sent back to their respective liconic incubators . the reformatting process was performed on a higres biosolution automated robotic deck featuring 2 stubli robotic arms , each with custom , end - effector , collision - sensing tools and a pair of pneumatic grippers that shuffles the labware components between stations . a dry nitrogen line also controlled humidity and oxygen levels in the chamber , preventing degradation of the chemicals by oxidation . throughout the reformatting process cellario software was used as a dynamic time scheduler , which assigns operational steps , controls timing during the continuous batched - reformatting process , and uses barcodes to track each labware s location . protocols created using cellario optimize the trafficking on the deck on the basis of particular timings , delay and priority logics , instrument availability , and input event sequences . compound tracking throughout the process was accomplished by tracking well numbers , custom bar codes on each plate , and the unique identifiers of each compound in the collection . the replating and compression stages from a single run of a vendor plate generated 1 remp96 copy for long - term storage , 2 pp384 copies for use directly in primary screens , and 3 remp384 copies for cherry - picking and follow - up experiments . all resulting copies were maintained in a fully automated and humidity - controlled 20c storage facility ( remp mss ) . the facility houses a one - aisle robot equipped with rack grippers , a handling platform , and a punching device , allowing plate delivery and retrieval . to minimize compound degradation , the reformatted plates are stored at -20 c under low humidity in the mss store , which is designed for long - term storage . in addition , the stbr96 and stbr384 rack systems allow for individual compound cherry - picking , minimizing the chances of compound degradation due to multiple freeze - thaw cycles . even though we utilize one of the most favorable conditions for long - term storage of compounds , degradation is still possible due to the labile nature of certain compounds . compounds that are known to be very reactive were filtered out and not included in our collection . because the software has real - time monitoring capabilities , the labware location indicated by cellario was confirmed by visually tracking the location of the physical items on the deck . additionally , the total volume of liquid delivered was monitored by measuring the weight of the transferred solutions . problems in the acd s capping and decapping steps could lead to system failure , resulting in cessation of all operations . to ensure that the caps were smoothly decapped and correctly recapped , each stbr96 and stbr384 plate was visually inspected for damage , and a dedicated stand - alone acd was used to manually perform the capping / decapping operations before loading the tube racks into the automation system many of the devices on the automation system require an air supply with steady pressure to ensure consistency during operations . to ensure continuous power during a reformatting process , an uninterruptible power supply battery for the automation system was wired in place to handle unexpected short - term power loss and sudden voltage fluctuations . the automated capper and decapper ( acd ) , central seal press ( csp ) , manual cap press , automated plate piercer , accumulator , mid size store ( mss ) , tube - based stbr96 , and stbr384 racks were acquired from remp ag ( switzerland , now part of nexus biosystems ) . the vprep conventional pipetting station , plateloc sealer , and 96lt 200 l pipette tips were obtained from velocity 11 ( now part of agilent technologies , santa clara , ca ) . the compound reformatting process involves several operations that can be divided as follows : stage 1 ( replating ) and stage 2 ( compression and replication ) ( fig . the first stage entails generating replicates from each vendor plate and transferring them to a long - term storage format with an identical layout . to accelerate the workflow and minimize cost , the same pipette tips used in stage 1 were re - used in stage 2 to dispense their corresponding compounds ( 10 l ) into pp384 plates ( for primary screening ) and stbr384 tube racks ( for cherry - picking ) . in the stbr384 tube racks , after the transfer , a csp sealed each individual tube in the stbr384 racks with pierceable aluminum sheets . this compression stage condensed 4 vendor plates into a single 384-well plate with a checkered pattern ( fig . ( 2 ) shows the various instrument setups , particularly how we established a communication network between the devices and the scheduler s ( ie , cellario s ) control environment . the trial - and - error process involves the manual adjustment and calibration of the robots , the various automation components and instruments to ensure the proper labware movement on the deck . to enable this , an alphanumeric check character was included to ensure the uniqueness of the bar code so that no repeated bar code was used in the sequence . 3 ) . the stbr96 racks had to be inspected off - deck ( see below ) , and the samples / labware were scanned offline to be registered into an alternate database , which was then compared with the log file generated during the automation process . at the beginning of the run , the scheduler generated a unique run order number based on selected protocols that were previously created . the vendor plates , the stbr96 racks , and the tip boxes were placed in 3 separate liconic incubators . incubators 3 and 4 performed the additional step of loading back the stbr96 , stbr384 , and pp384 copies . because liconic incubators can hold 11 stackers each , different densities of liconic stackers were used to hold different consumables : 22-level stackers for stbr384 tube racks and pp384 plates , 17-level stackers for stbr96 racks , and 8-level stackers for deep - well plates and pipette tip boxes . to initiate the reformatting process , the empty stbr96 racks were first moved to the acd where the tubes were decapped and scanned to detect bar codes before being transferred to the vprep pipetting station . after the liquid - pipetting cycle , the empty vendor plates and used tips were disposed . the stbr384 racks and the pp384 were sealed by the csp and the plateloc , respectively . finally , the vessels containing the reformatted compounds were sent back to their respective liconic incubators . the reformatting process was performed on a higres biosolution automated robotic deck featuring 2 stubli robotic arms , each with custom , end - effector , collision - sensing tools and a pair of pneumatic grippers that shuffles the labware components between stations . a dry nitrogen line also controlled humidity and oxygen levels in the chamber , preventing degradation of the chemicals by oxidation . throughout the reformatting process cellario software was used as a dynamic time scheduler , which assigns operational steps , controls timing during the continuous batched - reformatting process , and uses barcodes to track each labware s location . protocols created using cellario optimize the trafficking on the deck on the basis of particular timings , delay and priority logics , instrument availability , and input event sequences . compound tracking throughout the process was accomplished by tracking well numbers , custom bar codes on each plate , and the unique identifiers of each compound in the collection . the replating and compression stages from a single run of a vendor plate generated 1 remp96 copy for long - term storage , 2 pp384 copies for use directly in primary screens , and 3 remp384 copies for cherry - picking and follow - up experiments . the facility houses a one - aisle robot equipped with rack grippers , a handling platform , and a punching device , allowing plate delivery and retrieval . to minimize compound degradation , the reformatted plates are stored at -20 c under low humidity in the mss store , which is designed for long - term storage . in addition , the stbr96 and stbr384 rack systems allow for individual compound cherry - picking , minimizing the chances of compound degradation due to multiple freeze - thaw cycles . even though we utilize one of the most favorable conditions for long - term storage of compounds , degradation is still possible due to the labile nature of certain compounds . compounds that are known to be very reactive were filtered out and not included in our collection . runs without compounds or reagents ) . because the software has real - time monitoring capabilities , the labware location indicated by cellario was confirmed by visually tracking the location of the physical items on the deck . additionally , the total volume of liquid delivered was monitored by measuring the weight of the transferred solutions . problems in the acd s capping and decapping steps could lead to system failure , resulting in cessation of all operations . to ensure that the caps were smoothly decapped and correctly recapped , each stbr96 and stbr384 plate was visually inspected for damage , and a dedicated stand - alone acd was used to manually perform the capping / decapping operations before loading the tube racks into the automation system many of the devices on the automation system require an air supply with steady pressure to ensure consistency during operations . to ensure continuous power during a reformatting process , an uninterruptible power supply battery for the automation system was wired in place to handle unexpected short - term power loss and sudden voltage fluctuations . in a span of 42 weeks , we were able to reformat approximately 5.2 10 compounds ( fig . for a reformatting run involving 88 vendor plates , the entire reformatting process took 9 h. cellario s dynamic scheduling capabilities reduced the process by 6 h compared to a static method , which would have taken 15 h for completion ( fig . the specific operations follow a linear succession , and the system is on hold until a specific task is concluded . in contrast , in a dynamic environment , the workflow progresses based on resource allocation ( eg , when labware is being handled by a specific device , the robotic arms shuffle other items to optimize time ) . the time saved by using a dynamic approach instead of a static one increases as the number of plates increases from 24 to 88 ( fig . however , knowing that a system failure could lead to loss of a significant number of compounds , it is advisable to have somebody monitor the entire process to enable prompt troubleshooting and error recovery . therefore , the throughput is limited to some extent by the availability of such a designated monitor . out of a total of 8251 vendor plates , 23 plates were lost or discarded due to automation error , resulting in an overall failure rate of 0.28% . time in incubators 1 and 2 accounted for only 4% of the total time , and time in incubators 3 and 4 accounted for 8% . notably , capping / decapping of the stbr96 by the acd , sealing of stbr384 by the csp , and sealing of the pp384 screening plates by the plateloc occupied only a small fraction of the total time . one stbr96 copy , 2 pp384 copies , and 3 stbr384 copies were generated from each vendor plate . although only 1 copy of stbr96 was created , this step took 27 % of the total process time ( fig . because four 96-well vendor plates were compressed into a single 384-well plate or rack , 96-well labware were shuffled by the robotic arms to the vprep station 4 times for every 384-well vessel , which remained on designated vprep shelves until the 4-cycle process was completed . in our 384-well , screening - ready formats , columns 1 , 2 , 13 , and 14 correspond with empty wells of the original vendor plates and are reserved for control or reference samples needed for subsequent biologic assays . preserving compound integrity is vital for the quality and reliability of screening campaigns and follow - up confirmations [ 5 , 7 ] . therefore , our approach was to store each sample in individual tubes by using remp tube technology consumables : only those compounds of interest are taken out of the 20c store facility . in comparison to the use of traditional microtiter plates , the tube system reduces multiple freeze - thaw cycles of irrelevant compounds and decreases the chance of cross - contamination . therefore , compounds for long - term storage and cherry - picking are stocked in stbr96 and stbr384 , respectively . this standard reformatting protocol was also developed to generate compound copies to be used directly for screening in a 384-well format ( pp384 ) . condensing compounds from 96-well plates to higher density vessels conserves reagents , compounds , and labware while reducing screening time . therefore , the effort invested in compressing compounds can be advantageous in the long term , particularly in the case of a major project involving millions of compounds . concomitantly executing the replating , compression , and replication stages second , operating costs can be reduced by reusing the pipette tips when the same compounds are transferred during different stages . additionally , having all the compounds readily available in a consistent layout facilitates the screening process , data analysis , and interpretation . in general however , this would be at the expense of increasing the chances of dropping plates or racks from the grippers , which would squander precious chemicals . additionally , the vprep pipetting station had a limited number of shelves , constraining the amount of labware that it could handle . however , this approach would introduce the risk of triggering system deadlock or a traffic jam . the routes between these different devices , as well as robot acceleration and deceleration can be further optimized . in addition , changes in the layout of the stations on the deck can increase process speed . to increase the throughput , we used a dynamic software environment and concurrently executed the replating , compression , and replication processes . system maintenance , compound registration into the database , and labware restocking were some of the factors that prevented a constant throughput . most academic institutions perform the reformatting process in a semi - automation and static mode , generating only 384pp plates for long - term and immediate usage . this approach is more economically feasible , but it increases the likelihood for human error , cross - contamination , compound degradation , while increasing process time . some of the screening centers in the private sector employ strategies that focus on the long - term stability of the compounds by storing them solely in individual tubes as solutions or dry powder , which result in significant costs and the lack of screening copies for immediate use . our strategy incorporates elements from the above - mentioned methods to generate concomitantly multiple copies for short and long - term use , while minimizing cost and process time . the compound repository of st . jude children s research hospital is composed of over half a million compounds , with a growing number from both external and internal sources . reformatted compound plates resulting from the procedure described in this report have been used in 9 full - deck ( ie , entire library ) and 12 partial - deck screening campaigns . this method can be applied in institutions with limited resources if the dynamic process used simulates a priority to optimize the timing of events ; therefore , our experience completing this major task is of use to emerging screening facilities , particularly those in academic institutions .

human metapneumovirus ( hmpv ) is a paramyxovirus first identified in 2001 as a pathogen associated with upper and lower respiratory tract infections in children . seroprevalence studies show that almost all children acquire infection by 5 years of age , and that frequent reinfections occur through life [ 2 , 3 ] . the prevalence of hmpv infection among hospitalized children with acute respiratory infection or fever is similar to that of other respiratory viruses . clinical disease most closely resembles that associated with respiratory syncytial virus infection ( rsv ) , a related paramyxovirus . a prospective cohort study of hmpv revealed a 9.4% seroprevalence in children hospitalized with respiratory tract infections , with 63% of patients requiring supplemental oxygen and 3% requiring intensive care unit ( icu ) admission . lower respiratory tract illnesses ( lrti ) caused by hmpv includes bronchiolitis , pneumonia , and croup . hmpv is also associated with acute otitis media , as well as more rarely conjunctivitis , gastroenteritis , and rash [ 6 , 7 ] . in adults with hematologic malignancy , hmpv is associated with high rates of progression from upper to lower respiratory tract disease and substantial mortality [ 8 , 9 ] . animal data support the use of bronchodilators and corticosteroids , but no controlled trials have been done to assess their efficacy in human populations . ribavirin is a nucleoside analogue shown in in - vitro studies to have activity against hmpv . ribavirin is approved for use in treatment of respiratory syncytial virus ( rsv ) , and is often used in combination with intravenous immunoglobulin ( ivig ) in severely immunocompromised individuals . few prior descriptions of the clinical management and treatment of hmpv respiratory tract infections in a pediatric immunocompromised population , including children with leukemia and solid organ transplant recipients , are available . we describe hmpv respiratory tract infections in 55 pediatric immunocompromised patients at seattle children 's hospital , reporting on their clinical presentation , management , and outcomes . screening of all positive laboratory results for hmpv by both direct fluorescent antibody ( dfa ) and real - time reverse - transcriptase polymerase chain reaction ( rt - qpcr ) was performed to identify patients between the ages of newborn to 19 years who were diagnosed at seattle children 's hospital in seattle , wa during the years 20062010 . of 368 total patients with positive hmpv laboratory results , we performed retrospective chart review to identify a subset of 55 patients with immunocompromised conditions . we defined an immunocompromised condition as presence of hematologic malignancy , solid tumor , rheumatologic disease receiving immunosuppressive therapy , solid organ transplant , primary immunodeficiency , receipt of chronic immunosuppressive therapy , or receipt of a hematopoietic stem cell transplant ( hsct ) . using electronic chart review , we abstracted sociodemographic variables , symptoms , laboratory and radiologic values , clinical course , and treatment outcomes for these patients . respiratory specimens were obtained by nasal washes for patients with suspected respiratory viral infections by attending physicians , or from bronchoalveolar lavage fluid ( bal ) when this was performed . dfa was performed using virus - specific mouse monoclonal antibodies ( chemicon , temecula , ca ) , and rt - qpcr was performed using previously published methods at the university of washington virology laboratories . viral load values were obtained on a subset of patients whose samples were tested by rt - qpcr . estimated viral load data were calculated from cycle threshold values on rt - qpcr analysis using stored standard curve data for hmpv . upper respiratory tract infection ( urti ) was defined as hmpv documented in an upper respiratory tract specimen in a patient with compatible symptoms in the absence of radiographic or clinical evidence of pneumonia . lrti was defined as a new pulmonary infiltrate or presence of lower respiratory tract symptoms ( wheezing or hypoxia ) in association with a positive lower respiratory tract specimen or a positive upper respiratory tract specimen if the patient did not undergo bal . neutropenia was defined as absolute neutrophil count < 1000 cells / ml . lymphopenia was defined as lymphocyte count < 300 cells / ml ( not age - adjusted ) , and severe lymphopenia as lymphocyte count < 100 cells / ml . infections were defined as nosocomial if the patient was hospitalized within 7 days of diagnosis , and otherwise classified as community acquired . severe disease was defined as requiring icu stay and/or use of supplemental oxygen fio2 0.28 . mortality attributable to hmpv was defined as death due to respiratory failure from pneumonia during the hospitalization where hmpv was diagnosed , and hmpv was considered a contributor to lung injury . data were entered into a password - protected excel spreadsheet , and analyzed using stata 11.0 ( stata corp , college station , tx ) . comparison of clinical characteristics was performed using fisher 's exact tests for categorical variables and t tests with unequal variance for continuous variables . overall , 55 hmpv - infected immunocompromised patients were identified from may 29 , 2006 to march 26 , 2010 . the median age was 5 years ( range , 5 months19 years ) , and the majority had a hematologic malignancy as an underlying condition . of these , most had acute lymphoblastic leukemia ( all ) ( n = 21 ; 88% ) . the nine solid tumors included wilms tumor ( n = 3 ; 33% ) , osteosarcoma ( n = 1 ; 11% ) , and ovarian cancer ( n = 1 ; 11% ) . indications for transplant included aplastic anemia ( n = 4 ; 44% ) and severe combined immunodeficiency ( scid ) ( n = 2 ; 22% ) . eight ( 15% ) patients were solid organ transplant recipients , and all were receiving immunosuppressive therapy . eight ( 15% ) hmpv infections were acquired nosocomially , while 46 ( 85% ) were community acquired . of these 46 cases , 24 ( 52% ) data are shown as number ( n [ % ] ) unless otherwise indicatedcharacteristicsn = 5age in years , median ( range)5 ( 0.419)female sex26 ( 47)underlying condition hematologic malignancy24 ( 44 ) all21 ( 88 ) hsct recipient9 ( 16 ) pre - transplant3 ( 33 ) post - transplant6 ( 67 ) solid organ transplant8 ( 15 ) heart4 ( 50 ) kidney1 ( 13 ) liver3 ( 38 ) solid tumors9 ( 16 ) primary immunodeficiency3 ( 5 ) other3 ( 5)chemotherapy recipient ( n = 29)29 ( 100)steroid use12 ( 22)season of infection winter ( dec feb)19 ( 35 ) spring ( march may)32 ( 58)community acquired46 ( 85)nosocomial8 ( 15)in patients with hematologic malignancy or solid tumors.defined as outpatients at the time of hmpv diagnosis . data are shown as number ( n [ % ] ) unless otherwise indicated in patients with hematologic malignancy or solid tumors . defined as outpatients at the time of hmpv diagnosis . the overall characteristics of the hmpv illness episodes are listed in table 2 . the majority of patients presented with fever ( n = 44 ; 80% ) and/or cough ( n = 42 ; 82% ) . lymphopenia was present in 7 ( 13% ) patients at diagnosis , and neutropenia was present in 19 ( 35% ) . of the 35 patients who presented with urti symptoms alone , only 2 ( 6% ) progressed to lrti . as compared to those presenting with fever or urti , children presenting with lrtis did not differ by age ( p = .80 ) or presence of fever ( p = .70 ) , cough ( p = 1.00 ) , neutropenia ( p = .21 ) , or lymphopenia ( p = .09 ) . table 2.characteristics of human metapneumovirus illness episodesnumber of infections55severe disease8 ( 15)neutropenia ( anc < 1000)19 ( 35)lymphopenia ( alc < 300 ) [ n = 54]7 ( 13)severe lymphopenia ( alc < 100 ) [ n = 54]5 ( 9)clinical symptoms cough [ n = 51]42 ( 86 ) fever44 ( 80)stage at presentation asymptomatic1 ( 2 ) fever3 ( 5 ) upper respiratory tract infection35 ( 64 ) lower respiratory tract infection16 ( 29)abnormal chest imaging [ n = 36]16 ( 44)median initial viral load in log10 copies / ml ( range ) [ n = 6]6.96 ( 2.757.22)diagnostic modality direct fluorescent antibody54 ( 98 ) polymerase chain reaction6 ( 11)co - infections12 ( 22 ) adenovirus3 ( 5 ) cytomegalovirus3 ( 5 ) epstein barr virus1 ( 2 ) influenza b1 ( 2 ) parainfluenza1(2 ) human herpes virus81 ( 2 ) rhinovirus1 ( 2 ) staphylococcus3 ( 7 ) other3 ( 7)hospitalization required [ n = 46]24 ( 52)treatment received ribavirin and intravenous immunoglobulin5 ( 9 ) ribavirin alone2 ( 4 ) intravenous immunoglobulin alone2 ( 4)receipt of antibiotics22 ( 40)intensive care unit stay [ n = 52]6 ( 12)receipt of supplemental oxygen [ n = 54]9 ( 17)severe disease [ n = 52]12 ( 23)death attributed to human metapneumovirus infection3 ( 5)of the 46 patients who were outpatient at the time of diagnosis.severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . characteristics of human metapneumovirus illness episodes of the 46 patients who were outpatient at the time of diagnosis . severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . samples tested included nasal wash ( n = 43 ; 91% ) , bal ( n = 1 ; 2% ) , or both ( n = 3 ; 6% ) . diagnosis of hmpv was made by dfa alone in 49 ( 89% ) patients , by pcr alone in 1 patient ( 2% ) , and by dfa and pcr in 5 ( 9% ) patients . twelve ( 23% ) patients met criteria for severe disease , 6 ( 50% ) of whom were admitted to the intensive care unit ( icu ) . those with severe disease were more likely to be neutropenic ( p = .02 ) , but otherwise did not differ by age ( p = .27 ) , hsct recipient status ( p = .19 ) , or presence of lymphopenia ( p = .09 ) . icu stay alone was not associated with age ( p = .59 ) or presence of neutropenia ( p = .38 ) , or lymphopenia ( p = .54 ) , though hsct recipients were more likely to require icu stay ( p < .01 ) . nine ( 16% ) patients were hsct recipients ; indications for transplant included aplastic anemia ( n = 4 , 44% ) , scid ( n = 2 , 22% ) , ewing sarcoma ( n = 1 , 11% ) , acute lymphoblastic leukemia ( all ) ( n = 1 , 11% ) , and osteopetrosis ( n = 1 , 11% ) . in this subset , 4 ( 50% ) had abnormal chest imaging , and 1 ( 11% ) required supplemental oxygen . the chest radiograph and computed tomography imaging of 3 representative patients are illustrated in figure 1 ( patients 1 , 2 , and 3 ) . three ( 33% ) of these patients had a diagnosis of hmpv prior to transplant , including 2 children with scid and 1 with aplastic anemia . in general , the policy at our institution is to delay transplant if patients have respiratory tract infections ; however , in these 3 cases , it was felt that due to their underlying disease and lack of functioning immune system , these patients would not be able to clear their viral infection without hsct . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . ( b ) patient 2 is a 5-month - old girl with scid who died of respiratory failure secondary to hmpv pneumonia . ( c ) patient 3 is a 2-year - old boy with aplastic anemia who died of respiratory failure secondary to hmpv pneumonia . chest imaging findings of 3 patients . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . ( b ) patient 2 is a 5-month - old girl with scid who died of respiratory failure secondary to hmpv pneumonia . ( c ) patient 3 is a 2-year - old boy with aplastic anemia who died of respiratory failure secondary to hmpv pneumonia . the median time from transplant to hmpv infection was 12 months ( range , 557 months ) . three ( 50% ) of the 6 who had chest imaging performed had abnormalities on chest radiograph . none received treatment with ribavirin or ivig , and though there was 1 death , it was not attributed to hmpv infection . as compared to other immunocompromised patients , solid organ transplant recipients were no more likely to have fever ( p = .19 ) , cough ( p = .62 ) , abnormal chest imaging ( p = 1.00 ) , or to be classified as lrti disease on initial clinical presentation ( p = .35 ) . the median number of samples collected was 7 ( range , 244 samples ) , and the median duration of follow - up was 37 days ( range , 4562 days ) . types of samples were primarily nasal wash samples , but also included two bal , one sinus biopsy , and one lung biopsy sample in patient 1 , as well as 1 bal sample in patient 2 . median initial viral load was estimated as 6.96 log10 copies / ml ( range , 2.757.22 log10 copies / ml ) . the viral load data for all 6 patients are illustrated in figures 2a ( patient 1 ) and 2b ( patients 26 ) . in the 3 patients who died , the median time between initial and final sample was 7 days ( range , 220 days ) , and the median change in viral load was 1.31 log10 copies / ml ( range , 6.23 to 0.36 log10 copies / ml ) . for the 3 patients who survived , the median time to viral clearance was 43 days ( range , 4511 days ) . figure 2.(a ) viral loads in patient 1 ( 11-month - old with severe combined immunodeficiency ) over time . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . acute lymphoblastic leukemia ; mo , month ; scid , severe combined immunodeficiency syndrome ; yo , year old . ( a ) viral loads in patient 1 ( 11-month - old with severe combined immunodeficiency ) over time . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . acute lymphoblastic leukemia ; mo , month ; scid , severe combined immunodeficiency syndrome ; yo , year old . nine patients were treated for their hmpv infections ( median age 9 years ; range , 5 months19 years ) . these patients included 5 hsct recipients , 2 patients with hematologic malignancy , and 2 patients with solid tumors . ( n = 2 , 22% ) , scid ( n = 2 , 22% ) , and ewing sarcoma ( n = 1 , 11% ) . eight ( 89% ) of these treated patients had fever , 6 ( 75% ) had cough , and 7 ( 78% ) had abnormal chest imaging . five ( 56% ) were hospitalized in the icu , and 5 ( 56% ) received supplemental oxygen . five ( 56% ) patients received both ribavirin and ivig , 2 ( 22% ) received ribavirin alone , and 2 ( 22% ) received ivig alone . of the 5 hsct recipients , 3 ( 60% ) had hmpv detected prior to transplant ( median time prior to transplant : 43 days ; range , 9321 days ) , and 1 detected 11 days after transplant . treatment with ribavirin and ivig was initiated a median of 5 days after hmpv diagnosis ( range , 246 days ) , and several patients received multiple courses of ribavirin and ivig . treatment with ribavirin alone ( n = 2 ) was initiated 2 and 4 days after diagnosis , respectively . of the 7 patients who received ribavirin , 6 ( 86% ) received inhaled ribavirin at a dose of 2 g 3 times daily for a 5-day course , and 1 ( 14% ) received an 11-day course of intravenous ribavirin . two of these deaths occurred in hsct recipients who were diagnosed with hmpv prior to transplant ( figures 1 and 2b : patients 2 and 3 ) . one hsct recipient had not engrafted at time of death , while the other engrafted the day prior to death . the third death occurred in a patient with all and secondary acute myelogenous leukemia on salvage chemotherapy who did not receive hmpv - specific treatment ( figure 2b : patient 5 ) . hmpv was considered to be a contributing factor to her death , though the primary cause of death was thought to be progression of all . overall , 55 hmpv - infected immunocompromised patients were identified from may 29 , 2006 to march 26 , 2010 . the median age was 5 years ( range , 5 months19 years ) , and the majority had a hematologic malignancy as an underlying condition . of these , most had acute lymphoblastic leukemia ( all ) ( n = 21 ; 88% ) . the nine solid tumors included wilms tumor ( n = 3 ; 33% ) , osteosarcoma ( n = 1 ; 11% ) , and ovarian cancer ( n = 1 ; 11% ) . indications for transplant included aplastic anemia ( n = 4 ; 44% ) and severe combined immunodeficiency ( scid ) ( n = 2 ; 22% ) . eight ( 15% ) patients were solid organ transplant recipients , and all were receiving immunosuppressive therapy . eight ( 15% ) hmpv infections were acquired nosocomially , while 46 ( 85% ) were community acquired . of these 46 cases , 24 ( 52% ) data are shown as number ( n [ % ] ) unless otherwise indicatedcharacteristicsn = 5age in years , median ( range)5 ( 0.419)female sex26 ( 47)underlying condition hematologic malignancy24 ( 44 ) all21 ( 88 ) hsct recipient9 ( 16 ) pre - transplant3 ( 33 ) post - transplant6 ( 67 ) solid organ transplant8 ( 15 ) heart4 ( 50 ) kidney1 ( 13 ) liver3 ( 38 ) solid tumors9 ( 16 ) primary immunodeficiency3 ( 5 ) other3 ( 5)chemotherapy recipient ( n = 29)29 ( 100)steroid use12 ( 22)season of infection winter ( dec feb)19 ( 35 ) spring ( march may)32 ( 58)community acquired46 ( 85)nosocomial8 ( 15)in patients with hematologic malignancy or solid tumors.defined as outpatients at the time of hmpv diagnosis . data are shown as number ( n [ % ] ) unless otherwise indicated in patients with hematologic malignancy or solid tumors . defined as outpatients at the time of hmpv diagnosis . the overall characteristics of the hmpv illness episodes are listed in table 2 . the majority of patients presented with fever ( n = 44 ; 80% ) and/or cough ( n = 42 ; 82% ) . lymphopenia was present in 7 ( 13% ) patients at diagnosis , and neutropenia was present in 19 ( 35% ) . of the 35 patients who presented with urti symptoms alone , only 2 ( 6% ) progressed to lrti . as compared to those presenting with fever or urti , children presenting with lrtis did not differ by age ( p = .80 ) or presence of fever ( p = .70 ) , cough ( p = 1.00 ) , neutropenia ( p = .21 ) , or lymphopenia ( p = .09 ) . table 2.characteristics of human metapneumovirus illness episodesnumber of infections55severe disease8 ( 15)neutropenia ( anc < 1000)19 ( 35)lymphopenia ( alc < 300 ) [ n = 54]7 ( 13)severe lymphopenia ( alc < 100 ) [ n = 54]5 ( 9)clinical symptoms cough [ n = 51]42 ( 86 ) fever44 ( 80)stage at presentation asymptomatic1 ( 2 ) fever3 ( 5 ) upper respiratory tract infection35 ( 64 ) lower respiratory tract infection16 ( 29)abnormal chest imaging [ n = 36]16 ( 44)median initial viral load in log10 copies / ml ( range ) [ n = 6]6.96 ( 2.757.22)diagnostic modality direct fluorescent antibody54 ( 98 ) polymerase chain reaction6 ( 11)co - infections12 ( 22 ) adenovirus3 ( 5 ) cytomegalovirus3 ( 5 ) epstein barr virus1 ( 2 ) influenza b1 ( 2 ) parainfluenza1(2 ) human herpes virus81 ( 2 ) rhinovirus1 ( 2 ) staphylococcus3 ( 7 ) other3 ( 7)hospitalization required [ n = 46]24 ( 52)treatment received ribavirin and intravenous immunoglobulin5 ( 9 ) ribavirin alone2 ( 4 ) intravenous immunoglobulin alone2 ( 4)receipt of antibiotics22 ( 40)intensive care unit stay [ n = 52]6 ( 12)receipt of supplemental oxygen [ n = 54]9 ( 17)severe disease [ n = 52]12 ( 23)death attributed to human metapneumovirus infection3 ( 5)of the 46 patients who were outpatient at the time of diagnosis.severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . characteristics of human metapneumovirus illness episodes of the 46 patients who were outpatient at the time of diagnosis . severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . samples tested included nasal wash ( n = 43 ; 91% ) , bal ( n = 1 ; 2% ) , or both ( n = 3 ; 6% ) . diagnosis of hmpv was made by dfa alone in 49 ( 89% ) patients , by pcr alone in 1 patient ( 2% ) , and by dfa and pcr in 5 ( 9% ) patients . twelve ( 23% ) patients met criteria for severe disease , 6 ( 50% ) of whom were admitted to the intensive care unit ( icu ) . those with severe disease were more likely to be neutropenic ( p = .02 ) , but otherwise did not differ by age ( p = .27 ) , hsct recipient status ( p = .19 ) , or presence of lymphopenia ( p = .09 ) . icu stay alone was not associated with age ( p = .59 ) or presence of neutropenia ( p = .38 ) , or lymphopenia ( p = .54 ) , though hsct recipients were more likely to require icu stay ( p < .01 ) . nine ( 16% ) patients were hsct recipients ; indications for transplant included aplastic anemia ( n = 4 , 44% ) , scid ( n = 2 , 22% ) , ewing sarcoma ( n = 1 , 11% ) , acute lymphoblastic leukemia ( all ) ( n = 1 , 11% ) , and osteopetrosis ( n = 1 , 11% ) . in this subset , 4 ( 50% ) had abnormal chest imaging , and 1 ( 11% ) required supplemental oxygen . the chest radiograph and computed tomography imaging of 3 representative patients are illustrated in figure 1 ( patients 1 , 2 , and 3 ) . three ( 33% ) of these patients had a diagnosis of hmpv prior to transplant , including 2 children with scid and 1 with aplastic anemia . in general , the policy at our institution is to delay transplant if patients have respiratory tract infections ; however , in these 3 cases , it was felt that due to their underlying disease and lack of functioning immune system , these patients would not be able to clear their viral infection without hsct . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . ( b ) patient 2 is a 5-month - old girl with scid who died of respiratory failure secondary to hmpv pneumonia . ( c ) patient 3 is a 2-year - old boy with aplastic anemia who died of respiratory failure secondary to hmpv pneumonia . chest imaging findings of 3 patients . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . ( b ) patient 2 is a 5-month - old girl with scid who died of respiratory failure secondary to hmpv pneumonia . ( c ) patient 3 is a 2-year - old boy with aplastic anemia who died of respiratory failure secondary to hmpv pneumonia . the median time from transplant to hmpv infection was 12 months ( range , 557 months ) . three ( 50% ) of the 6 who had chest imaging performed had abnormalities on chest radiograph . none received treatment with ribavirin or ivig , and though there was 1 death , it was not attributed to hmpv infection . as compared to other immunocompromised patients , solid organ transplant recipients were no more likely to have fever ( p = .19 ) , cough ( p = .62 ) , abnormal chest imaging ( p = 1.00 ) , or to be classified as lrti disease on initial clinical presentation ( p = .35 ) . the median number of samples collected was 7 ( range , 244 samples ) , and the median duration of follow - up was 37 days ( range , 4562 days ) . types of samples were primarily nasal wash samples , but also included two bal , one sinus biopsy , and one lung biopsy sample in patient 1 , as well as 1 bal sample in patient 2 . median initial viral load was estimated as 6.96 log10 copies / ml ( range , 2.757.22 log10 copies / ml ) . the viral load data for all 6 patients are illustrated in figures 2a ( patient 1 ) and 2b ( patients 26 ) . in the 3 patients who died , the median time between initial and final sample was 7 days ( range , 220 days ) , and the median change in viral load was 1.31 log10 copies / ml ( range , 6.23 to 0.36 log10 copies / ml ) . for the 3 patients who survived , the median time to viral clearance was 43 days ( range , 4511 days ) . figure 2.(a ) viral loads in patient 1 ( 11-month - old with severe combined immunodeficiency ) over time . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . acute lymphoblastic leukemia ; mo , month ; scid , severe combined immunodeficiency syndrome ; yo , year old . ( a ) viral loads in patient 1 ( 11-month - old with severe combined immunodeficiency ) over time . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . acute lymphoblastic leukemia ; mo , month ; scid , severe combined immunodeficiency syndrome ; yo , year old . nine patients were treated for their hmpv infections ( median age 9 years ; range , 5 months19 years ) . these patients included 5 hsct recipients , 2 patients with hematologic malignancy , and 2 patients with solid tumors . indications for hsct included aplastic anemia ( n = 2 , 22% ) , scid ( n = 2 , 22% ) , and ewing sarcoma ( n = 1 , 11% ) . eight ( 89% ) of these treated patients had fever , 6 ( 75% ) had cough , and 7 ( 78% ) had abnormal chest imaging . five ( 56% ) were hospitalized in the icu , and 5 ( 56% ) received supplemental oxygen . five ( 56% ) patients received both ribavirin and ivig , 2 ( 22% ) received ribavirin alone , and 2 ( 22% ) received ivig alone . of the 5 hsct recipients , 3 ( 60% ) had hmpv detected prior to transplant ( median time prior to transplant : 43 days ; range , 9321 days ) , and 1 detected 11 days after transplant . treatment with ribavirin and ivig was initiated a median of 5 days after hmpv diagnosis ( range , 246 days ) , and several patients received multiple courses of ribavirin and ivig . treatment with ribavirin alone ( n = 2 ) was initiated 2 and 4 days after diagnosis , respectively . of the 7 patients who received ribavirin , 6 ( 86% ) received inhaled ribavirin at a dose of 2 g 3 times daily for a 5-day course , and 1 ( 14% ) received an 11-day course of intravenous ribavirin . two of these deaths occurred in hsct recipients who were diagnosed with hmpv prior to transplant ( figures 1 and 2b : patients 2 and 3 ) . one hsct recipient had not engrafted at time of death , while the other engrafted the day prior to death . the third death occurred in a patient with all and secondary acute myelogenous leukemia on salvage chemotherapy who did not receive hmpv - specific treatment ( figure 2b : patient 5 ) . hmpv was considered to be a contributing factor to her death , though the primary cause of death was thought to be progression of all . human metapneumovirus infections in our immunocompromised patient population was associated with high rates of lrti at presentation , and an attributable mortality of 5% , rates substantially higher than that in the general population where hmpv is most commonly a self - limited urti . in our study , 12 ( 23% ) of immunocompromised children with hmpv were classified as having severe disease , and neutropenia was identified as a significant risk factor for disease severity . this rate is comparable or even higher than previous rates reported in seriously immunocompromised adult patients . similar retrospective studies performed in pediatric cancer patients have looked at morbidity and mortality related to other respiratory viruses . parainfluenza virus was associated with lrti in 2036% of patients with a mortality rate of 07% in a mixed population of pediatric cancer and transplant patients [ 14 , 15 ] . similarly , respiratory syncytial virus ( rsv ) has been associated with lrti in 28% and mortality in 8% of a mixed group of pediatric and transplant patients . more recently , pandemic h1n1 and seasonal influenza a were associated with lrti rates of 2042% and 427% and mortality rates of 1012% and 24% , respectively , despite treatment of the majority of patients with antiviral drugs [ 17 , 18 ] . an additional study performed in a larger group of patients described a lower rate of lrti ( 16% ) associated with pandemic h1n1 with only 1 severe case , with no related mortality . prospective studies of hmpv in adults with hematologic malignancies demonstrated that 9% of respiratory tract infections were attributed to hmpv , and that the majority of these were in hsct recipients . among hsct recipients , our study is one of the first to describe the presentation and management of symptomatic hmpv infection in immunocompromised children . the presentation of hmpv in pediatric solid organ transplant recipients was shown to be similar to patients with other immunocompromising conditions , with fever and cough as the most common symptoms . we also show that the majority of immunocompromised patients diagnosed with hmpv are subsequently admitted to the hospital for further evaluation , but that only 2% of those initially presenting with urti symptoms go on to develop lrti . however , there were no clear differences in the presentation of patients with urti or lrti in terms of age , symptoms ( fever or cough ) , laboratory abnormalities , or underlying condition that would clearly identify risk factors for lrti disease and guide decision - making . interestingly , 2 of the 3 patient deaths were in patients with hmpv pneumonia diagnosed prior to hsct . both of these patients , however , had underlying diseases ( aplastic anemia and scid ) , which made it extremely unlikely that they would be able to clear their viral infection without reconstitution of their immune system through hsct . these patients both underwent hsct , and subsequently died of hmpv pneumonia despite use of ribavirin and ivig and supportive care including mechanical ventilation and icu stay . these 2 patients continued to have detectable virus within days of death . at our institution as well as other transplant centers , pretransplant screening for rsv and delay of transplant in adults is effective in reducing rates of pneumonia . our policy in pediatric patients is to delay transplant if patients have evidence of infection with rsv , influenza , adenovirus , or hmpv , even if patients are asymptomatic . transplantation is not generally delayed for asymptomatic shedding of rhinovirus , bocavirus , or coronavirus . this study further enforces the risks associated with transplantation during documented hmpv viral shedding or disease . because infection may result in such serious disease , potential modalities to treat or at least stabilize patients while white blood cells and/or immune function can return are considered and utilized in these patients . treatment with ivig and ribavirin is currently being utilized at our institution for severely immunocompromised individuals [ 21 , 22 ] . among the patients who received treatment , we demonstrated a 22% mortality rate as compared to a 2% mortality rate in untreated individuals . this , however , is almost certainly a reflection of clinical decision - making to initiate treatment in individuals with more severe disease . in this retrospective study , we identified hmpv - infected immunocompromised children through screening of laboratory results from our virology laboratory . we did not screen asymptomatic children and may not have captured complete data on children who were evaluated solely as outpatients at other private clinics or hospitalized in other institutions . as the regional pediatric referral center , most immunocompromised patients are seen and followed at our institution but because of our large geographic referral area , it is possible that some children were seen elsewhere . therefore , our study could be biased towards detection of more severe hmpv disease . also , it is possible that a subset of earlier hmpv - positive cases were missed through use of dfa as compared to pcr , a more sensitive detection technique . furthermore , we were unable to capture all data at the time of clinical presentation , or to obtain full information regarding the clinical decision - making for the administration of ribavirin and/or ivig . therefore , the comparison of outcomes in patients who did and did not receive treatment is problematic , and the potential benefits of therapy are not readily evaluable . unlike a previous hmpv study conducted in adult hsct recipient patients in italy , we did not assess hmpv shedding in asymptomatic patients and unfortunately can not comment on rates of symptomatic versus asymptomatic shedding of hmpv in our patients . finally , we were limited in our ability to classify disease severity based on hypoxemia , as some patients did not have oxygen saturations charted . we suspect that a higher proportion of our patients would be classified as having severe disease using criteria such as that used by papenburg et al . although only 8 ( 15% ) of our patients were identified as having acquired their hmpv infection nosocomially , the vast majority of the patients were seen frequently on an outpatient basis in clinic for blood draws , interventions , and chemotherapy , making it difficult to differentiate true community - acquired disease from that acquired in the hospital setting . a prospective randomized study of ribavirin and ivig administration , or of newer therapeutic agents under development for treatment of paramyxovirus infections [ 24 , 25 ] , would be helpful given the high morbidity associated with hmpv in the immunocompromised population . in particular , a study of the use of novel agents , including fusion inhibitors , could be studied in this population given their potential to serve as postexposure prophylaxis in high - risk individuals . the documentation of severe hmpv disease in immunocompromised children supports previous data documenting severe and fatal hmpv disease reported in adult hsct recipients [ 9 , 13 ] and highlights the need for treatment options in this population .

backgroundthe clinical presentation and management of human metapneumovirus ( hmpv ) infections in immunocompromised children is not well understood.methodswe performed a retrospective evaluation of pediatric patients with laboratory - confirmed hmpv infections and underlying hematologic malignancy , solid tumors , solid organ transplant , rheumatologic disease , and/or receipt of chronic immunosuppressants . data were analyzed using t tests and fisher 's exact tests.resultsoverall , 55 patients ( median age : 5 years ; range : 5 months19 years ) with hmpv infection documented between 2006 and 2010 were identified , including 24 ( 44% ) with hematologic malignancy , 9 ( 16% ) undergoing hematopoietic stem cell transplant , 9 ( 16% ) with solid tumors , and 8 ( 15% ) with solid organ transplants . three ( 5% ) presented with fever alone , 35 ( 64% ) presented with upper respiratory tract infections , and 16 ( 29% ) presented with lower respiratory tract infections ( lrti ) . twelve ( 23% ) patients required intensive care unit admission and/or supplemental oxygen 28% fio2 . those with severe disease were more likely to be neutropenic ( p = .02 ) , but otherwise did not differ by age ( p = .27 ) , hematopoietic stem cell transplant recipient status ( p = .19 ) , or presence of lymphopenia ( p = .09 ) . nine ( 16% ) patients received treatment with ribavirin , intravenous immunoglobulin , or both . three children ( 5% ) died of hmpv pneumonia.conclusionsimmunocompromised pediatric patients with hmpv infection have high rates of lrti and mortality . the benefits of treatment with ribavirin and intravenous immunoglobulin in this patient population require further evaluation .

BACKGROUND METHODS RESULTS Clinical Characteristics Virologic Characteristics of hMPV Episodes Treatment of hMPV Mortality Attributed to hMPV CONCLUSIONS

human metapneumovirus ( hmpv ) is a paramyxovirus first identified in 2001 as a pathogen associated with upper and lower respiratory tract infections in children . a prospective cohort study of hmpv revealed a 9.4% seroprevalence in children hospitalized with respiratory tract infections , with 63% of patients requiring supplemental oxygen and 3% requiring intensive care unit ( icu ) admission . lower respiratory tract illnesses ( lrti ) caused by hmpv includes bronchiolitis , pneumonia , and croup . in adults with hematologic malignancy , hmpv is associated with high rates of progression from upper to lower respiratory tract disease and substantial mortality [ 8 , 9 ] . few prior descriptions of the clinical management and treatment of hmpv respiratory tract infections in a pediatric immunocompromised population , including children with leukemia and solid organ transplant recipients , are available . we describe hmpv respiratory tract infections in 55 pediatric immunocompromised patients at seattle children 's hospital , reporting on their clinical presentation , management , and outcomes . we defined an immunocompromised condition as presence of hematologic malignancy , solid tumor , rheumatologic disease receiving immunosuppressive therapy , solid organ transplant , primary immunodeficiency , receipt of chronic immunosuppressive therapy , or receipt of a hematopoietic stem cell transplant ( hsct ) . lrti was defined as a new pulmonary infiltrate or presence of lower respiratory tract symptoms ( wheezing or hypoxia ) in association with a positive lower respiratory tract specimen or a positive upper respiratory tract specimen if the patient did not undergo bal . comparison of clinical characteristics was performed using fisher 's exact tests for categorical variables and t tests with unequal variance for continuous variables . the median age was 5 years ( range , 5 months19 years ) , and the majority had a hematologic malignancy as an underlying condition . the nine solid tumors included wilms tumor ( n = 3 ; 33% ) , osteosarcoma ( n = 1 ; 11% ) , and ovarian cancer ( n = 1 ; 11% ) . eight ( 15% ) patients were solid organ transplant recipients , and all were receiving immunosuppressive therapy . of these 46 cases , 24 ( 52% ) data are shown as number ( n [ % ] ) unless otherwise indicatedcharacteristicsn = 5age in years , median ( range)5 ( 0.419)female sex26 ( 47)underlying condition hematologic malignancy24 ( 44 ) all21 ( 88 ) hsct recipient9 ( 16 ) pre - transplant3 ( 33 ) post - transplant6 ( 67 ) solid organ transplant8 ( 15 ) heart4 ( 50 ) kidney1 ( 13 ) liver3 ( 38 ) solid tumors9 ( 16 ) primary immunodeficiency3 ( 5 ) other3 ( 5)chemotherapy recipient ( n = 29)29 ( 100)steroid use12 ( 22)season of infection winter ( dec feb)19 ( 35 ) spring ( march may)32 ( 58)community acquired46 ( 85)nosocomial8 ( 15)in patients with hematologic malignancy or solid tumors.defined as outpatients at the time of hmpv diagnosis . data are shown as number ( n [ % ] ) unless otherwise indicated in patients with hematologic malignancy or solid tumors . as compared to those presenting with fever or urti , children presenting with lrtis did not differ by age ( p = .80 ) or presence of fever ( p = .70 ) , cough ( p = 1.00 ) , neutropenia ( p = .21 ) , or lymphopenia ( p = .09 ) . table 2.characteristics of human metapneumovirus illness episodesnumber of infections55severe disease8 ( 15)neutropenia ( anc < 1000)19 ( 35)lymphopenia ( alc < 300 ) [ n = 54]7 ( 13)severe lymphopenia ( alc < 100 ) [ n = 54]5 ( 9)clinical symptoms cough [ n = 51]42 ( 86 ) fever44 ( 80)stage at presentation asymptomatic1 ( 2 ) fever3 ( 5 ) upper respiratory tract infection35 ( 64 ) lower respiratory tract infection16 ( 29)abnormal chest imaging [ n = 36]16 ( 44)median initial viral load in log10 copies / ml ( range ) [ n = 6]6.96 ( 2.757.22)diagnostic modality direct fluorescent antibody54 ( 98 ) polymerase chain reaction6 ( 11)co - infections12 ( 22 ) adenovirus3 ( 5 ) cytomegalovirus3 ( 5 ) epstein barr virus1 ( 2 ) influenza b1 ( 2 ) parainfluenza1(2 ) human herpes virus81 ( 2 ) rhinovirus1 ( 2 ) staphylococcus3 ( 7 ) other3 ( 7)hospitalization required [ n = 46]24 ( 52)treatment received ribavirin and intravenous immunoglobulin5 ( 9 ) ribavirin alone2 ( 4 ) intravenous immunoglobulin alone2 ( 4)receipt of antibiotics22 ( 40)intensive care unit stay [ n = 52]6 ( 12)receipt of supplemental oxygen [ n = 54]9 ( 17)severe disease [ n = 52]12 ( 23)death attributed to human metapneumovirus infection3 ( 5)of the 46 patients who were outpatient at the time of diagnosis.severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . diagnosis of hmpv was made by dfa alone in 49 ( 89% ) patients , by pcr alone in 1 patient ( 2% ) , and by dfa and pcr in 5 ( 9% ) patients . twelve ( 23% ) patients met criteria for severe disease , 6 ( 50% ) of whom were admitted to the intensive care unit ( icu ) . those with severe disease were more likely to be neutropenic ( p = .02 ) , but otherwise did not differ by age ( p = .27 ) , hsct recipient status ( p = .19 ) , or presence of lymphopenia ( p = .09 ) . icu stay alone was not associated with age ( p = .59 ) or presence of neutropenia ( p = .38 ) , or lymphopenia ( p = .54 ) , though hsct recipients were more likely to require icu stay ( p < .01 ) . nine ( 16% ) patients were hsct recipients ; indications for transplant included aplastic anemia ( n = 4 , 44% ) , scid ( n = 2 , 22% ) , ewing sarcoma ( n = 1 , 11% ) , acute lymphoblastic leukemia ( all ) ( n = 1 , 11% ) , and osteopetrosis ( n = 1 , 11% ) . three ( 33% ) of these patients had a diagnosis of hmpv prior to transplant , including 2 children with scid and 1 with aplastic anemia . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . none received treatment with ribavirin or ivig , and though there was 1 death , it was not attributed to hmpv infection . as compared to other immunocompromised patients , solid organ transplant recipients were no more likely to have fever ( p = .19 ) , cough ( p = .62 ) , abnormal chest imaging ( p = 1.00 ) , or to be classified as lrti disease on initial clinical presentation ( p = .35 ) . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . nine patients were treated for their hmpv infections ( median age 9 years ; range , 5 months19 years ) . these patients included 5 hsct recipients , 2 patients with hematologic malignancy , and 2 patients with solid tumors . five ( 56% ) patients received both ribavirin and ivig , 2 ( 22% ) received ribavirin alone , and 2 ( 22% ) received ivig alone . of the 5 hsct recipients , 3 ( 60% ) had hmpv detected prior to transplant ( median time prior to transplant : 43 days ; range , 9321 days ) , and 1 detected 11 days after transplant . treatment with ribavirin and ivig was initiated a median of 5 days after hmpv diagnosis ( range , 246 days ) , and several patients received multiple courses of ribavirin and ivig . the median age was 5 years ( range , 5 months19 years ) , and the majority had a hematologic malignancy as an underlying condition . eight ( 15% ) patients were solid organ transplant recipients , and all were receiving immunosuppressive therapy . of these 46 cases , 24 ( 52% ) data are shown as number ( n [ % ] ) unless otherwise indicatedcharacteristicsn = 5age in years , median ( range)5 ( 0.419)female sex26 ( 47)underlying condition hematologic malignancy24 ( 44 ) all21 ( 88 ) hsct recipient9 ( 16 ) pre - transplant3 ( 33 ) post - transplant6 ( 67 ) solid organ transplant8 ( 15 ) heart4 ( 50 ) kidney1 ( 13 ) liver3 ( 38 ) solid tumors9 ( 16 ) primary immunodeficiency3 ( 5 ) other3 ( 5)chemotherapy recipient ( n = 29)29 ( 100)steroid use12 ( 22)season of infection winter ( dec feb)19 ( 35 ) spring ( march may)32 ( 58)community acquired46 ( 85)nosocomial8 ( 15)in patients with hematologic malignancy or solid tumors.defined as outpatients at the time of hmpv diagnosis . data are shown as number ( n [ % ] ) unless otherwise indicated in patients with hematologic malignancy or solid tumors . as compared to those presenting with fever or urti , children presenting with lrtis did not differ by age ( p = .80 ) or presence of fever ( p = .70 ) , cough ( p = 1.00 ) , neutropenia ( p = .21 ) , or lymphopenia ( p = .09 ) . table 2.characteristics of human metapneumovirus illness episodesnumber of infections55severe disease8 ( 15)neutropenia ( anc < 1000)19 ( 35)lymphopenia ( alc < 300 ) [ n = 54]7 ( 13)severe lymphopenia ( alc < 100 ) [ n = 54]5 ( 9)clinical symptoms cough [ n = 51]42 ( 86 ) fever44 ( 80)stage at presentation asymptomatic1 ( 2 ) fever3 ( 5 ) upper respiratory tract infection35 ( 64 ) lower respiratory tract infection16 ( 29)abnormal chest imaging [ n = 36]16 ( 44)median initial viral load in log10 copies / ml ( range ) [ n = 6]6.96 ( 2.757.22)diagnostic modality direct fluorescent antibody54 ( 98 ) polymerase chain reaction6 ( 11)co - infections12 ( 22 ) adenovirus3 ( 5 ) cytomegalovirus3 ( 5 ) epstein barr virus1 ( 2 ) influenza b1 ( 2 ) parainfluenza1(2 ) human herpes virus81 ( 2 ) rhinovirus1 ( 2 ) staphylococcus3 ( 7 ) other3 ( 7)hospitalization required [ n = 46]24 ( 52)treatment received ribavirin and intravenous immunoglobulin5 ( 9 ) ribavirin alone2 ( 4 ) intravenous immunoglobulin alone2 ( 4)receipt of antibiotics22 ( 40)intensive care unit stay [ n = 52]6 ( 12)receipt of supplemental oxygen [ n = 54]9 ( 17)severe disease [ n = 52]12 ( 23)death attributed to human metapneumovirus infection3 ( 5)of the 46 patients who were outpatient at the time of diagnosis.severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . severe disease is defined as intensive care unit stay and/or use of supplemental oxygen ( fio2 0.28 ) . diagnosis of hmpv was made by dfa alone in 49 ( 89% ) patients , by pcr alone in 1 patient ( 2% ) , and by dfa and pcr in 5 ( 9% ) patients . twelve ( 23% ) patients met criteria for severe disease , 6 ( 50% ) of whom were admitted to the intensive care unit ( icu ) . those with severe disease were more likely to be neutropenic ( p = .02 ) , but otherwise did not differ by age ( p = .27 ) , hsct recipient status ( p = .19 ) , or presence of lymphopenia ( p = .09 ) . icu stay alone was not associated with age ( p = .59 ) or presence of neutropenia ( p = .38 ) , or lymphopenia ( p = .54 ) , though hsct recipients were more likely to require icu stay ( p < .01 ) . nine ( 16% ) patients were hsct recipients ; indications for transplant included aplastic anemia ( n = 4 , 44% ) , scid ( n = 2 , 22% ) , ewing sarcoma ( n = 1 , 11% ) , acute lymphoblastic leukemia ( all ) ( n = 1 , 11% ) , and osteopetrosis ( n = 1 , 11% ) . three ( 33% ) of these patients had a diagnosis of hmpv prior to transplant , including 2 children with scid and 1 with aplastic anemia . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . ( a ) patient 1 is an 11-month - old boy with severe combined immunodeficiency ( scid ) who survived and cleared human metapneumovirus ( hmpv ) 511 days after diagnosis . none received treatment with ribavirin or ivig , and though there was 1 death , it was not attributed to hmpv infection . as compared to other immunocompromised patients , solid organ transplant recipients were no more likely to have fever ( p = .19 ) , cough ( p = .62 ) , abnormal chest imaging ( p = 1.00 ) , or to be classified as lrti disease on initial clinical presentation ( p = .35 ) . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . ivig , intravenous immunoglobulin ; hsct , hematopoietic stem cell transplant ; hmpv , human metapneumovirus . nine patients were treated for their hmpv infections ( median age 9 years ; range , 5 months19 years ) . these patients included 5 hsct recipients , 2 patients with hematologic malignancy , and 2 patients with solid tumors . five ( 56% ) patients received both ribavirin and ivig , 2 ( 22% ) received ribavirin alone , and 2 ( 22% ) received ivig alone . of the 5 hsct recipients , 3 ( 60% ) had hmpv detected prior to transplant ( median time prior to transplant : 43 days ; range , 9321 days ) , and 1 detected 11 days after transplant . treatment with ribavirin and ivig was initiated a median of 5 days after hmpv diagnosis ( range , 246 days ) , and several patients received multiple courses of ribavirin and ivig . human metapneumovirus infections in our immunocompromised patient population was associated with high rates of lrti at presentation , and an attributable mortality of 5% , rates substantially higher than that in the general population where hmpv is most commonly a self - limited urti . in our study , 12 ( 23% ) of immunocompromised children with hmpv were classified as having severe disease , and neutropenia was identified as a significant risk factor for disease severity . prospective studies of hmpv in adults with hematologic malignancies demonstrated that 9% of respiratory tract infections were attributed to hmpv , and that the majority of these were in hsct recipients . among hsct recipients , our study is one of the first to describe the presentation and management of symptomatic hmpv infection in immunocompromised children . the presentation of hmpv in pediatric solid organ transplant recipients was shown to be similar to patients with other immunocompromising conditions , with fever and cough as the most common symptoms . however , there were no clear differences in the presentation of patients with urti or lrti in terms of age , symptoms ( fever or cough ) , laboratory abnormalities , or underlying condition that would clearly identify risk factors for lrti disease and guide decision - making . these patients both underwent hsct , and subsequently died of hmpv pneumonia despite use of ribavirin and ivig and supportive care including mechanical ventilation and icu stay . unlike a previous hmpv study conducted in adult hsct recipient patients in italy , we did not assess hmpv shedding in asymptomatic patients and unfortunately can not comment on rates of symptomatic versus asymptomatic shedding of hmpv in our patients . although only 8 ( 15% ) of our patients were identified as having acquired their hmpv infection nosocomially , the vast majority of the patients were seen frequently on an outpatient basis in clinic for blood draws , interventions , and chemotherapy , making it difficult to differentiate true community - acquired disease from that acquired in the hospital setting . a prospective randomized study of ribavirin and ivig administration , or of newer therapeutic agents under development for treatment of paramyxovirus infections [ 24 , 25 ] , would be helpful given the high morbidity associated with hmpv in the immunocompromised population . the documentation of severe hmpv disease in immunocompromised children supports previous data documenting severe and fatal hmpv disease reported in adult hsct recipients [ 9 , 13 ] and highlights the need for treatment options in this population .

atrial fibrillation ( af ) is the most common major cardiac arrhythmia and affects a large number of people , particularly the elderly . early detection of paroxysmal atrial fibrillation ( paf ) in electrocardiography will enable the patient to obtain timely treatment to manage and prevent further complications . in this communication , heart rate data , unlike ecg traces , are easily monitored using many commercially available wearable devices . it is the purpose of this communication to determine the feasibility of using heart rate data to examine whether they could be used to alert a paf subject before a sudden attack of af . such a warning system would help a paf subject to seek preventive treatment immediately before such an attack occurs . before we proceed to develop such a system , let us examine the results of some relevant studies which involved analysis of rr intervals from paf subjects . the rr interval is the inverse of the heart rate . in a study of a large group of paf patients , rr interval data collected in the hour preceding paf and the twenty minutes before paf were divided into four 5-min period , and were analyzed . it was observed that there was a significant increase in the standard deviation ( sd ) of the rr intervals from 654 to 704 ms ( p<0.02 ) before the onset of paf . the symbol p is the probability of observing the result under the assumption of the null hypothesis . there was also a linear decrease in mean rr interval from 92516 to 90616 ms ( p<0.0002 ) before the onset of paf . in the frequency domain , there was a significant increase in high - frequency ( hf ) components before paf ( p<0.001 ) , and a decrease in low - frequency ( lf ) components ( p<0.0001 ) . the low / high frequency ratio also showed a linear increase until 10 min before paf , followed by a sharp decrease immediately before paf . another study on the onset of arrhythmia using holter monitoring also showed higher values in the sd of rr intervals in the last 5-min period before the start of paf , compared with the other intervals . the analysis was conducted using six 5-min periods before the onset , and a control 5-min period . in the frequency domain there was significant lowering in the lf region in the last 5 min before onset , while the hf was similar in all intervals , as well as in the control period . dimmer et al . , in their analysis of paf , also observed an increase in the sd of rr intervals in the last 5 min before af occurred . however , heart rate variability analysis in the frequency domain did not reveal any significant changes before the onset of af . on the other hand , rr interval analysis by vickman et al . in 22 paf subjects , which involved using segments of 20 min , showed no significant changes in the traditional time and frequency variability indices . however , their study showed that nonlinear hr variability measures , the approximate entropy , and the altered fractal properties ( short term scaling exponent ) , showed significant changes before the onset of paf . there was a decrease in the approximate entropy , from 1.090.26 , 120 to 100 min before af , to 0.880.24 , 20 to 0 min before af , with p<0.001 ; in the short term scaling exponent , there was a decrease from 1.010.28 , 120 to 100 min before af , to 0.890.28 , 20 to 0 min before af . it is possible that their inability to observe the changes in sd , hf , and lf could be due to the choice of large segment length used in this study . examination of the results of all of the above studies showed that , except for the work of vickman et al . , the sd of the rr interval showed a significant increase when an af attack was imminent . that nonlinear changes accompany the onset of af is a hint to search for a nonlinear index , which is suitable for short segments of data . this led to the analysis of rr data in this paper to be carried out using sd of the rr intervals , a linear time domain index , and spectral entropy ( se ) , a non - linear index . sd and se are measures of variability and disorder of a time series , respectively . approximate entropy ( apen ) and sample entropy ( sampen ) are some of the indices that have been used to measure the disorder of a system . however , unlike apen or sampen , the evaluation of se does not require a large amount of rr interval data , which is a distinct advantage in the development of a fast response warning system . se is a measure of the disorder in the frequency domain of a time series , and is evaluated using power spectra . although the work here on se uses some of the parameter choices from the work of staniczenko et al . the use of the randomness property of the rr intervals in this study was prompted by the fact that the distribution of the rr intervals exhibit random behavior in the af region . since se is not commonly used in rr interval analysis , a brief summary of its evaluation is given in section 2 . in this paper , both sd and se are examined with the view that they will complement each other and provide confidence to the paf subject that an af episode is imminent . let us now briefly describe the manner in which the data are analyzed in this paper . the rr interval data in the first time period of thirty minutes , referred to as a in this paper , represent the time period during which there are no instances of paf , for 45 min on either side of that time range . the rr intervals in the second time period of thirty minutes , referred to as b in this paper , represent the time period just preceding an af attack . immediately following time period b , this third period , referred to as c in this paper , when paf occurs , has a duration of about five minutes . in this communication , the rr interval data in time periods a , b , and c are concatenated before analysis . this is done with a view to determine whether one could observe changes in sd and se in sufficient time so that the paf subject can take appropriate action before the onset of paf . both sd and se are examined in this paper instead of alone , with the view that they will complement each other and provide confidence to the paf subject that an af episode is imminent . section 2 gives a brief description of the se method , the data used , and the analysis procedure . the evaluation of se of the rr interval time series is carried by first symbolizing the rr data . one problem that is encountered in obtaining the power spectra of the rr data is that the time intervals between beats are not uniform . this is overcome by using a constant sampling interval and symbolizing the rr data , where the rr data are converted into a binary string . initially a suitable sampling interval ( ) , a window length ( l samples ) , and a window separation length ( ls samples ) are chosen . each of the sampling intervals present in a window is categorized as either 1 or 0 , depending on whether it contains a beat or not . thus , each window contains a uniformly - sampled time series of 1 s and 0 s . after obtaining the binary string , the power spectrum of this segment of data is obtained using the discrete fourier transform ( dft ) with a rectangular window . if the power of the ith frequency is ci then the probability of having that power can be defined as(1)pi = cii=1l/2ciwhere the number of frequency bins is ( l/2 ) , assuming l is even and the mean is removed . the entropy of this probability distribution referred to as the se is then given by(2)h=i=1l/2pilog2pi white noise has power at all frequencies , and has a flat power spectrum . such a spectrum gives the maximum value for the se:(3)hmax = log2(l/2 ) in order to have the range of se in [ 0 , 1 ] , the value of h in eq . the choice used for the sampling interval ( ) , the window length ( l ) , the shape of the window function in dft , and window separation length ( ls ) was guided by the choices of staniczenko et al . . the sampling interval was chosen by the criterion that there are no useful frequencies above ( 1/2 ) . such a criterion led to the choice of the sampling interval of 0.03 s , . the choice of the window length l differed from that chosen by staniczenko et al . . this was achieved with the window length being chosen to contain at least 10 beats . in the present study , window lengths with different beats were examined . it was found that a window length with an average between 40 and 60 beats provided the best distinction between different regions of the rr data . finally , a window length of 40 beats was chosen . since the number of beats in the window is small compared to the length of samples in the segment , the shape of the window was chosen to be rectangular . this window shape has a low value for the equivalent noise band width ( enbw ) , . in order to obtain a large number of se values the shape and the overlap between windows are the same as that chosen by staniczenko et al . the overlap between windows was set at 0.75l , giving a window separation ls = l/4 . the study employs the learning data set made available in the cardiology 2001 challenge and found in the physionet data bank . the sex , age , and medical history of the subjects , and whether data were obtained during the day or at night , were not made available to the public . the first 30-min sample is distant from any paf episode , while the second 30-min sample directly precedes an episode . the third 5-min sample is a continuation of the second record , when the paf episode occurs . the first sample is distant from any paf episode , and has no paf episodes within 45 min on either side of the excerpt . the rr intervals obtained from such a heart rate time series were further de - trended and ectopic beats removed before being used for analysis . ectopic beats were removed since they introduce bias in heart rate analysis , and the origins of some are not completely physiological . the vertical lines separate the periods a , b , and c. the focus of this paper is to develop a procedure that will alert the paf subject before the onset of the attack in c. in order to study and develop a warning system to alert a paf subject before the onset of an af attack , the rr interval time series of regions a , b , and c were concatenated , so that each subject was associated with just one rr interval time series . region a is further away from any af attack , region b precedes the attack , and region c is when the af attack occurs . in the manner described in section 2.1 , a time series of se samples suppose se ( i ) and t ( i ) are the se and the time in minutes corresponding to each window . let us digress at this point , and consider the evaluation of the sd time series , which is much easier , before a procedure is outlined how these se values can be used in the development of an alert system . let us consider an initial time interval containing nrm rr intervals , starting from the first rr interval in region a , and let sd(1 ) be the sd in the time interval that contains the range of rr intervals from [ 0 , nrm ] . an sd time series is then constructed { sd(1 ) , sd(2 ) , , sd(nl ) } , where sd(k ) corresponds to the sd in the time interval that contains the range of rr intervals [ 0 ( nrm+k1 ) ] . thus a time series of sd values is constructed where the first sd corresponds to nrm rr intervals , followed by a series of sd values corresponding to ( nrm+1 ) , ( nrm+2 ) , rr intervals . thus , the difference in the sd values between successive terms in the series arises from a single rr interval . let nra , nra+b and nra+b+c be the number of rr intervals in regions a , ( a+b ) , and ( a+b+c ) respectively . the last term sd(nl ) in the sd series corresponds to the sd containing nra+b+c rr intervals . now that a time series of sd values is evaluated , let us consider a similar type of series , using the sequence of se values that were evaluated before . let se(nsm ) , se(nsa ) , se(nsa+b ) , se(nsa+b+c ) be the se sample that is closest to the time corresponding to nrm , nra , nra+b and nra+b+c rr intervals . using the set { se(1 ) , se(2 ) , se(nsm ) } , various statistical indices were evaluated , namely the mean ( semn(1 ) ) , 90% quantile value ( se90(1 ) ) , and 99% quantile value(se99(1 ) ) . subsequent samples semn(i ) , se90(i ) , se99(i ) were evaluated using the set { se(1 ) , se(2 ) , , thus , a time series similar to sd is obtained for semn , se90 , and se99 . in the latter three series , the separation in time is the same as se . in each of the time series of sd , semn , se90 , and se99 , the sample size of the rr intervals , and se values that are used to evaluate the samples in these series , increase with time . thus , unless there is a marked increase in the rr and se values with time , one would not expect the values in the constructed time series to show an increasing trend . these time series , which reflect the time variation of variability and disorder of the rr interval series , will be tested using the data described in section 2.2 , to determine their usefulness in providing prior warning to a paf subject of an impending af attack . the evaluation of se of the rr interval time series is carried by first symbolizing the rr data . one problem that is encountered in obtaining the power spectra of the rr data is that the time intervals between beats are not uniform . this is overcome by using a constant sampling interval and symbolizing the rr data , where the rr data are converted into a binary string . initially a suitable sampling interval ( ) , a window length ( l samples ) , and a window separation length ( ls samples ) are chosen . each of the sampling intervals present in a window is categorized as either 1 or 0 , depending on whether it contains a beat or not . thus , each window contains a uniformly - sampled time series of 1 s and 0 s . after obtaining the binary string , the power spectrum of this segment of data is obtained using the discrete fourier transform ( dft ) with a rectangular window . if the power of the ith frequency is ci then the probability of having that power can be defined as(1)pi = cii=1l/2ciwhere the number of frequency bins is ( l/2 ) , assuming l is even and the mean is removed . the entropy of this probability distribution referred to as the se is then given by(2)h=i=1l/2pilog2pi white noise has power at all frequencies , and has a flat power spectrum . such a spectrum gives the maximum value for the se:(3)hmax = log2(l/2 ) in order to have the range of se in [ 0 , 1 ] , the value of h in eq . the choice used for the sampling interval ( ) , the window length ( l ) , the shape of the window function in dft , and window separation length ( ls ) was guided by the choices of staniczenko et al . . the sampling interval was chosen by the criterion that there are no useful frequencies above ( 1/2 ) . such a criterion led to the choice of the sampling interval of 0.03 s , . the choice of the window length l differed from that chosen by staniczenko et al . . this was achieved with the window length being chosen to contain at least 10 beats . in the present study , window lengths with different beats were examined . it was found that a window length with an average between 40 and 60 beats provided the best distinction between different regions of the rr data . finally , a window length of 40 beats was chosen . since the number of beats in the window is small compared to the length of samples in the segment , the shape of the window was chosen to be rectangular . this window shape has a low value for the equivalent noise band width ( enbw ) , . in order to obtain a large number of se values the shape and the overlap between windows are the same as that chosen by staniczenko et al . the overlap between windows was set at 0.75l , giving a window separation ls = l/4 . the study employs the learning data set made available in the cardiology 2001 challenge and found in the physionet data bank . the sex , age , and medical history of the subjects , and whether data were obtained during the day or at night , were not made available to the public . for each subject , two 30-min samples and one 5-min sample are used . the first 30-min sample is distant from any paf episode , while the second 30-min sample directly precedes an episode . the third 5-min sample is a continuation of the second record , when the paf episode occurs . the first sample is distant from any paf episode , and has no paf episodes within 45 min on either side of the excerpt . the rr intervals obtained from such a heart rate time series were further de - trended and ectopic beats removed before being used for analysis . ectopic beats were removed since they introduce bias in heart rate analysis , and the origins of some are not completely physiological . the vertical lines separate the periods a , b , and c. the focus of this paper is to develop a procedure that will alert the paf subject before the onset of the attack in c. in order to study and develop a warning system to alert a paf subject before the onset of an af attack , the rr interval time series of regions a , b , and c were concatenated , so that each subject was associated with just one rr interval time series . region a is further away from any af attack , region b precedes the attack , and region c is when the af attack occurs . in the manner described in section 2.1 , a time series of se samples suppose se ( i ) and t ( i ) are the se and the time in minutes corresponding to each window . let us digress at this point , and consider the evaluation of the sd time series , which is much easier , before a procedure is outlined how these se values can be used in the development of an alert system . let us consider an initial time interval containing nrm rr intervals , starting from the first rr interval in region a , and let sd(1 ) be the sd in the time interval that contains the range of rr intervals from [ 0 , nrm ] . an sd time series is then constructed { sd(1 ) , sd(2 ) , , sd(nl ) } , where sd(k ) corresponds to the sd in the time interval that contains the range of rr intervals [ 0 ( nrm+k1 ) ] . thus a time series of sd values is constructed where the first sd corresponds to nrm rr intervals , followed by a series of sd values corresponding to ( nrm+1 ) , ( nrm+2 ) , rr intervals . thus , the difference in the sd values between successive terms in the series arises from a single rr interval . let nra , nra+b and nra+b+c be the number of rr intervals in regions a , ( a+b ) , and ( a+b+c ) respectively . the last term sd(nl ) in now that a time series of sd values is evaluated , let us consider a similar type of series , using the sequence of se values that were evaluated before . let se(nsm ) , se(nsa ) , se(nsa+b ) , se(nsa+b+c ) be the se sample that is closest to the time corresponding to nrm , nra , nra+b and nra+b+c rr intervals . using the set { se(1 ) , se(2 ) , se(nsm ) } , various statistical indices were evaluated , namely the mean ( semn(1 ) ) , 90% quantile value ( se90(1 ) ) , and 99% quantile value(se99(1 ) ) . subsequent samples semn(i ) , se90(i ) , se99(i ) were evaluated using the set { se(1 ) , se(2 ) , , thus , a time series similar to sd is obtained for semn , se90 , and se99 . in the latter three series , the separation in time is the same as se . in each of the time series of sd , semn , se90 , and se99 , the sample size of the rr intervals , and se values that are used to evaluate the samples in these series , increase with time . thus , unless there is a marked increase in the rr and se values with time , one would not expect the values in the constructed time series to show an increasing trend . these time series , which reflect the time variation of variability and disorder of the rr interval series , will be tested using the data described in section 2.2 , to determine their usefulness in providing prior warning to a paf subject of an impending af attack . the number of rr intervals nrm to construct the time series was chosen to ensure that the sample size was sufficient to obtain a reliable estimate of sd(1 ) , semn(1),se90(1),se99(1 ) , and that the number of samples for these series in region a are not small . numerical experiments showed that a choice of 300 rr intervals in region a was sufficient . this corresponds to an approximate time interval of 4 min and nearly 15% of the rr intervals and se samples found in region a. after constructing the time series sd , semn , se90,se99 , the difference in the mean values of these samples in regions a , ( a+b ) , and ( a+b+c ) were evaluated for all 22 subjects . this was done to determine whether the mean value in region ( a+b ) is greater than in region a , and the mean value in region ( a+b+c ) is greater than in region ( a+b ) . if this is so , then one would expect that there is a steady increase in these indices with time . such a trend would make these indices suitable markers to indicate an impending af attack . after evaluating the mean values of these indices in the three regions for the 22 subjects , a paired t - test was carried to determine whether the differences were statistically significant . the p - value showed that sd and se99 provided the best statistic , where the mean value in region ( a+b ) was greater than in region ( a ) , and the mean value in region ( a+b+c ) was greater than the mean value in region ( a+b+c ) . the p - value for semn showed that the differences between region ( a+b ) and a were statistically insignificant ( > 0.05 ) , while for se90 it was > 0.01 . for differences between region ( a+b+c ) and ( a+b ) , the p - values for semn and se90 were much better ; the p - value for semn was > 0.01 and for se90 it was 2.8(4 ) . however , in comparison , for se99 and sd , the p - value for the differences between region ( a+b ) and a were 0.0011 and 3.7(5 ) , respectively ; for differences between ( a+b+c ) and ( a+b ) , the p - values for se99 and sd were 1.3(6 ) and 1.5(7 ) , respectively . for sd , the values in region ( a+b ) were greater than region a , and values in region ( a+b+c ) were greater than in region ( a+b ) in all 100% of the paf subjects ; for se99 , this occurred in 86% and 100% of the paf subjects , respectively . these results prompted this study to focus only on the two indices , sd and se99 , in the development of an alert system for paf subjects . in fig . 2 , a time series plot of sd and se99 is shown using the data shown in fig . 1 . the results indicate an increase in the values of the variables as a function of time . in order to overcome the discontinuities that occur , so as to obtain a smoother variation and thus make it easier to observe the underlying increase in the values of these variables , an approximation of these variables was obtained using wavelet analysis . db4 is used and the approximation is constructed at level 10 . such an analysis can be carried out as a function of time once the time series is available . 3 shows the results of this analysis for the time series sd and se99 given in fig . wavelet analysis allows the approximation to be plotted simultaneously with that of sd and se99 . using these wavelet approximations , the difference in the mean values of these samples in regions a , ( a+b ) , and ( a+b+c ) were reevaluated for all 22 subjects . the results showed that for sd , the p - value for the mean value in region ( a+b ) to be greater than in region ( a ) is 3.2(5 ) , and the p - value for the mean value in region ( a+b+c ) to be greater than the mean value in region ( a+b ) is 2.3(7 ) . furthermore , for these two approximations of sd and se99 , all 100% of the subjects showed values in region ( a+b ) to be greater than in region a , and values in region ( a+b+c ) to be greater than in region ( a+b).the results of the statistical tests for the wavelet approximations are also summarized in table 1 . the results show that when an af attack is imminent for a paf subject , both sd and se99 begin to increase . thus , both sd and se99 are useful indices to alert a paf subject of an impending af attack , and to seek appropriate treatment . the increase in these indices just before an af attack is seen clearly in all the paf subjects studied here . this is encouraging , and can provide a basis to develop an alert system for paf subjects . this study was carried out in order to develop an early warning system to alert a paf subject before the onset of a sudden af attack . with the availability of mobile technology to obtain heartbeat data in real time , such a method would benefit paf patients greatly . this study used rr interval data sets collected in three time periods from normal sinus rhythm in paf subjects . one data set is far removed from any paf episode , while the other directly precedes it . the third set is a continuation of the second set , and is the period when a paf episode occurs . the method employed for this analysis is based on the use of sd , a linear measure of variability , and se , a non - linear index , which measures the disorder of the time series . in the latter case , the se is not used directly , but instead a time series constructed from the 0.99 quantile values of the se is used . so that the method is easily adaptable for a continuous real - time application , the time series of these indices are constructed not using constant distinct intervals , but where the indices are evaluated over intervals that change continuously with time . both these properties are evaluated separately as a function of time , using the concatenated rr interval series in the three periods . in order to obtain a smooth variation of these values as a function of time , wavelet analysis was carried out to obtain an approximation free of details . the result is an increasing value in these properties as the time of a paf attack approaches . the study shows that although either of the properties can be used for detection , the use of both will provide more confidence to the paf subject as to when to seek help . the evaluation of se and the quantile values are computationally fast , and provide additional evidence of the paf attack . the implementation of this methodology for practical use would involve the rapid transfer of the heartbeat data into a suitable device , where the rr data are preprocessed and analyzed . such a device can be either a part of the rr collection unit or separate . since the method assumes that there is an initial period of about 4 min where there is no paf , there is a time lag of about 4 min before the results of the analysis are seen . this time lag of 4 min appears to be the only limitation , since after this the sd and se99 values are evaluated after each sample of rr and se . although the technique developed here to signal the onset of paf shows much promise , the study was limited by the data set used . since the data used were from the public domain , personal details of the subjects , in particular their medical history and data collection history , were not available . however , the positive results observed here should be an impetus for further studies to be undertaken with data sets having more samples and known subject and data collection history . this study shows that a time series constructed using the sd and the 0.99 quantile values of the se using heartbeat data offers a feasible method to alert a paf subject of an imminent af attack . the method was tested using rr interval data from 22 paf subjects , and the results indicate that in all cases there was a steady increase in these indices as the onset of the af attack approached . this signature of a steady increase in these values provides a useful marker to alert the paf subject to seek help . with advances in mobile technology that allow rr interval measurements to be made in real time from heartbeat data , this study demonstrates that the use of this technique in the analysis of rr data could provide much - needed help for paf patients to better manage the onset of episodes .

backgroundknowledge of the onset of atrial fibrillation ( af ) episodes in patients with paroxysmal atrial fibrillation ( paf ) will enable them to better manage this condition . current advances in mobile technology allow rr interval data to be obtained in real time . an analysis technique using rr interval data is presented with a view to alert a subject before a paf episode.methodthe method is based on a time series of standard deviation and 0.99 quantile values of the spectral entropy , constructed from rr data . the rr data are taken from three time periods . the first time period has no occurrences of af for 45 min to either side of the time period . the second time period just precedes an af attack . both of these are of thirty minutes duration . the third time period of approximately 5 min follows the second , and is when af occurs.resultstwenty-two paf subjects were studied and in all cases there was a steady increase in the values of these indices as the onset of the af attack approached.conclusionthis method of analysis of rr interval data shows potential use to alert a paf subject before the onset of an af episode .

Introduction Method and materials Evaluation of spectral entropy Data used Analysis procedure Results Discussion Conclusion Conflict of interest

atrial fibrillation ( af ) is the most common major cardiac arrhythmia and affects a large number of people , particularly the elderly . early detection of paroxysmal atrial fibrillation ( paf ) in electrocardiography will enable the patient to obtain timely treatment to manage and prevent further complications . it is the purpose of this communication to determine the feasibility of using heart rate data to examine whether they could be used to alert a paf subject before a sudden attack of af . such a warning system would help a paf subject to seek preventive treatment immediately before such an attack occurs . before we proceed to develop such a system , let us examine the results of some relevant studies which involved analysis of rr intervals from paf subjects . the rr interval is the inverse of the heart rate . in a study of a large group of paf patients , rr interval data collected in the hour preceding paf and the twenty minutes before paf were divided into four 5-min period , and were analyzed . it was observed that there was a significant increase in the standard deviation ( sd ) of the rr intervals from 654 to 704 ms ( p<0.02 ) before the onset of paf . there was also a linear decrease in mean rr interval from 92516 to 90616 ms ( p<0.0002 ) before the onset of paf . in the frequency domain , there was a significant increase in high - frequency ( hf ) components before paf ( p<0.001 ) , and a decrease in low - frequency ( lf ) components ( p<0.0001 ) . another study on the onset of arrhythmia using holter monitoring also showed higher values in the sd of rr intervals in the last 5-min period before the start of paf , compared with the other intervals . the analysis was conducted using six 5-min periods before the onset , and a control 5-min period . in the frequency domain there was significant lowering in the lf region in the last 5 min before onset , while the hf was similar in all intervals , as well as in the control period . , in their analysis of paf , also observed an increase in the sd of rr intervals in the last 5 min before af occurred . however , heart rate variability analysis in the frequency domain did not reveal any significant changes before the onset of af . however , their study showed that nonlinear hr variability measures , the approximate entropy , and the altered fractal properties ( short term scaling exponent ) , showed significant changes before the onset of paf . there was a decrease in the approximate entropy , from 1.090.26 , 120 to 100 min before af , to 0.880.24 , 20 to 0 min before af , with p<0.001 ; in the short term scaling exponent , there was a decrease from 1.010.28 , 120 to 100 min before af , to 0.890.28 , 20 to 0 min before af . , the sd of the rr interval showed a significant increase when an af attack was imminent . that nonlinear changes accompany the onset of af is a hint to search for a nonlinear index , which is suitable for short segments of data . this led to the analysis of rr data in this paper to be carried out using sd of the rr intervals , a linear time domain index , and spectral entropy ( se ) , a non - linear index . however , unlike apen or sampen , the evaluation of se does not require a large amount of rr interval data , which is a distinct advantage in the development of a fast response warning system . se is a measure of the disorder in the frequency domain of a time series , and is evaluated using power spectra . the use of the randomness property of the rr intervals in this study was prompted by the fact that the distribution of the rr intervals exhibit random behavior in the af region . in this paper , both sd and se are examined with the view that they will complement each other and provide confidence to the paf subject that an af episode is imminent . the rr interval data in the first time period of thirty minutes , referred to as a in this paper , represent the time period during which there are no instances of paf , for 45 min on either side of that time range . the rr intervals in the second time period of thirty minutes , referred to as b in this paper , represent the time period just preceding an af attack . in this communication , the rr interval data in time periods a , b , and c are concatenated before analysis . this is done with a view to determine whether one could observe changes in sd and se in sufficient time so that the paf subject can take appropriate action before the onset of paf . both sd and se are examined in this paper instead of alone , with the view that they will complement each other and provide confidence to the paf subject that an af episode is imminent . the evaluation of se of the rr interval time series is carried by first symbolizing the rr data . one problem that is encountered in obtaining the power spectra of the rr data is that the time intervals between beats are not uniform . this is overcome by using a constant sampling interval and symbolizing the rr data , where the rr data are converted into a binary string . thus , each window contains a uniformly - sampled time series of 1 s and 0 s . the choice used for the sampling interval ( ) , the window length ( l ) , the shape of the window function in dft , and window separation length ( ls ) was guided by the choices of staniczenko et al . it was found that a window length with an average between 40 and 60 beats provided the best distinction between different regions of the rr data . since the number of beats in the window is small compared to the length of samples in the segment , the shape of the window was chosen to be rectangular . the first 30-min sample is distant from any paf episode , while the second 30-min sample directly precedes an episode . the third 5-min sample is a continuation of the second record , when the paf episode occurs . the first sample is distant from any paf episode , and has no paf episodes within 45 min on either side of the excerpt . the rr intervals obtained from such a heart rate time series were further de - trended and ectopic beats removed before being used for analysis . the vertical lines separate the periods a , b , and c. the focus of this paper is to develop a procedure that will alert the paf subject before the onset of the attack in c. in order to study and develop a warning system to alert a paf subject before the onset of an af attack , the rr interval time series of regions a , b , and c were concatenated , so that each subject was associated with just one rr interval time series . region a is further away from any af attack , region b precedes the attack , and region c is when the af attack occurs . in the manner described in section 2.1 , a time series of se samples suppose se ( i ) and t ( i ) are the se and the time in minutes corresponding to each window . let us digress at this point , and consider the evaluation of the sd time series , which is much easier , before a procedure is outlined how these se values can be used in the development of an alert system . let us consider an initial time interval containing nrm rr intervals , starting from the first rr interval in region a , and let sd(1 ) be the sd in the time interval that contains the range of rr intervals from [ 0 , nrm ] . an sd time series is then constructed { sd(1 ) , sd(2 ) , , sd(nl ) } , where sd(k ) corresponds to the sd in the time interval that contains the range of rr intervals [ 0 ( nrm+k1 ) ] . thus a time series of sd values is constructed where the first sd corresponds to nrm rr intervals , followed by a series of sd values corresponding to ( nrm+1 ) , ( nrm+2 ) , rr intervals . thus , the difference in the sd values between successive terms in the series arises from a single rr interval . let nra , nra+b and nra+b+c be the number of rr intervals in regions a , ( a+b ) , and ( a+b+c ) respectively . now that a time series of sd values is evaluated , let us consider a similar type of series , using the sequence of se values that were evaluated before . subsequent samples semn(i ) , se90(i ) , se99(i ) were evaluated using the set { se(1 ) , se(2 ) , , thus , a time series similar to sd is obtained for semn , se90 , and se99 . in each of the time series of sd , semn , se90 , and se99 , the sample size of the rr intervals , and se values that are used to evaluate the samples in these series , increase with time . thus , unless there is a marked increase in the rr and se values with time , one would not expect the values in the constructed time series to show an increasing trend . these time series , which reflect the time variation of variability and disorder of the rr interval series , will be tested using the data described in section 2.2 , to determine their usefulness in providing prior warning to a paf subject of an impending af attack . the evaluation of se of the rr interval time series is carried by first symbolizing the rr data . one problem that is encountered in obtaining the power spectra of the rr data is that the time intervals between beats are not uniform . this is overcome by using a constant sampling interval and symbolizing the rr data , where the rr data are converted into a binary string . thus , each window contains a uniformly - sampled time series of 1 s and 0 s . the choice used for the sampling interval ( ) , the window length ( l ) , the shape of the window function in dft , and window separation length ( ls ) was guided by the choices of staniczenko et al . it was found that a window length with an average between 40 and 60 beats provided the best distinction between different regions of the rr data . since the number of beats in the window is small compared to the length of samples in the segment , the shape of the window was chosen to be rectangular . the first 30-min sample is distant from any paf episode , while the second 30-min sample directly precedes an episode . the third 5-min sample is a continuation of the second record , when the paf episode occurs . the first sample is distant from any paf episode , and has no paf episodes within 45 min on either side of the excerpt . the rr intervals obtained from such a heart rate time series were further de - trended and ectopic beats removed before being used for analysis . the vertical lines separate the periods a , b , and c. the focus of this paper is to develop a procedure that will alert the paf subject before the onset of the attack in c. in order to study and develop a warning system to alert a paf subject before the onset of an af attack , the rr interval time series of regions a , b , and c were concatenated , so that each subject was associated with just one rr interval time series . region a is further away from any af attack , region b precedes the attack , and region c is when the af attack occurs . in the manner described in section 2.1 , a time series of se samples suppose se ( i ) and t ( i ) are the se and the time in minutes corresponding to each window . let us digress at this point , and consider the evaluation of the sd time series , which is much easier , before a procedure is outlined how these se values can be used in the development of an alert system . let us consider an initial time interval containing nrm rr intervals , starting from the first rr interval in region a , and let sd(1 ) be the sd in the time interval that contains the range of rr intervals from [ 0 , nrm ] . an sd time series is then constructed { sd(1 ) , sd(2 ) , , sd(nl ) } , where sd(k ) corresponds to the sd in the time interval that contains the range of rr intervals [ 0 ( nrm+k1 ) ] . thus a time series of sd values is constructed where the first sd corresponds to nrm rr intervals , followed by a series of sd values corresponding to ( nrm+1 ) , ( nrm+2 ) , rr intervals . the last term sd(nl ) in now that a time series of sd values is evaluated , let us consider a similar type of series , using the sequence of se values that were evaluated before . subsequent samples semn(i ) , se90(i ) , se99(i ) were evaluated using the set { se(1 ) , se(2 ) , , thus , a time series similar to sd is obtained for semn , se90 , and se99 . in each of the time series of sd , semn , se90 , and se99 , the sample size of the rr intervals , and se values that are used to evaluate the samples in these series , increase with time . thus , unless there is a marked increase in the rr and se values with time , one would not expect the values in the constructed time series to show an increasing trend . these time series , which reflect the time variation of variability and disorder of the rr interval series , will be tested using the data described in section 2.2 , to determine their usefulness in providing prior warning to a paf subject of an impending af attack . the number of rr intervals nrm to construct the time series was chosen to ensure that the sample size was sufficient to obtain a reliable estimate of sd(1 ) , semn(1),se90(1),se99(1 ) , and that the number of samples for these series in region a are not small . this corresponds to an approximate time interval of 4 min and nearly 15% of the rr intervals and se samples found in region a. after constructing the time series sd , semn , se90,se99 , the difference in the mean values of these samples in regions a , ( a+b ) , and ( a+b+c ) were evaluated for all 22 subjects . if this is so , then one would expect that there is a steady increase in these indices with time . after evaluating the mean values of these indices in the three regions for the 22 subjects , a paired t - test was carried to determine whether the differences were statistically significant . for sd , the values in region ( a+b ) were greater than region a , and values in region ( a+b+c ) were greater than in region ( a+b ) in all 100% of the paf subjects ; for se99 , this occurred in 86% and 100% of the paf subjects , respectively . these results prompted this study to focus only on the two indices , sd and se99 , in the development of an alert system for paf subjects . 2 , a time series plot of sd and se99 is shown using the data shown in fig . the results indicate an increase in the values of the variables as a function of time . in order to overcome the discontinuities that occur , so as to obtain a smoother variation and thus make it easier to observe the underlying increase in the values of these variables , an approximation of these variables was obtained using wavelet analysis . such an analysis can be carried out as a function of time once the time series is available . using these wavelet approximations , the difference in the mean values of these samples in regions a , ( a+b ) , and ( a+b+c ) were reevaluated for all 22 subjects . the results showed that for sd , the p - value for the mean value in region ( a+b ) to be greater than in region ( a ) is 3.2(5 ) , and the p - value for the mean value in region ( a+b+c ) to be greater than the mean value in region ( a+b ) is 2.3(7 ) . furthermore , for these two approximations of sd and se99 , all 100% of the subjects showed values in region ( a+b ) to be greater than in region a , and values in region ( a+b+c ) to be greater than in region ( a+b).the results of the statistical tests for the wavelet approximations are also summarized in table 1 . the results show that when an af attack is imminent for a paf subject , both sd and se99 begin to increase . thus , both sd and se99 are useful indices to alert a paf subject of an impending af attack , and to seek appropriate treatment . the increase in these indices just before an af attack is seen clearly in all the paf subjects studied here . this study was carried out in order to develop an early warning system to alert a paf subject before the onset of a sudden af attack . with the availability of mobile technology to obtain heartbeat data in real time , such a method would benefit paf patients greatly . this study used rr interval data sets collected in three time periods from normal sinus rhythm in paf subjects . the third set is a continuation of the second set , and is the period when a paf episode occurs . the method employed for this analysis is based on the use of sd , a linear measure of variability , and se , a non - linear index , which measures the disorder of the time series . in the latter case , the se is not used directly , but instead a time series constructed from the 0.99 quantile values of the se is used . so that the method is easily adaptable for a continuous real - time application , the time series of these indices are constructed not using constant distinct intervals , but where the indices are evaluated over intervals that change continuously with time . the result is an increasing value in these properties as the time of a paf attack approaches . the evaluation of se and the quantile values are computationally fast , and provide additional evidence of the paf attack . the implementation of this methodology for practical use would involve the rapid transfer of the heartbeat data into a suitable device , where the rr data are preprocessed and analyzed . such a device can be either a part of the rr collection unit or separate . since the method assumes that there is an initial period of about 4 min where there is no paf , there is a time lag of about 4 min before the results of the analysis are seen . although the technique developed here to signal the onset of paf shows much promise , the study was limited by the data set used . this study shows that a time series constructed using the sd and the 0.99 quantile values of the se using heartbeat data offers a feasible method to alert a paf subject of an imminent af attack . the method was tested using rr interval data from 22 paf subjects , and the results indicate that in all cases there was a steady increase in these indices as the onset of the af attack approached . this signature of a steady increase in these values provides a useful marker to alert the paf subject to seek help . with advances in mobile technology that allow rr interval measurements to be made in real time from heartbeat data , this study demonstrates that the use of this technique in the analysis of rr data could provide much - needed help for paf patients to better manage the onset of episodes .

urinary calculi are commonly encountered in clinical practice and their composition or mineralogy has a crucial role in determining patient management . for example , uric acid ( ua ) calculi can be treated by oral chemolysis , thus avoiding more invasive therapies , whereas certain calculi like calcium oxalate monohydrate ( com ) calculi , which are denser in nature , are less amenable to extracorporeal shock wave lithotripsy . urine biochemical parameters such as urinary sodium , calcium , oxalate , and ua may provide clues to stone composition . the hounsfield unit ( hu ) value of urinary calculi obtained from noncontrast computed tomography ( ncct ) has been shown to affect treatment decisions in a few studies . however , the exact chemical composition of calculi can not be diagnosed by use of these methods . thus , in present clinical practice , stone composition is evaluated only after stone extraction . with the recent commercial availability of dual - energy computed tomography ( dect ) , many investigators have evaluated the ability of this scanning method to identify calculus composition before extraction , i.e. , in vivo . dect consists of scanning the same anatomic region at two different energy levels . because of inherent differences in atomic number , calculi of differing compositions exhibit disparate attenuation properties at different energy or kilovoltage levels moreover , dect also provides all anatomic information provided by conventional ncct . among the different types of dual - energy scanners , dual - source dect ( ds - dect ) scanners have been the most popular . the initial studies were conducted with dual - source scanners without added filtration on the x - ray tube ( known as first - generation dual - source scanners ) and were able to differentiate only ua - containing and calcium - containing calculi . with the addition of a tin filter in the second - generation dual - source scanner , a few phantom and in vivo studies have shown that differentiation even among the various non - ua - containing calculi is possible . at the same time , it is also important not to ignore the issue of the radiation dose delivered to the patient . traditionally , low - dose conventional ct for urolithiasis detection has been defined as ct imparting an effective dose less than 3 msv . in our study , we attempted to combine these two aspects of urolithiasis imaging , namely , accurately predicting the composition of urinary calculi and reducing the radiation dose imparted to the patient , by use of a second - generation ds - dect scanner . this was a single - institution prospective study conducted over a period of 2 years from january 2013 to december 2014 . it was approved by the ethics committee of post graduate institute of medical education and research ( pgimer ) , chandigarh , india . a total of 132 patients suspected of having a calculus in the kidney or ureter were included in the study . confirmatory stone analysis was not available in 80 of these 132 patients , because they did not undergo stone extraction . patients included in the study were scanned by use of a second - generation 128-slice ds - dect scanner ( somatom definition flash , siemens healthcare , forchheim , germany ) . the two x - ray tubes in the scanner were set to operate at 80 and 140 kv ( with added tin filtration ) with corresponding reference tube current - time products ( mas ) of 105 and 41 mas . these reference mas levels represent approximately one - fourth of the manufacturer - prescribed reference mas used for routine ds - dect . the caredose4d ( siemens healthcare ) was used to further adapt tube current to individual body habitus . collimation was 64 mm0.6 mm with z - flying focal spot and the pitch was 0.7 . weighted average 1-mm thick images ( weighting factor , 0.3 ) were reconstructed by using a standard soft - tissue kernel ( q30f ) . the dual - energy ( de ) ratio and weighted average hu value of each calculus were recorded by manually placing circular regions of interest ( rois ) on the calculi ( fig . the de ratio was obtained by dividing the attenuation value of the calculus at 80 kv by its attenuation value at 140 kv . circular rois were placed in the aortic lumen at the level of the renal pelvis and in the urinary bladder lumen at the level of the femoral head . the standard deviation ( sd ) was recorded in these rois as a quantitative measure of image noise . in addition , waist circumference was measured on axial sections at the level of the midpoint between the lower margin of the last rib and the top of the iliac crest . two radiologists blinded to the patient 's details were provided the images to assess the quality of the weighted average images subjectively . the subjective rating of study quality was based on the radiologists ' confidence in detecting the calculi , related soft tissue attenuation changes such as perinephric stranding and soft tissue ureteric rim , subjective assessment of the amount of image noise , and image sharpness . by use of these criteria , the two observers rated the studies as being good , average , or poor in quality . stones extracted during percutaneous nephrolithotomy or ureteroscopy were subjected to fourier transform infrared spectroscopy ( ftirs ) . the spectrum was recorded in the spectrumtwo ftirs device ( perkin elmer , buckinghamshire , uk ) . according to the ftirs results , the calculi were classed into five groups : mixed calcium oxalate monohydrate and dihydrate ( com+cod ) , com , carbonate apatite ( ca ) , struvite , and ua . the calculi were then retrospectively compared with their hu values and de ratios . analysis of variance ( anova ) was used to determine if significant differences existed between the de ratio values of each calculus group . receiver operating characteristic ( roc ) curve analysis was used to identify optimal cutoff de ratio values to differentiate the various groups of calculi . image noise and patient radiation dose were compared with patient waist circumference by use of correlation analysis . patients included in the study were scanned by use of a second - generation 128-slice ds - dect scanner ( somatom definition flash , siemens healthcare , forchheim , germany ) . the two x - ray tubes in the scanner were set to operate at 80 and 140 kv ( with added tin filtration ) with corresponding reference tube current - time products ( mas ) of 105 and 41 mas . these reference mas levels represent approximately one - fourth of the manufacturer - prescribed reference mas used for routine ds - dect . the caredose4d ( siemens healthcare ) was used to further adapt tube current to individual body habitus . collimation was 64 mm0.6 mm with z - flying focal spot and the pitch was 0.7 . weighted average 1-mm thick images ( weighting factor , 0.3 ) were reconstructed by using a standard soft - tissue kernel ( q30f ) . the dual - energy ( de ) ratio and weighted average hu value of each calculus were recorded by manually placing circular regions of interest ( rois ) on the calculi ( fig . 1 ) . the de ratio was obtained by dividing the attenuation value of the calculus at 80 kv by its attenuation value at 140 kv . circular rois were placed in the aortic lumen at the level of the renal pelvis and in the urinary bladder lumen at the level of the femoral head . the standard deviation ( sd ) in addition , waist circumference was measured on axial sections at the level of the midpoint between the lower margin of the last rib and the top of the iliac crest . two radiologists blinded to the patient 's details were provided the images to assess the quality of the weighted average images subjectively . the subjective rating of study quality was based on the radiologists ' confidence in detecting the calculi , related soft tissue attenuation changes such as perinephric stranding and soft tissue ureteric rim , subjective assessment of the amount of image noise , and image sharpness . by use of these criteria , the two observers rated the studies as being good , average , or poor in quality . stones extracted during percutaneous nephrolithotomy or ureteroscopy were subjected to fourier transform infrared spectroscopy ( ftirs ) . the spectrum was recorded in the spectrumtwo ftirs device ( perkin elmer , buckinghamshire , uk ) . according to the ftirs results , the calculi were classed into five groups : mixed calcium oxalate monohydrate and dihydrate ( com+cod ) , com , carbonate apatite ( ca ) , struvite , and ua . the calculi were then retrospectively compared with their hu values and de ratios . analysis of variance ( anova ) was used to determine if significant differences existed between the de ratio values of each calculus group . receiver operating characteristic ( roc ) curve analysis was used to identify optimal cutoff de ratio values to differentiate the various groups of calculi . image noise and patient radiation dose were compared with patient waist circumference by use of correlation analysis . the study population consisted of 37 males ( 71.2% ) and 15 females ( 28.8% ) with a mean age of 39 years ( sd , 12.17 ; range , 20 - 63 years ) . ftirs analysis showed that 29% of the calculi ( n=40 ) were pure stones , whereas the remaining stones were mixed stones . all of the calculi of mixed composition had a principal constituent stone type contributing to at least two - thirds ( > 66% ) of the stone on the basis of the ftirs analysis . the number of calculi in each group was as follows : ua ( n=17 ) , struvite ( n=3 ) , com+cod ( n=84 ) , com ( n=28 ) , and ca ( n=5 ) . the mean calculus size was 15.2 mm ( range , 4 - 31 mm ; sd , 5.9 mm ) . there were 29 calculi measuring < 10 mm in diameter , of which 28 were calcium oxalate calculi and one was a ca calculus . all of these calculi were correctly classified as calcium - containing calculi by using de ratio values . the majority of the stones ( ~97% ) were located in the kidneys ; five stones were located in the ureters . all stones in the ureteric location were calcium oxalate stones and were classified correctly by using the de ratio . the distribution of the hu values and de ratios of the calculi is depicted in table 1 . anova analysis showed that hu values could differentiate only ua calculi and calcium - containing calculi confidently ( p<0.001 ) . roc analysis also showed a significant difference between ua calculi and other calculi ( area under curve=0.952 ) with a cutoff of < 615 hu having 94.1% sensitivity and 94.2% specificity for identifying ua calculi . anova analysis showed that there was a significant difference in the de ratio between the groups ( p<0.001 ) . uric acid calculi had the lowest de ratio value , followed by progressively higher values in struvite , calcium oxalate , and ca calculi . there was no overlap in the de ratio of ua and other calculi , with a de ratio of < 1.22 having 100% sensitivity and specificity for identifying ua calculi . the results of the roc curve analysis of the de ratios used to differentiate between struvite , calcium oxalate , and calcium carbonate and the further attempted subclassification of calcium oxalate calculi are depicted in table 2 . the mean waist circumference of the study population was 85.25 cm ( sd , 11.2 ; range , 66 - 110 cm ) . the image quality was assessed by two observers independently and was scored as good , average , or poor . none of the studies were rated as being poor or uninterpretable in quality by either observer . we note that high waist circumference did not have a detrimental effect on study quality . the noise measured in the aortic lumen showed only a weak positive correlation with the waist circumference ( pearson correlation coefficient=0.283 , p<0.05 ) . noise measured in the urinary bladder did not show a significant correlation with waist circumference . there was a significant correlation between the waist circumference and the volumetric computed tomography dose index ( ctdivol ) with a pearson correlation coefficient of 0.917 ( p<0.01 ) . the mean dose length product ( dlp ) was 103.13 mgycm ( sd , 27.9 ) . the mean effective dose was obtained by multiplying the dlp by a weighting factor of 0.018 msv / mgycm , which yielded a mean effective dose of 1.85 msv ( sd , 0.5 ) . the mean calculus size was 15.2 mm ( range , 4 - 31 mm ; sd , 5.9 mm ) . there were 29 calculi measuring < 10 mm in diameter , of which 28 were calcium oxalate calculi and one was a ca calculus . all of these calculi were correctly classified as calcium - containing calculi by using de ratio values . the majority of the stones ( ~97% ) were located in the kidneys ; five stones were located in the ureters . all stones in the ureteric location were calcium oxalate stones and were classified correctly by using the de ratio . the distribution of the hu values and de ratios of the calculi is depicted in table 1 . anova analysis showed that hu values could differentiate only ua calculi and calcium - containing calculi confidently ( p<0.001 ) . roc analysis also showed a significant difference between ua calculi and other calculi ( area under curve=0.952 ) with a cutoff of < 615 hu having 94.1% sensitivity and 94.2% specificity for identifying ua calculi . anova analysis showed that there was a significant difference in the de ratio between the groups ( p<0.001 ) . uric acid calculi had the lowest de ratio value , followed by progressively higher values in struvite , calcium oxalate , and ca calculi . there was no overlap in the de ratio of ua and other calculi , with a de ratio of < 1.22 having 100% sensitivity and specificity for identifying ua calculi . the results of the roc curve analysis of the de ratios used to differentiate between struvite , calcium oxalate , and calcium carbonate and the further attempted subclassification of calcium oxalate calculi are depicted in table 2 . the mean waist circumference of the study population was 85.25 cm ( sd , 11.2 ; range , 66 - 110 cm ) . the image quality was assessed by two observers independently and was scored as good , average , or poor . none of the studies were rated as being poor or uninterpretable in quality by either observer . we note that high waist circumference did not have a detrimental effect on study quality . the noise measured in the aortic lumen showed only a weak positive correlation with the waist circumference ( pearson correlation coefficient=0.283 , p<0.05 ) . noise measured in the urinary bladder did not show a significant correlation with waist circumference . there was a significant correlation between the waist circumference and the volumetric computed tomography dose index ( ctdivol ) with a pearson correlation coefficient of 0.917 ( p<0.01 ) . the mean dose length product ( dlp ) was 103.13 mgycm ( sd , 27.9 ) . the mean effective dose was obtained by multiplying the dlp by a weighting factor of 0.018 msv / mgycm , which yielded a mean effective dose of 1.85 msv ( sd , 0.5 ) . in this study , dect was accurate in differentiating ua , struvite , and ca calculi from calcium oxalate calculi . both struvite and ca stones are associated with urinary tract infections . for stones associated with infection , the risk of posttreatment sepsis and recurrence of calculi is higher . hence , identification of these calculi in vivo may help in directing appropriate therapy even before stone extraction . the discriminant de ratio values obtained in our study for identifying calculus composition are depicted in fig . these values are similar to those reported by a phantom study performed by qu et al . ( < 1.19 for ua , 1.19 - 1.46 for cystine , 1.46 - 1.60 for struvite , 1.60 - 1.71 calcium oxalate and struvite , and > 1.71 for ca and hydroxyapatite ) except for struvite stones . this may have been due to the small sample size of struvite calculi ( n=3 in our study and n=4 in the phantom study ) . the study by acharya et al . is the only previous in vivo study ( to the best of our knowledge ) performed in the second - generation ds - dect scanner that has shown the possibility of subdifferentiating non - ua calculi . however , the tube voltage setting used in their study was 100/140 sn ( tin filter ) compared with 80/140 sn used in our study . whereas acharya et al . showed that the cutoff de ratio value of < 1.335 had 100% specificity for detection of com calculi resistant to extracorporeal shock wave lithotripsy , we could not differentiate pure com from mixed com+cod calculi in our study . in our study , we included calculi of mixed composition , with the caveat that the major constituent should contribute to at least two - thirds of the total mass of the calculus . most studies done earlier included only pure stones or mixed stones in which the major constituent contributed to at least 90% of the total , which does not reflect clinical reality . thus , our study sample is likely to be more representative of the patient population with urolithiasis . radiation exposure in routine ct studies of the abdomen varies between 3 and 25 msv . it has been shown that radiation exposure in range of 10 to 100 msv in atomic bomb survivors is associated with increased risk of carcinogenesis . with epidemiological data lacking for the effects of radiation doses below 10 msv , the linear no threshold model , which states that there is no safe threshold radiation dose below which radiation - induced carcinogenesis does not occur , has been followed . relatively few studies have assessed the role of low - dose dect for predicting urinary calculus composition , and non - ua calculi could not be confidently differentiated in those studies . acquired a routine single - energy low - dose ct followed by a targeted de acquisition of the region containing the calculus . this protocol is difficult to implement in patients with multiple calculi and moreover requires the presence of a radiologist to review the images while the patient is in the scanner . to the best of our knowledge , our study is the first to assess a low - dose dect protocol in the second - generation ds - dect scanner . the radiation doses obtained in our study represent a 3-fold reduction in radiation dose compared to standard - dose dect ( 4.5 - 7 msv ) and a 2- to 3-fold reduction compared to standard - dose single - energy ncct ( 4 - 5 msv ) . compared to low - dose single - energy ncct ( 0.9 - 1.9 msv ) -the recommended mode for urinary calculus evaluation - thus , this low - dose protocol can be relied upon to produce images of at least reasonable quality even in obese patients the limitations of our study were as follows . the number of struvite and ca calculi encountered in our study was low and hence the de ratio values of these calculi need further validation by studies with larger sample size . moreover , we could not assess firsthand the impact of calculus composition detection using dect on patient management . this would require further studies in which the management decisions in patients with urolithiasis are made on the basis of the composition obtained by dect analysis . in this study , dect was accurate in differentiating ua , struvite , and ca calculi from calcium oxalate calculi . both struvite and ca stones are associated with urinary tract infections . for stones associated with infection , the risk of posttreatment sepsis and recurrence of calculi is higher . hence , identification of these calculi in vivo may help in directing appropriate therapy even before stone extraction . the discriminant de ratio values obtained in our study for identifying calculus composition are depicted in fig . these values are similar to those reported by a phantom study performed by qu et al . ( < 1.19 for ua , 1.19 - 1.46 for cystine , 1.46 - 1.60 for struvite , 1.60 - 1.71 calcium oxalate and struvite , and > 1.71 for ca and hydroxyapatite ) except for struvite stones . this may have been due to the small sample size of struvite calculi ( n=3 in our study and n=4 in the phantom study ) . the study by acharya et al . is the only previous in vivo study ( to the best of our knowledge ) performed in the second - generation ds - dect scanner that has shown the possibility of subdifferentiating non - ua calculi . however , the tube voltage setting used in their study was 100/140 sn ( tin filter ) compared with 80/140 sn used in our study . whereas acharya et al . showed that the cutoff de ratio value of < 1.335 had 100% specificity for detection of com calculi resistant to extracorporeal shock wave lithotripsy , we could not differentiate pure com from mixed com+cod calculi in our study . in our study , we included calculi of mixed composition , with the caveat that the major constituent should contribute to at least two - thirds of the total mass of the calculus . most studies done earlier included only pure stones or mixed stones in which the major constituent contributed to at least 90% of the total , which does not reflect clinical reality . thus , our study sample is likely to be more representative of the patient population with urolithiasis . radiation exposure in routine ct studies of the abdomen varies between 3 and 25 msv . it has been shown that radiation exposure in range of 10 to 100 msv in atomic bomb survivors is associated with increased risk of carcinogenesis . with epidemiological data lacking for the effects of radiation doses below 10 msv , the linear no threshold model , which states that there is no safe threshold radiation dose below which radiation - induced carcinogenesis does not occur , has been followed . relatively few studies have assessed the role of low - dose dect for predicting urinary calculus composition , and non - ua calculi could not be confidently differentiated in those studies . acquired a routine single - energy low - dose ct followed by a targeted de acquisition of the region containing the calculus . this protocol is difficult to implement in patients with multiple calculi and moreover requires the presence of a radiologist to review the images while the patient is in the scanner . to the best of our knowledge , our study is the first to assess a low - dose dect protocol in the second - generation ds - dect scanner . the radiation doses obtained in our study represent a 3-fold reduction in radiation dose compared to standard - dose dect ( 4.5 - 7 msv ) and a 2- to 3-fold reduction compared to standard - dose single - energy ncct ( 4 - 5 msv ) . compared to low - dose single - energy ncct ( 0.9 - 1.9 msv ) -the recommended mode for urinary calculus evaluation - thus , this low - dose protocol can be relied upon to produce images of at least reasonable quality even in obese patients the number of struvite and ca calculi encountered in our study was low and hence the de ratio values of these calculi need further validation by studies with larger sample size . moreover , we could not assess firsthand the impact of calculus composition detection using dect on patient management . this would require further studies in which the management decisions in patients with urolithiasis are made on the basis of the composition obtained by dect analysis . low - dose dect can identify calcium oxalate , ua , struvite , and ca urinary calculi in vivo with a high degree of accuracy while reducing patient radiation exposure significantly and without compromising study quality . low - dose dect could possibly replace conventional ncct as the diagnostic test of choice for urolithiasis .

purposethis study aimed to assess the accuracy of low - dose dual - energy computed tomography ( dect ) in predicting the composition of urinary calculi.materials and methodsa total of 52 patients with urinary calculi were scanned with a 128-slice dual - source dect scanner by use of a low - dose protocol . dual - energy ( de ) ratio , weighted average hounsfield unit ( hu ) of calculi , radiation dose , and image noise levels were recorded . two radiologists independently rated study quality . stone composition was assessed after extraction by fourier transform infrared spectroscopy ( ftirs ) . analysis of variance was used to determine if the differences in hu values and de ratios between the various calculus groups were significant . threshold cutoff values to classify the calculi into separate groups were identified by receiver operating characteristic curve analysis.resultsa total of 137 calculi were detected . ftirs analysis differentiated the calculi into five groups : uric acid ( n=17 ) , struvite ( n=3 ) , calcium oxalate monohydrate and dihydrate ( com - cod , n=84 ) , calcium oxalate monohydrate ( com , n=28 ) , and carbonate apatite ( n=5 ) . the hu value could differentiate only uric acid calculi from calcified calculi ( p<0.001 ) . the de ratio could confidently differentiate uric acid , struvite , calcium oxalate , and carbonate apatite calculi ( p<0.001 ) with cutoff values of 1.12 , 1.34 , and 1.66 , respectively , giving > 80% sensitivity and specificity to differentiate them . the de ratio could not differentiate com from com - cod calculi . no study was rated poor in quality by either of the observers . the mean radiation dose was 1.8 msv.conclusionslow-dose dect accurately predicts urinary calculus composition in vivo while simultaneously reducing radiation exposure without compromising study quality .

INTRODUCTION MATERIALS AND METHODS 1. Image acquisition 2. Image interpretation and rating of study quality 3. Confirmatory analysis of stone composition 4. Statistical analysis RESULTS 1. Size and location of calculi 2. HU value and DE ratio 3. Waist circumference, image quality, and radiation dose DISCUSSION 1. Calculus composition 2. Radiation dose, patient size, and image quality 3. Study limitations CONCLUSIONS

for example , uric acid ( ua ) calculi can be treated by oral chemolysis , thus avoiding more invasive therapies , whereas certain calculi like calcium oxalate monohydrate ( com ) calculi , which are denser in nature , are less amenable to extracorporeal shock wave lithotripsy . urine biochemical parameters such as urinary sodium , calcium , oxalate , and ua may provide clues to stone composition . the hounsfield unit ( hu ) value of urinary calculi obtained from noncontrast computed tomography ( ncct ) has been shown to affect treatment decisions in a few studies . however , the exact chemical composition of calculi can not be diagnosed by use of these methods . thus , in present clinical practice , stone composition is evaluated only after stone extraction . with the recent commercial availability of dual - energy computed tomography ( dect ) , many investigators have evaluated the ability of this scanning method to identify calculus composition before extraction , i.e. , in vivo . among the different types of dual - energy scanners , dual - source dect ( ds - dect ) scanners have been the most popular . the initial studies were conducted with dual - source scanners without added filtration on the x - ray tube ( known as first - generation dual - source scanners ) and were able to differentiate only ua - containing and calcium - containing calculi . with the addition of a tin filter in the second - generation dual - source scanner , a few phantom and in vivo studies have shown that differentiation even among the various non - ua - containing calculi is possible . at the same time , it is also important not to ignore the issue of the radiation dose delivered to the patient . traditionally , low - dose conventional ct for urolithiasis detection has been defined as ct imparting an effective dose less than 3 msv . in our study , we attempted to combine these two aspects of urolithiasis imaging , namely , accurately predicting the composition of urinary calculi and reducing the radiation dose imparted to the patient , by use of a second - generation ds - dect scanner . it was approved by the ethics committee of post graduate institute of medical education and research ( pgimer ) , chandigarh , india . patients included in the study were scanned by use of a second - generation 128-slice ds - dect scanner ( somatom definition flash , siemens healthcare , forchheim , germany ) . the two x - ray tubes in the scanner were set to operate at 80 and 140 kv ( with added tin filtration ) with corresponding reference tube current - time products ( mas ) of 105 and 41 mas . the caredose4d ( siemens healthcare ) was used to further adapt tube current to individual body habitus . the dual - energy ( de ) ratio and weighted average hu value of each calculus were recorded by manually placing circular regions of interest ( rois ) on the calculi ( fig . the de ratio was obtained by dividing the attenuation value of the calculus at 80 kv by its attenuation value at 140 kv . circular rois were placed in the aortic lumen at the level of the renal pelvis and in the urinary bladder lumen at the level of the femoral head . the standard deviation ( sd ) was recorded in these rois as a quantitative measure of image noise . in addition , waist circumference was measured on axial sections at the level of the midpoint between the lower margin of the last rib and the top of the iliac crest . two radiologists blinded to the patient 's details were provided the images to assess the quality of the weighted average images subjectively . the subjective rating of study quality was based on the radiologists ' confidence in detecting the calculi , related soft tissue attenuation changes such as perinephric stranding and soft tissue ureteric rim , subjective assessment of the amount of image noise , and image sharpness . by use of these criteria , the two observers rated the studies as being good , average , or poor in quality . stones extracted during percutaneous nephrolithotomy or ureteroscopy were subjected to fourier transform infrared spectroscopy ( ftirs ) . according to the ftirs results , the calculi were classed into five groups : mixed calcium oxalate monohydrate and dihydrate ( com+cod ) , com , carbonate apatite ( ca ) , struvite , and ua . the calculi were then retrospectively compared with their hu values and de ratios . analysis of variance ( anova ) was used to determine if significant differences existed between the de ratio values of each calculus group . receiver operating characteristic ( roc ) curve analysis was used to identify optimal cutoff de ratio values to differentiate the various groups of calculi . image noise and patient radiation dose were compared with patient waist circumference by use of correlation analysis . patients included in the study were scanned by use of a second - generation 128-slice ds - dect scanner ( somatom definition flash , siemens healthcare , forchheim , germany ) . the two x - ray tubes in the scanner were set to operate at 80 and 140 kv ( with added tin filtration ) with corresponding reference tube current - time products ( mas ) of 105 and 41 mas . these reference mas levels represent approximately one - fourth of the manufacturer - prescribed reference mas used for routine ds - dect . the caredose4d ( siemens healthcare ) was used to further adapt tube current to individual body habitus . weighted average 1-mm thick images ( weighting factor , 0.3 ) were reconstructed by using a standard soft - tissue kernel ( q30f ) . the dual - energy ( de ) ratio and weighted average hu value of each calculus were recorded by manually placing circular regions of interest ( rois ) on the calculi ( fig . the de ratio was obtained by dividing the attenuation value of the calculus at 80 kv by its attenuation value at 140 kv . the standard deviation ( sd ) in addition , waist circumference was measured on axial sections at the level of the midpoint between the lower margin of the last rib and the top of the iliac crest . two radiologists blinded to the patient 's details were provided the images to assess the quality of the weighted average images subjectively . the subjective rating of study quality was based on the radiologists ' confidence in detecting the calculi , related soft tissue attenuation changes such as perinephric stranding and soft tissue ureteric rim , subjective assessment of the amount of image noise , and image sharpness . by use of these criteria , the two observers rated the studies as being good , average , or poor in quality . stones extracted during percutaneous nephrolithotomy or ureteroscopy were subjected to fourier transform infrared spectroscopy ( ftirs ) . according to the ftirs results , the calculi were classed into five groups : mixed calcium oxalate monohydrate and dihydrate ( com+cod ) , com , carbonate apatite ( ca ) , struvite , and ua . the calculi were then retrospectively compared with their hu values and de ratios . analysis of variance ( anova ) was used to determine if significant differences existed between the de ratio values of each calculus group . receiver operating characteristic ( roc ) curve analysis was used to identify optimal cutoff de ratio values to differentiate the various groups of calculi . image noise and patient radiation dose were compared with patient waist circumference by use of correlation analysis . the study population consisted of 37 males ( 71.2% ) and 15 females ( 28.8% ) with a mean age of 39 years ( sd , 12.17 ; range , 20 - 63 years ) . ftirs analysis showed that 29% of the calculi ( n=40 ) were pure stones , whereas the remaining stones were mixed stones . all of the calculi of mixed composition had a principal constituent stone type contributing to at least two - thirds ( > 66% ) of the stone on the basis of the ftirs analysis . the number of calculi in each group was as follows : ua ( n=17 ) , struvite ( n=3 ) , com+cod ( n=84 ) , com ( n=28 ) , and ca ( n=5 ) . all of these calculi were correctly classified as calcium - containing calculi by using de ratio values . the majority of the stones ( ~97% ) were located in the kidneys ; five stones were located in the ureters . all stones in the ureteric location were calcium oxalate stones and were classified correctly by using the de ratio . the distribution of the hu values and de ratios of the calculi is depicted in table 1 . anova analysis showed that hu values could differentiate only ua calculi and calcium - containing calculi confidently ( p<0.001 ) . roc analysis also showed a significant difference between ua calculi and other calculi ( area under curve=0.952 ) with a cutoff of < 615 hu having 94.1% sensitivity and 94.2% specificity for identifying ua calculi . anova analysis showed that there was a significant difference in the de ratio between the groups ( p<0.001 ) . uric acid calculi had the lowest de ratio value , followed by progressively higher values in struvite , calcium oxalate , and ca calculi . there was no overlap in the de ratio of ua and other calculi , with a de ratio of < 1.22 having 100% sensitivity and specificity for identifying ua calculi . the results of the roc curve analysis of the de ratios used to differentiate between struvite , calcium oxalate , and calcium carbonate and the further attempted subclassification of calcium oxalate calculi are depicted in table 2 . the mean waist circumference of the study population was 85.25 cm ( sd , 11.2 ; range , 66 - 110 cm ) . none of the studies were rated as being poor or uninterpretable in quality by either observer . we note that high waist circumference did not have a detrimental effect on study quality . there was a significant correlation between the waist circumference and the volumetric computed tomography dose index ( ctdivol ) with a pearson correlation coefficient of 0.917 ( p<0.01 ) . the mean effective dose was obtained by multiplying the dlp by a weighting factor of 0.018 msv / mgycm , which yielded a mean effective dose of 1.85 msv ( sd , 0.5 ) . the mean calculus size was 15.2 mm ( range , 4 - 31 mm ; sd , 5.9 mm ) . all of these calculi were correctly classified as calcium - containing calculi by using de ratio values . all stones in the ureteric location were calcium oxalate stones and were classified correctly by using the de ratio . the distribution of the hu values and de ratios of the calculi is depicted in table 1 . anova analysis showed that hu values could differentiate only ua calculi and calcium - containing calculi confidently ( p<0.001 ) . roc analysis also showed a significant difference between ua calculi and other calculi ( area under curve=0.952 ) with a cutoff of < 615 hu having 94.1% sensitivity and 94.2% specificity for identifying ua calculi . anova analysis showed that there was a significant difference in the de ratio between the groups ( p<0.001 ) . uric acid calculi had the lowest de ratio value , followed by progressively higher values in struvite , calcium oxalate , and ca calculi . there was no overlap in the de ratio of ua and other calculi , with a de ratio of < 1.22 having 100% sensitivity and specificity for identifying ua calculi . the results of the roc curve analysis of the de ratios used to differentiate between struvite , calcium oxalate , and calcium carbonate and the further attempted subclassification of calcium oxalate calculi are depicted in table 2 . the mean waist circumference of the study population was 85.25 cm ( sd , 11.2 ; range , 66 - 110 cm ) . none of the studies were rated as being poor or uninterpretable in quality by either observer . there was a significant correlation between the waist circumference and the volumetric computed tomography dose index ( ctdivol ) with a pearson correlation coefficient of 0.917 ( p<0.01 ) . the mean dose length product ( dlp ) was 103.13 mgycm ( sd , 27.9 ) . the mean effective dose was obtained by multiplying the dlp by a weighting factor of 0.018 msv / mgycm , which yielded a mean effective dose of 1.85 msv ( sd , 0.5 ) . in this study , dect was accurate in differentiating ua , struvite , and ca calculi from calcium oxalate calculi . the discriminant de ratio values obtained in our study for identifying calculus composition are depicted in fig . ( < 1.19 for ua , 1.19 - 1.46 for cystine , 1.46 - 1.60 for struvite , 1.60 - 1.71 calcium oxalate and struvite , and > 1.71 for ca and hydroxyapatite ) except for struvite stones . this may have been due to the small sample size of struvite calculi ( n=3 in our study and n=4 in the phantom study ) . is the only previous in vivo study ( to the best of our knowledge ) performed in the second - generation ds - dect scanner that has shown the possibility of subdifferentiating non - ua calculi . showed that the cutoff de ratio value of < 1.335 had 100% specificity for detection of com calculi resistant to extracorporeal shock wave lithotripsy , we could not differentiate pure com from mixed com+cod calculi in our study . most studies done earlier included only pure stones or mixed stones in which the major constituent contributed to at least 90% of the total , which does not reflect clinical reality . radiation exposure in routine ct studies of the abdomen varies between 3 and 25 msv . it has been shown that radiation exposure in range of 10 to 100 msv in atomic bomb survivors is associated with increased risk of carcinogenesis . relatively few studies have assessed the role of low - dose dect for predicting urinary calculus composition , and non - ua calculi could not be confidently differentiated in those studies . acquired a routine single - energy low - dose ct followed by a targeted de acquisition of the region containing the calculus . this protocol is difficult to implement in patients with multiple calculi and moreover requires the presence of a radiologist to review the images while the patient is in the scanner . to the best of our knowledge , our study is the first to assess a low - dose dect protocol in the second - generation ds - dect scanner . the radiation doses obtained in our study represent a 3-fold reduction in radiation dose compared to standard - dose dect ( 4.5 - 7 msv ) and a 2- to 3-fold reduction compared to standard - dose single - energy ncct ( 4 - 5 msv ) . compared to low - dose single - energy ncct ( 0.9 - 1.9 msv ) -the recommended mode for urinary calculus evaluation - thus , this low - dose protocol can be relied upon to produce images of at least reasonable quality even in obese patients the limitations of our study were as follows . the number of struvite and ca calculi encountered in our study was low and hence the de ratio values of these calculi need further validation by studies with larger sample size . moreover , we could not assess firsthand the impact of calculus composition detection using dect on patient management . this would require further studies in which the management decisions in patients with urolithiasis are made on the basis of the composition obtained by dect analysis . in this study , dect was accurate in differentiating ua , struvite , and ca calculi from calcium oxalate calculi . both struvite and ca stones are associated with urinary tract infections . the discriminant de ratio values obtained in our study for identifying calculus composition are depicted in fig . ( < 1.19 for ua , 1.19 - 1.46 for cystine , 1.46 - 1.60 for struvite , 1.60 - 1.71 calcium oxalate and struvite , and > 1.71 for ca and hydroxyapatite ) except for struvite stones . this may have been due to the small sample size of struvite calculi ( n=3 in our study and n=4 in the phantom study ) . is the only previous in vivo study ( to the best of our knowledge ) performed in the second - generation ds - dect scanner that has shown the possibility of subdifferentiating non - ua calculi . showed that the cutoff de ratio value of < 1.335 had 100% specificity for detection of com calculi resistant to extracorporeal shock wave lithotripsy , we could not differentiate pure com from mixed com+cod calculi in our study . in our study , we included calculi of mixed composition , with the caveat that the major constituent should contribute to at least two - thirds of the total mass of the calculus . radiation exposure in routine ct studies of the abdomen varies between 3 and 25 msv . it has been shown that radiation exposure in range of 10 to 100 msv in atomic bomb survivors is associated with increased risk of carcinogenesis . relatively few studies have assessed the role of low - dose dect for predicting urinary calculus composition , and non - ua calculi could not be confidently differentiated in those studies . acquired a routine single - energy low - dose ct followed by a targeted de acquisition of the region containing the calculus . this protocol is difficult to implement in patients with multiple calculi and moreover requires the presence of a radiologist to review the images while the patient is in the scanner . to the best of our knowledge , our study is the first to assess a low - dose dect protocol in the second - generation ds - dect scanner . the radiation doses obtained in our study represent a 3-fold reduction in radiation dose compared to standard - dose dect ( 4.5 - 7 msv ) and a 2- to 3-fold reduction compared to standard - dose single - energy ncct ( 4 - 5 msv ) . compared to low - dose single - energy ncct ( 0.9 - 1.9 msv ) -the recommended mode for urinary calculus evaluation - thus , this low - dose protocol can be relied upon to produce images of at least reasonable quality even in obese patients the number of struvite and ca calculi encountered in our study was low and hence the de ratio values of these calculi need further validation by studies with larger sample size . moreover , we could not assess firsthand the impact of calculus composition detection using dect on patient management . this would require further studies in which the management decisions in patients with urolithiasis are made on the basis of the composition obtained by dect analysis . low - dose dect can identify calcium oxalate , ua , struvite , and ca urinary calculi in vivo with a high degree of accuracy while reducing patient radiation exposure significantly and without compromising study quality . low - dose dect could possibly replace conventional ncct as the diagnostic test of choice for urolithiasis .

anaphylactic food allergies are rapid in onset and can be fatal if treatment is delayed ; thus , complete dietary avoidance of the allergen and timely access to treatment are essential . three studies found that children from low - income families face barriers to appropriate anaphylaxis management [ 57 ] . conversely , one study found that associations between allergy severity and health care utilization did not differ by poverty status and another study found no conclusive associations between the epinephrine autoinjectors ( eais ) availability and sociodemographic factors . a recent study on the economic impact of childhood food allergy in the united states found the overall economic cost of food allergy to be almost $ 25 billion annually . this study found that costs borne by families affected by food allergies include lost labour productivity , out - of - pocket costs , and opportunity costs . costs associated with food allergies are described as direct , indirect , or intangible [ 1113 ] . direct costs are financial costs incurred as a result of the food allergy ( including inpatient admissions , outpatient visits , medication costs , and the cost of suitable foods ) . indirect costs reflect time spent in various activities as a result of food allergy , including lost productivity and opportunity costs . intangible costs are losses of utility , such as experiences of pain , suffering , and grief , and can be measured by self - reported quality of life and well - being [ 12 , 15 ] . one recent study on the intangible costs of food allergy in four european countries found that patients with food hypersensitivity reported lower well - being than the control group . the authors note , however , that well - being was positively related to income and , thus , income compensation might improve welfare among food hypersensitive individuals . low - income individuals are differentially affected by the costs of food allergy , since people with allergy who also have low incomes may have greater difficulties in access to [ allergen - free ] food . it is intuitive that absolute costs of allergy medication or allergen - free foods pose a disproportionate burden to low - income families . although these costs are conventionally assessed quantitatively , exploring qualitatively assessed direct , indirect , and intangible costs of food allergies among low - income families is important for three reasons . first , low - income individuals may have poorer access to anaphylaxis treatment [ 5 , 6 ] and may therefore be at high risk of allergy - related harms . second , understanding barriers to anaphylaxis management or treatment faced by low - income individuals can facilitate the development of valid quantitative surveys intended to describe the overall scope of food allergies and treatment accessibility . third , exploring how low - income families cope with or manage anaphylactic allergies has both clinical and policy implications . qualitative research has enormous potential to make a distinctive contribution to knowledge about allergy management and to provide insights that quantitative studies can not . although extant research assesses direct , indirect , and intangible costs quantitatively , this study explores these costs qualitatively from the perspective of low - income families , which is a current gap in the allergy literature . findings from in - depth interviews with key informants and low - income families affected by food allergy begin to fill the gaps in our understanding around coping and management for this vulnerable population . the purpose of this study was to take an in - depth look at the direct , indirect and intangible costs that are particularly burdensome for low - income families in terms of anaphylactic food allergy management and treatment . in - depth , semi - structured interviews with key informants ( n = 10 ) and low - income individuals affected by food allergy ( n = 13 ) were conducted in southwestern ontario , canada . in ontario , the provincial health insurance plan covers the cost of physician and other medically necessary services . of note , however , individuals who are receiving social assistance or are 65 years of age or older are eligible for drug coverage through the provincial government . individuals who are not receiving social assistance either have private drug insurance through their employer or pay out of pocket for drugs . key informants were recruited from agencies working with low - income families in a context relevant to food allergy ( i.e. , food procurement or medication access ) ( table 1 ) . low - income participants were recruited via posters placed in these targeted agencies . over half ( 54% ) of participants were parents of allergic children ; the remainder were allergic adults . most ( 77% ) participants were on social assistance , which included drug benefits ( table 2 ) . verification of diagnosis was not requested from participants ' doctors because many participants reported not having a family doctor . all participants self - identified as being low income and were using community services offered specifically to low - income people . interrater reliability assessment between two transcripts achieved reliability of 0.70 . after a theme code set was developed , two researchers conducted intercoder tests to determine levels of coding agreement ( 70% agreement or higher is considered acceptable ) to ensure confidence in the coding scheme . over half ( 54% ) of low - income participants and 20% of key informants noted the high cost of eais as a barrier to anaphylaxis treatment for low - income families . eais cost about usd$100 , which many allergists consider to be high in relation to minimum wage and average daily earnings . participants often kept expired eais rather than replacing them promptly ; cost also affected their decision whether to purchase at all : when you have an anaphylactic reaction you do n't just have the fear in that whole process and then your body feeling terrible for two to five days after , you have also just lost $ 100 or $ 200 . ( allergic female ) when you have an anaphylactic reaction you do n't just have the fear in that whole process and then your body feeling terrible for two to five days after , you have also just lost $ 100 or $ 200 . ( allergic female ) one mother , unsure of her son 's eai expiry date , spoke of the impact her financial situation had on replacing expired eais : so coming up with a hundred dollars to replace it again , that is probably one of the reasons why i haven't checked to see if it is expired . ( mother of allergic toddler ) so coming up with a hundred dollars to replace it again , that is probably one of the reasons why i haven't checked to see if it is expired . ( mother of allergic toddler ) keeping expired eais was reported by 31% of low - income participants . one woman described her experience of living as a homeless youth with an anaphylactic allergy thus , they expire . i always carry around all of mine , and the first ones i ever try would be the expired ones , just to try it will save like money , while you are saving your life , right . sounds terrible to say it that way but it is really true . just with the cost of an epipen , like that is a lot of money especially if you are like fifteen and living on the street . so it is like take the expired ones first and then usually they work and if they do n't take the other one . i always carry around all of mine , and the first ones i ever try would be the expired ones , just to try it will save like money , while you are saving your life , right . sounds terrible to say it that way but it is really true . just with the cost of an epipen , like that is a lot of money especially if you are like fifteen and living on the street . so it is like take the expired ones first and then usually they work and if they do n't take the other one . ( allergic female ) some participants reported retaining an inadequate number of eais or not having one at all due to cost . when asked how many eais she had for her tree - nut - allergic son , a low - income mother said , just the one . these experiences were echoed by the perceptions of an allergist : i think there is a considerable amount especially in the lower income , that will make a decision based on , if i use that now , i am not going to have it for the next ten months . and a $ 100 item may be a big deal for somebody who is generating income below the poverty line . so i think that their reliance would still be on an antihistamine and prolonged care at home . ( allergist ) i think there is a considerable amount especially in the lower income , that will make a decision based on , if i use that now , i am not going to have it for the next ten months . and a $ 100 item may be a big deal for somebody who is generating income below the poverty line . so i think that their reliance would still be on an antihistamine and prolonged care at home . ( allergist ) this is problematic because the first line of anaphylaxis treatment is epinephrine ; antihistamines are not a recommended treatment , although they may be perceived as a low - cost alternative . i use liquid , so that way it gets into your system faster , as opposed to pill form . i use liquid , so that way it gets into your system faster , as opposed to pill form . ( allergic female ) a peanut - allergic woman who had been hospitalized numerous times for anaphylactic reactions and who considered eais to be a waste of money considered antihistamines a life line for treating her peanut allergy . less than half ( 43% ) of respondents perceived no price differential for allergen - free foods , while 35% did : i think they are more expensive because the factories have to make sure that you know there is no allergens in it because we had to figure out how to make this without eggs , we are going to charge you two bucks more . ( allergic female ) i think they are more expensive because the factories have to make sure that you know there is no allergens in it because we had to figure out how to make this without eggs , we are going to charge you two bucks more . ( allergic female ) five participants ( 22% ) thought that cost was dependent on the type of allergen - free food : it depends what you are looking for wheat stuff like wheat free stuff is expensive . ( mother of an allergic child ) it depends what you are looking for wheat stuff like wheat free stuff is expensive . ( mother of an allergic child ) beyond the direct cost of specialty food items , participants differed in their opinions of how their overall grocery bill changed subsequent to an allergy diagnosis . participants who reported that their grocery bill remained unchanged generally referred to the allergen being a previously infrequent purchase with no overall impact on their grocery bill . overall , no clear consensus on costs associated with procuring allergen - free foods emerged among participants . about a quarter ( 23% ) of low - income participants reported that their weekly grocery bill decreased since the allergy diagnosis due to a reduction in purchasing more expensive processed ( and potentially cross contaminated ) foods or due to the elimination of purchasing expensive , allergenic foods ( e.g. , cashews ) . regarding prediagnosis grocery bills , one woman explained , probably more , just because there were more things that i could buy . and then i had to start like making my own i guess you know , which could be cheaper . ( mother of an allergic child ) probably more , just because there were more things that i could buy . and then i had to start like making my own i guess you know , which could be cheaper . ( mother of an allergic child ) finally , two participants ( 15% ) described increased grocery costs after diagnosis : i would say our grocery budget i would have to estimate we ca n't buy bulk being a low - income family that is where it affects us ( father of an allergic child ) we ca n't buy bulk being a low - income family that is where it affects us ( father of an allergic child ) a significant proportion ( 46% ) of low - income participants reported misinformation related to social support programs and medical insurance coverage . some misinformation resulted in direct costs to participants : ontario works [ ontario 's welfare system ] wo n't pay for an epi - pen . ( allergic female ) ontario works [ ontario 's welfare system ] wo n't pay for an epi - pen . ( allergic female ) this statement was in direct contradiction to information provided by a key informant employed by ontario works who noted how common misinformation was : we ca n't tell each person everything that they can qualify for . you say a drug card and you hope they kind of put it all together . so with allergies and everything you hope people will come to you and say , and then we can talk about just that one specific one , but it is hard to get enough of the information out there ( social assistance key informant ) we ca n't tell each person everything that they can qualify for . you say a drug card and you hope they kind of put it all together . so with allergies and everything you hope people will come to you and say , and then we can talk about just that one specific one , but it is hard to get enough of the information out there ( social assistance key informant ) one participant who knew she required an eai but was unaware that it would be covered by her social assistance drug plan rationalized her lack of eai by exaggerating the direct costs , and i haven't gotten one now , because they are like a hundred and fifty bucks a piece every six months you have to renew it . and i haven't gotten one now , because they are like a hundred and fifty bucks a piece every six months you have to renew it . other participants who were unaware that they qualified for eia coverage through ontario works spoke of taking care of themselves so they would not require an eia : and so it is sometimes like wasting money but i think as an adult now i will take much better care of myself and i just do n't eat nothing [ if ] i do n't know who cooked it . but i think as an adult now i will take much better care of myself and i just do n't eat nothing [ if ] i do n't know who cooked it . ( allergic female ) in the event of an unintentional exposure to an allergen , two participants who did not have eais due to the perceived high cost reported using emergency services instead of an eai . there were few indirect costs associated with anaphylactic food allergies that were unique to low - income families . this is because indirect costs , including lost labour productivity , obtaining health care , and time spent shopping for safe foods , are actually higher for people who earn more money [ 10 , 12 ] . only one indirect cost emerged as unique to low - income individuals with anaphylactic food allergies : 17% of participants referred to low - income groups having poorer continuity of care by family doctors , which may result in increased opportunity costs in terms of being unnecessarily re - tested for allergies . for folks who are dependent on urgent care , which is a lot of people , they just do n't have family doctors that would be terrible to try and navigate . ( employment centre key informant ) for folks who are dependent on urgent care , which is a lot of people , they just do n't have family doctors that would be terrible to try and navigate . ( employment centre key informant ) one woman said , you have to have some sort of identification proving your allergies . we have a new family doctor , and i have never had a scratch test done with him , so he does not have anything really on record about my allergies ( allergic female ) you have to have some sort of identification proving your allergies . we have a new family doctor , and i have never had a scratch test done with him , so he does not have anything really on record about my allergies ( allergic female ) almost 40% of participants ( 5 key informants and 4 low - income respondents ) reflected on the challenge of eating an adequate , healthy diet due to a lack of money . many participants who relied on food banks felt stress due to the additional difficulty of obtaining allergen - free foods from the food bank ( 48% of participants):well a grocery store , you can pick and choose what you are getting . when it comes to the food hamper , you have got to take what you get , right even though you put down specifically in a yellow highlight pencil , or whatever , that [ i 'm ] severely allergic to nuts , i still get it . ( allergic male ) well a grocery store , you can pick and choose what you are getting . when it comes to the food hamper , you have got to take what you get , right even though you put down specifically in a yellow highlight pencil , or whatever , that [ i 'm ] severely allergic to nuts , i still get it . ( allergic male ) one woman further explained how reporting allergies can actually decrease the overall amount of food that can be obtained from a food bank , they do n't substitute it with anything , because they can only give you what they have , right we have never said okay we are allergic to this , and they will give us something else . they do n't substitute it with anything , because they can only give you what they have , right we have never said okay we are allergic to this , and they will give us something else . ( allergic female ) almost one - third of participants reported feeling less safe at discount supermarkets relative to regular supermarkets because of perceived poor food availability and cross - contamination that participants thought was worse in discount supermarkets . in terms of product availability : i know they are going to have a wide variety of brands to choose from , so it makes it easier for me . whereas [ discount supermarket ] might get stuff from the states , so it takes much longer to go through all the labels at [ discount supermarket ] than it would at [ regular supermarket ] . ( mother of allergic child ) i know they are going to have a wide variety of brands to choose from , so it makes it easier for me . whereas [ discount supermarket ] might get stuff from the states , so it takes much longer to go through all the labels at [ discount supermarket ] than it would at [ regular supermarket ] . ( mother of allergic child ) respondents also pointed to employees practices : because i do n't feel that [ discount supermarket ] probably deals with their produce as well as [ regular supermarket ] does . like at [ regular supermarket ] you can see that how often the employees wash their hands . at [ discount supermarket ] they just touch this and they go touch that . you know they are less worried about what kind of produce they have , and what they 're touching and what they are doing with it . ( allergic female ) because i do n't feel that [ discount supermarket ] probably deals with their produce as well as [ regular supermarket ] does . like at [ regular supermarket ] you can see that how often the employees wash their hands . at [ discount supermarket ] they just touch this and they go touch that . you know they are less worried about what kind of produce they have , and what they 're touching and what they are doing with it . ( allergic female ) these data reveal trade - offs between direct and intangible risks : participants who shopped at regular grocery stores recognized that spending more on groceries ( increased direct costs ) reduced their fear of cross - contamination ( decreased intangible costs ) . four distinct low - income groups were described by participants as facing substantial barriers to proper anaphylaxis management and treatment . of note , groups identified by participants were not groups with which the participants self - identified ( e.g. , only canadian - born participants identified that newcomers to canada may face additional barriers to food allergy management ) . these were the working poor with no or inadequate health insurance ( 22% of participants ) , newcomers to canada ( i.e. , foreign immigrants ) ( 17% ) , food bank users ( 13% ) , and youth living in poverty ( 9% ) . both allergists and several low - income participants identified the working poor as being likely unable to afford eais . one allergist said , if you are a working poor person that is making minimum wage or a little bit more , they may not have the drug benefits that the government offers and their jobs might not be good enough to have a drug plan . so i suspect that the majority of these people will not buy themselves epipens because they are just too expensive . if you are a working poor person that is making minimum wage or a little bit more , they may not have the drug benefits that the government offers and their jobs might not be good enough to have a drug plan . so i suspect that the majority of these people will not buy themselves epipens because they are just too expensive . newcomers to canada were another low - income group considered to be especially at risk : new canadians would be on very limited incomes , but they might have like food sensitivities and like , my child is very , very sick and has to go to the hospital , but anaphylactic just is n't a familiar word and allergy even is not like a familiar word . ( employment centre key informant ) new canadians would be on very limited incomes , but they might have like food sensitivities and like , my child is very , very sick and has to go to the hospital , but anaphylactic just is n't a familiar word and allergy even is not like a familiar word . ( employment centre key informant ) a newcomer who participated in this research had difficulty articulating her son 's allergies : to eggs , some of the nuts cashews , and there is another one . ( mother of allergic child ) to eggs , some of the nuts cashews , and there is another one . ( mother of allergic child ) this mother knew no one in her home country with a food allergy and had an eai trainer device but was unsure what it was or how to use it . participants ' stress related to the difficulty of obtaining allergen - free foods at the food bank ( discussed above ) explains why several participants noted that food bank users qualify as another at risk group . in addition to the perceived potential for cross - contamination at the food bank , one woman said , it frustrates me that the food bank never says nut - free products when they are looking for food . ( mother of an allergic child ) it frustrates me that the food bank never says nut - free products when they are looking for food ( mother of an allergic child ) finally , two key informants related their concerns about youth with anaphylactic allergies who lived in poverty . one key informant was concerned about youth living in poverty being embarrassed to vocalize their allergy - related concerns , i think it is embarrassing for a lot of the kids . i do n't want to ask for food from you , because i do n't want you to know i am hungry , like that i have no money , and that my parents have kicked me out . a lot of them do n't know a nice way of saying , just tell me what is in this , you know , so they do n't . ( social assistance key informant ) i think it is embarrassing for a lot of the kids . i do n't want to ask for food from you , because i do n't want you to know i am hungry , like that i have no money , and that my parents have kicked me out . a lot of them do n't know a nice way of saying , just tell me what is in this , you know , so they do n't . ( social assistance key informant ) another key informant shared knowledge of how a street youth with anaphylactic food allergies might cope : i think too , people on the street share epipens or sell expired ones or like there is kind of a small black market . ( employment centre key informant ) i think too , people on the street share epipens or sell expired ones or like there is kind of a small black market . ( employment centre key informant ) youth in extreme poverty who have no access to drug insurance may have to rely on drastic measures to access medication in this case , medication that is much more affordable once it has expired . the economic impact of food allergies is significant [ 10 , 14 , 15 ] . no studies to date have provided an in - depth exploration of how low - income families experience the direct , indirect , or intangible costs associated with food allergy management or treatment . complementing the economic analyses that have described costs of food allergy , our qualitative study has described the meaning of the costs of food allergy ( including how they cope with and manage the high cost of eais ) among a group of low - income families . the qualitative approach we adopted highlighted discrepancies between low - income families ' perceptions about barriers to managing food allergies and the actual availability of support ( e.g. , while ontario 's social assistance drug plan covers the cost of eais , many families perceived that the social assistance drug plan did not cover eais ) . low - income families and key informants perceive significant financial barriers to appropriate management and treatment of anaphylactic food allergies . our findings that low - income individuals are perceived to have low access to family doctors are contradicted by empirical evidence suggesting that , in countries where income - related inequities exist in the distribution of family doctor visits , it is often a propoor distribution . that said , prorich income - related inequalities in access to specialists ( such as allergists ) are common [ 21 , 22 ] . in terms of access to medication , in many jurisdictions , pharmaceuticals are not publicly insured . therefore , working poor families ( which comprise 25% of low - income canadians under 65 years ) may be particularly at risk of having inadequate access to medication . almost 70% of our low - income sample reported having an up - to - date eai ; in another study , 87% of a fairly well - educated sample had an up - to - date eai and 75% of children of wealthier families registered in a peanut allergy registry reported having up - to - date eais ( a. clarke , unpublished observation ) . although retaining expired eais is not practiced exclusively by low - income families , financial barriers to medication access should be addressed alongside educational campaigns for the broader allergic population . indeed , the fact that some families would chance an anaphylactic reaction for themselves or their children due to the cost of an eai is quite concerning . limitations of this study include the fact that all participants were from ontario : experiences with allergy management and access to medication and allergen - free foods may differ in other jurisdictions . although there were only 23 participants , the goal of qualitative research is to explore individuals ' experiences rather than to generalize or verify hypotheses about causal relationships , and thus the small sample was not necessarily a limitation of the current study . strengths of this study are the in - depth interviews employed to gain insight into the experiences of low - income families affected by food allergies as well as the inclusion of both affected individuals and a number of key informants who represented a variety of stakeholder perspectives . media reports suggest that food allergies greatly increase a family 's food budget ; empirical evidence from the uk indicates that nut - allergic individuals pay an average of 11% more for a grocery basket than non - nut - allergic individuals . these findings may explain increased grocery bills observed in finland for families with allergic infants . finally , food bank use is increasing in canada , the united states , and the uk ; these trends have implications for the safety of low - income individuals with anaphylactic food allergies . the lack of robust findings around indirect costs of food allergy unique to low - income families reflects the fact that indirect costs like labour productivity are higher for people who make more money per hour relative to people who make less money per hour . at risk groups , including the working poor , newcomers , food bank users , and youth in poverty , are especially important groups to target in terms of education and medication assistance . in terms of newcomers , however , it is likely that competency in the host country 's language would moderate experiences of difficulty managing anaphylactic food allergies . although many clinicians do not collect income data from participants , encouraging patients on social assistance to confirm their drug benefits to procure eais at low or no cost may be an important practice implication from this research . clinicians , advocacy groups , policy makers , and eai manufacturers all have a role to play in ensuring more equitable access to required medication for low - income individuals with anaphylactic food allergies . in addition , from a prevention perspective , securing allergen - free foods is equivalent to a treatment . thus , providing additional funds to families to ensure their ability to secure allergen - free foods for their families may be another potential policy option .

objectives . low - income families may face financial barriers to management and treatment of chronic illnesses . no studies have explored how low - income individuals and families with anaphylactic food allergies cope with financial barriers to anaphylaxis management and/or treatment . this study explores qualitatively assessed direct , indirect , and intangible costs of anaphylaxis management and treatment faced by low - income families . methods . in - depth , semistructured interviews with 23 participants were conducted to gain insight into income - related barriers to managing and treating anaphylactic food allergies . results . perceived direct costs included the cost of allergen - free foods and allergy medication and costs incurred as a result of misinformation about social support programs . perceived indirect costs included those associated with lack of continuity of health care . perceived intangible costs included the stress related to the difficulty of obtaining allergen - free foods at the food bank and feeling unsafe at discount grocery stores . these perceived costs represented barriers that were perceived as especially salient for the working poor , immigrants , youth living in poverty , and food bank users . discussion . low - income families report significant financial barriers to food allergy management and anaphylaxis preparedness . clinicians , advocacy groups , and eai manufacturers all have a role to play in ensuring equitable access to medication for low - income individuals with allergies .

1. Introduction 2. Methods 3. Results 4. Discussion

anaphylactic food allergies are rapid in onset and can be fatal if treatment is delayed ; thus , complete dietary avoidance of the allergen and timely access to treatment are essential . three studies found that children from low - income families face barriers to appropriate anaphylaxis management [ 57 ] . conversely , one study found that associations between allergy severity and health care utilization did not differ by poverty status and another study found no conclusive associations between the epinephrine autoinjectors ( eais ) availability and sociodemographic factors . a recent study on the economic impact of childhood food allergy in the united states found the overall economic cost of food allergy to be almost $ 25 billion annually . this study found that costs borne by families affected by food allergies include lost labour productivity , out - of - pocket costs , and opportunity costs . costs associated with food allergies are described as direct , indirect , or intangible [ 1113 ] . direct costs are financial costs incurred as a result of the food allergy ( including inpatient admissions , outpatient visits , medication costs , and the cost of suitable foods ) . indirect costs reflect time spent in various activities as a result of food allergy , including lost productivity and opportunity costs . intangible costs are losses of utility , such as experiences of pain , suffering , and grief , and can be measured by self - reported quality of life and well - being [ 12 , 15 ] . one recent study on the intangible costs of food allergy in four european countries found that patients with food hypersensitivity reported lower well - being than the control group . the authors note , however , that well - being was positively related to income and , thus , income compensation might improve welfare among food hypersensitive individuals . low - income individuals are differentially affected by the costs of food allergy , since people with allergy who also have low incomes may have greater difficulties in access to [ allergen - free ] food . it is intuitive that absolute costs of allergy medication or allergen - free foods pose a disproportionate burden to low - income families . although these costs are conventionally assessed quantitatively , exploring qualitatively assessed direct , indirect , and intangible costs of food allergies among low - income families is important for three reasons . first , low - income individuals may have poorer access to anaphylaxis treatment [ 5 , 6 ] and may therefore be at high risk of allergy - related harms . second , understanding barriers to anaphylaxis management or treatment faced by low - income individuals can facilitate the development of valid quantitative surveys intended to describe the overall scope of food allergies and treatment accessibility . third , exploring how low - income families cope with or manage anaphylactic allergies has both clinical and policy implications . qualitative research has enormous potential to make a distinctive contribution to knowledge about allergy management and to provide insights that quantitative studies can not . although extant research assesses direct , indirect , and intangible costs quantitatively , this study explores these costs qualitatively from the perspective of low - income families , which is a current gap in the allergy literature . findings from in - depth interviews with key informants and low - income families affected by food allergy begin to fill the gaps in our understanding around coping and management for this vulnerable population . the purpose of this study was to take an in - depth look at the direct , indirect and intangible costs that are particularly burdensome for low - income families in terms of anaphylactic food allergy management and treatment . in - depth , semi - structured interviews with key informants ( n = 10 ) and low - income individuals affected by food allergy ( n = 13 ) were conducted in southwestern ontario , canada . in ontario , the provincial health insurance plan covers the cost of physician and other medically necessary services . key informants were recruited from agencies working with low - income families in a context relevant to food allergy ( i.e. low - income participants were recruited via posters placed in these targeted agencies . over half ( 54% ) of participants were parents of allergic children ; the remainder were allergic adults . most ( 77% ) participants were on social assistance , which included drug benefits ( table 2 ) . all participants self - identified as being low income and were using community services offered specifically to low - income people . over half ( 54% ) of low - income participants and 20% of key informants noted the high cost of eais as a barrier to anaphylaxis treatment for low - income families . ( mother of allergic toddler ) keeping expired eais was reported by 31% of low - income participants . one woman described her experience of living as a homeless youth with an anaphylactic allergy thus , they expire . i always carry around all of mine , and the first ones i ever try would be the expired ones , just to try it will save like money , while you are saving your life , right . just with the cost of an epipen , like that is a lot of money especially if you are like fifteen and living on the street . i always carry around all of mine , and the first ones i ever try would be the expired ones , just to try it will save like money , while you are saving your life , right . just with the cost of an epipen , like that is a lot of money especially if you are like fifteen and living on the street . when asked how many eais she had for her tree - nut - allergic son , a low - income mother said , just the one . these experiences were echoed by the perceptions of an allergist : i think there is a considerable amount especially in the lower income , that will make a decision based on , if i use that now , i am not going to have it for the next ten months . ( allergist ) i think there is a considerable amount especially in the lower income , that will make a decision based on , if i use that now , i am not going to have it for the next ten months . ( allergist ) this is problematic because the first line of anaphylaxis treatment is epinephrine ; antihistamines are not a recommended treatment , although they may be perceived as a low - cost alternative . less than half ( 43% ) of respondents perceived no price differential for allergen - free foods , while 35% did : i think they are more expensive because the factories have to make sure that you know there is no allergens in it because we had to figure out how to make this without eggs , we are going to charge you two bucks more . ( allergic female ) five participants ( 22% ) thought that cost was dependent on the type of allergen - free food : it depends what you are looking for wheat stuff like wheat free stuff is expensive . ( mother of an allergic child ) beyond the direct cost of specialty food items , participants differed in their opinions of how their overall grocery bill changed subsequent to an allergy diagnosis . participants who reported that their grocery bill remained unchanged generally referred to the allergen being a previously infrequent purchase with no overall impact on their grocery bill . overall , no clear consensus on costs associated with procuring allergen - free foods emerged among participants . about a quarter ( 23% ) of low - income participants reported that their weekly grocery bill decreased since the allergy diagnosis due to a reduction in purchasing more expensive processed ( and potentially cross contaminated ) foods or due to the elimination of purchasing expensive , allergenic foods ( e.g. ( mother of an allergic child ) finally , two participants ( 15% ) described increased grocery costs after diagnosis : i would say our grocery budget i would have to estimate we ca n't buy bulk being a low - income family that is where it affects us ( father of an allergic child ) we ca n't buy bulk being a low - income family that is where it affects us ( father of an allergic child ) a significant proportion ( 46% ) of low - income participants reported misinformation related to social support programs and medical insurance coverage . some misinformation resulted in direct costs to participants : ontario works [ ontario 's welfare system ] wo n't pay for an epi - pen . so with allergies and everything you hope people will come to you and say , and then we can talk about just that one specific one , but it is hard to get enough of the information out there ( social assistance key informant ) we ca n't tell each person everything that they can qualify for . so with allergies and everything you hope people will come to you and say , and then we can talk about just that one specific one , but it is hard to get enough of the information out there ( social assistance key informant ) one participant who knew she required an eai but was unaware that it would be covered by her social assistance drug plan rationalized her lack of eai by exaggerating the direct costs , and i haven't gotten one now , because they are like a hundred and fifty bucks a piece every six months you have to renew it . there were few indirect costs associated with anaphylactic food allergies that were unique to low - income families . this is because indirect costs , including lost labour productivity , obtaining health care , and time spent shopping for safe foods , are actually higher for people who earn more money [ 10 , 12 ] . only one indirect cost emerged as unique to low - income individuals with anaphylactic food allergies : 17% of participants referred to low - income groups having poorer continuity of care by family doctors , which may result in increased opportunity costs in terms of being unnecessarily re - tested for allergies . we have a new family doctor , and i have never had a scratch test done with him , so he does not have anything really on record about my allergies ( allergic female ) you have to have some sort of identification proving your allergies . we have a new family doctor , and i have never had a scratch test done with him , so he does not have anything really on record about my allergies ( allergic female ) almost 40% of participants ( 5 key informants and 4 low - income respondents ) reflected on the challenge of eating an adequate , healthy diet due to a lack of money . many participants who relied on food banks felt stress due to the additional difficulty of obtaining allergen - free foods from the food bank ( 48% of participants):well a grocery store , you can pick and choose what you are getting . when it comes to the food hamper , you have got to take what you get , right even though you put down specifically in a yellow highlight pencil , or whatever , that [ i 'm ] severely allergic to nuts , i still get it . when it comes to the food hamper , you have got to take what you get , right even though you put down specifically in a yellow highlight pencil , or whatever , that [ i 'm ] severely allergic to nuts , i still get it . ( allergic male ) one woman further explained how reporting allergies can actually decrease the overall amount of food that can be obtained from a food bank , they do n't substitute it with anything , because they can only give you what they have , right we have never said okay we are allergic to this , and they will give us something else . in terms of product availability : i know they are going to have a wide variety of brands to choose from , so it makes it easier for me . ( mother of allergic child ) i know they are going to have a wide variety of brands to choose from , so it makes it easier for me . you know they are less worried about what kind of produce they have , and what they 're touching and what they are doing with it . you know they are less worried about what kind of produce they have , and what they 're touching and what they are doing with it . ( allergic female ) these data reveal trade - offs between direct and intangible risks : participants who shopped at regular grocery stores recognized that spending more on groceries ( increased direct costs ) reduced their fear of cross - contamination ( decreased intangible costs ) . four distinct low - income groups were described by participants as facing substantial barriers to proper anaphylaxis management and treatment . of note , groups identified by participants were not groups with which the participants self - identified ( e.g. , only canadian - born participants identified that newcomers to canada may face additional barriers to food allergy management ) . these were the working poor with no or inadequate health insurance ( 22% of participants ) , newcomers to canada ( i.e. , foreign immigrants ) ( 17% ) , food bank users ( 13% ) , and youth living in poverty ( 9% ) . both allergists and several low - income participants identified the working poor as being likely unable to afford eais . one allergist said , if you are a working poor person that is making minimum wage or a little bit more , they may not have the drug benefits that the government offers and their jobs might not be good enough to have a drug plan . if you are a working poor person that is making minimum wage or a little bit more , they may not have the drug benefits that the government offers and their jobs might not be good enough to have a drug plan . newcomers to canada were another low - income group considered to be especially at risk : new canadians would be on very limited incomes , but they might have like food sensitivities and like , my child is very , very sick and has to go to the hospital , but anaphylactic just is n't a familiar word and allergy even is not like a familiar word . ( employment centre key informant ) new canadians would be on very limited incomes , but they might have like food sensitivities and like , my child is very , very sick and has to go to the hospital , but anaphylactic just is n't a familiar word and allergy even is not like a familiar word . ( employment centre key informant ) a newcomer who participated in this research had difficulty articulating her son 's allergies : to eggs , some of the nuts cashews , and there is another one . ( mother of allergic child ) to eggs , some of the nuts cashews , and there is another one . ( mother of allergic child ) this mother knew no one in her home country with a food allergy and had an eai trainer device but was unsure what it was or how to use it . participants ' stress related to the difficulty of obtaining allergen - free foods at the food bank ( discussed above ) explains why several participants noted that food bank users qualify as another at risk group . in addition to the perceived potential for cross - contamination at the food bank , one woman said , it frustrates me that the food bank never says nut - free products when they are looking for food . ( mother of an allergic child ) it frustrates me that the food bank never says nut - free products when they are looking for food ( mother of an allergic child ) finally , two key informants related their concerns about youth with anaphylactic allergies who lived in poverty . one key informant was concerned about youth living in poverty being embarrassed to vocalize their allergy - related concerns , i think it is embarrassing for a lot of the kids . i do n't want to ask for food from you , because i do n't want you to know i am hungry , like that i have no money , and that my parents have kicked me out . ( social assistance key informant ) another key informant shared knowledge of how a street youth with anaphylactic food allergies might cope : i think too , people on the street share epipens or sell expired ones or like there is kind of a small black market . no studies to date have provided an in - depth exploration of how low - income families experience the direct , indirect , or intangible costs associated with food allergy management or treatment . complementing the economic analyses that have described costs of food allergy , our qualitative study has described the meaning of the costs of food allergy ( including how they cope with and manage the high cost of eais ) among a group of low - income families . the qualitative approach we adopted highlighted discrepancies between low - income families ' perceptions about barriers to managing food allergies and the actual availability of support ( e.g. , while ontario 's social assistance drug plan covers the cost of eais , many families perceived that the social assistance drug plan did not cover eais ) . low - income families and key informants perceive significant financial barriers to appropriate management and treatment of anaphylactic food allergies . our findings that low - income individuals are perceived to have low access to family doctors are contradicted by empirical evidence suggesting that , in countries where income - related inequities exist in the distribution of family doctor visits , it is often a propoor distribution . that said , prorich income - related inequalities in access to specialists ( such as allergists ) are common [ 21 , 22 ] . in terms of access to medication , in many jurisdictions , pharmaceuticals are not publicly insured . therefore , working poor families ( which comprise 25% of low - income canadians under 65 years ) may be particularly at risk of having inadequate access to medication . almost 70% of our low - income sample reported having an up - to - date eai ; in another study , 87% of a fairly well - educated sample had an up - to - date eai and 75% of children of wealthier families registered in a peanut allergy registry reported having up - to - date eais ( a. clarke , unpublished observation ) . although retaining expired eais is not practiced exclusively by low - income families , financial barriers to medication access should be addressed alongside educational campaigns for the broader allergic population . indeed , the fact that some families would chance an anaphylactic reaction for themselves or their children due to the cost of an eai is quite concerning . limitations of this study include the fact that all participants were from ontario : experiences with allergy management and access to medication and allergen - free foods may differ in other jurisdictions . although there were only 23 participants , the goal of qualitative research is to explore individuals ' experiences rather than to generalize or verify hypotheses about causal relationships , and thus the small sample was not necessarily a limitation of the current study . strengths of this study are the in - depth interviews employed to gain insight into the experiences of low - income families affected by food allergies as well as the inclusion of both affected individuals and a number of key informants who represented a variety of stakeholder perspectives . media reports suggest that food allergies greatly increase a family 's food budget ; empirical evidence from the uk indicates that nut - allergic individuals pay an average of 11% more for a grocery basket than non - nut - allergic individuals . these findings may explain increased grocery bills observed in finland for families with allergic infants . finally , food bank use is increasing in canada , the united states , and the uk ; these trends have implications for the safety of low - income individuals with anaphylactic food allergies . the lack of robust findings around indirect costs of food allergy unique to low - income families reflects the fact that indirect costs like labour productivity are higher for people who make more money per hour relative to people who make less money per hour . at risk groups , including the working poor , newcomers , food bank users , and youth in poverty , are especially important groups to target in terms of education and medication assistance . in terms of newcomers , however , it is likely that competency in the host country 's language would moderate experiences of difficulty managing anaphylactic food allergies . clinicians , advocacy groups , policy makers , and eai manufacturers all have a role to play in ensuring more equitable access to required medication for low - income individuals with anaphylactic food allergies . in addition , from a prevention perspective , securing allergen - free foods is equivalent to a treatment . thus , providing additional funds to families to ensure their ability to secure allergen - free foods for their families may be another potential policy option .

stress is a biologically important and ubiquitous circumstance that can influence brain functions . because of the importance of both the beneficial and deleterious effects of acute and chronic stresses on cognitive functions , they have been the subject of numerous studies during the past 8 decades . the response to stress involves the activation of the hypothalamic - pituitary - adrenocortical ( hpa ) axis and its final product glucocorticoids . the hippocampus , which has the highest density of glucocorticoid receptors in the brain , is involved in the regulation of the hpa and the behavioral respon - ses to stress . this sea - horse - shaped structure is a part of a medial temporal lobe system necessary for the formation of stable declarative memory in humans ( 1 - 3 ) and spatial memory in rodents ( 4 , 5 ) . focusing on the effects of stress on the hippocampus , a large body of work has uncovered effects of chronic and short - term stress on learning and memory ( 6 - 8 ) . these effects have been accompanied by morpho - logical changes : specifically , reduced dendritic arbors after chronic stress ( 9 - 11 ) and reduced spine density after acute stress ( 12 - 14 ) have been described . numerous studies have shown that stressful experiences and/or corticosterone can dramatically impair subsequent cognitive processes ( 15 ) , such as the acquisition ( 16 , 17 ) or the retrieval of informa - tion ( 18 , 20 ) . previous findings indicated that acute administration of glucocorticoids impairs memory retrieval ( 20 - 22 ) systemic injections of stress - level doses of corticosterone administered to rats shortly before retention testing impair retrieval in tasks that rely on spatial or contextual information , including water - maze and inhibitory avoidance ( 20 ) . furthermore , stress - level glucocorticoid adminis - tration to human subjects shortly before retention testing impairs hippocampus - dependent recall of previously learned verbal material ( 23 , 24 ) on the other hand , chronic stress is known to increase levels of adrenal glucocorticoids resulting in deleterious cognitive functioning ( 25 , 26 ) . chronic restraint stress causes alterations in biochemistry , pharmacology , and morphology within the hippo - campus , especially in ca1 and ca3 ( 27 - 29 ) , and cognitive alterations have been systematically reported after repeated exposures to stress ( 29 - 34 ) we are exposed to various forms of stress daily , a common occurrence in the lives of most individuals , which have both positive and negative effects on brain function . in its acute form , stress may be a necessary adaptive mechanism for survival and with only transient changes in the brain . although , prolonged stress causes overactivation and dysre - gulation of the hpa axis thus induces detrimental changes in the brain structure and function . therefore , chronic stress is often considered a negative modulator of the cognitive functions including the learning and memory processes . exposure to long - lasting stress reduces health and increases vulnerability to mental disorders ( 35 ) . the mechanisms of impairment of cognition and synaptic plasticity following stress are largely unknown . however , based on studies in adult and older animals and humans , a glucocorticoid cascade hypothesis is suggested : there is a relationship between cumulative exposures to high glucocorticoid levels and hippocampal atrophy ( 36 ) . in an attempt to clarify the mechanism by which glucocorticoid levels correlate with hippocampal atrophy , a this hypothesis suggests that prolonged exposure to glucocorticoids diminishes the ability of neurons to resist insults , thus increasing the rate at which they are damaged ( 37 ) . several studies have shown that chronic and acute stress produce adverse effects on learning and memory ( 20 , 38 - 40 ) . since two type of stress influence plasma glucocorticoid levels that are involved in learning and memory impairments , we , therefore , hypothesized that the combination of acute and chronic stresses could exert a greater deleterious effect on learning and memory than either factor alone . in the current study , this hypothesis was tested on hippocampus - dependent learning and memory using the morris water maze tasks . male wistar rats ( weighing 20020 g ) were purchased from pasteur institute of iran . animals were kept under standard laboratory conditions with a 12-hr light / dark cycle with ad libitum food and water throughout the experiments . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . during restraint stress , control animals were handled . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . it is important to note that this really examines the effects of heterotypic stress and that the responses to heterotypic stress may not exhibit habituation as is often observed during exposure to homotypic stress . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . timelines of experiments the mwm used in our study was a black circular pool ( 140 cm diameter , 45 cm high ) filled with water ( 30 cm depth ) at 242 c . an invisible escape platform ( 10 cm diameter ) was placed in the middle of one of the quadrants ( 1.5 cm below the water surface ) equidistant from the side wall and middle of the pool . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . a trial began by placing the rat into the water facing the wall of the pool at one of the starting points . when a rat failed to escape within 60 sec , it was guided to the platform by the experimenter . once the rat reached the platform , it was allowed to remain for 30 sec and then placed in a holding cage for an inter - trial interval of 30 sec . after the last trial , each animal was towel dried and returned to its home cage . retention of the spatial training was assessed 22 days after the last training session with a 60 sec free - swim probe trial using a new starting position . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . corticosterone levels were determined by an elisa assay ( cayman chemical , item number 500655 ) . data is expressed as the mean standard error of the mean ( sem ) . behavioral data were analyzed by one - way and two - way ( anova ) , followed by tukey s test for post hoc comparison of the means . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . all analyses were performed using the statistical package for the social science ( spss ) software in a pc - compatible computer . male wistar rats ( weighing 20020 g ) were purchased from pasteur institute of iran . animals were kept under standard laboratory conditions with a 12-hr light / dark cycle with ad libitum food and water throughout the experiments . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . during restraint stress , control animals were handled . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . it is important to note that this really examines the effects of heterotypic stress and that the responses to heterotypic stress may not exhibit habituation as is often observed during exposure to homotypic stress . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . timelines of experiments the mwm used in our study was a black circular pool ( 140 cm diameter , 45 cm high ) filled with water ( 30 cm depth ) at 242 c . an invisible escape platform ( 10 cm diameter ) was placed in the middle of one of the quadrants ( 1.5 cm below the water surface ) equidistant from the side wall and middle of the pool . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . a trial began by placing the rat into the water facing the wall of the pool at one of the starting points . when a rat failed to escape within 60 sec , it was guided to the platform by the experimenter . once the rat reached the platform , it was allowed to remain for 30 sec and then placed in a holding cage for an inter - trial interval of 30 sec . after the last trial , each animal was towel dried and returned to its home cage . retention of the spatial training was assessed 22 days after the last training session with a 60 sec free - swim probe trial using a new starting position . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . corticosterone levels were determined by an elisa assay ( cayman chemical , item number 500655 ) . data is expressed as the mean standard error of the mean ( sem ) . behavioral data were analyzed by one - way and two - way ( anova ) , followed by tukey s test for post hoc comparison of the means . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . all analyses were performed using the statistical package for the social science ( spss ) software in a pc - compatible computer . male wistar rats ( weighing 20020 g ) were purchased from pasteur institute of iran . animals were kept under standard laboratory conditions with a 12-hr light / dark cycle with ad libitum food and water throughout the experiments . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . during restraint stress , control animals were handled . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . it is important to note that this really examines the effects of heterotypic stress and that the responses to heterotypic stress may not exhibit habituation as is often observed during exposure to homotypic stress . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . the mwm used in our study was a black circular pool ( 140 cm diameter , 45 cm high ) filled with water ( 30 cm depth ) at 242 c . an invisible escape platform ( 10 cm diameter ) was placed in the middle of one of the quadrants ( 1.5 cm below the water surface ) equidistant from the side wall and middle of the pool . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . a trial began by placing the rat into the water facing the wall of the pool at one of the starting points . when a rat failed to escape within 60 sec , it was guided to the platform by the experimenter . once the rat reached the platform , it was allowed to remain for 30 sec and then placed in a holding cage for an inter - trial interval of 30 sec . after the last trial , each animal was towel dried and returned to its home cage . retention of the spatial training was assessed 22 days after the last training session with a 60 sec free - swim probe trial using a new starting position . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . corticosterone levels were determined by an elisa assay ( cayman chemical , item number 500655 ) . data is expressed as the mean standard error of the mean ( sem ) . behavioral data were analyzed by one - way and two - way ( anova ) , followed by tukey s test for post hoc comparison of the means . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . all analyses were performed using the statistical package for the social science ( spss ) software in a pc - compatible computer . distance traveled , escape latency and swimming speed data of the animals during the 6 days of training in water maze are illustrated in figure 2 a , two - way anova ( group training days ) was used to analyze the escape latencies during training . all animals were able to improve their performance as shown by the reduction of escape latencies ( figure 2a ) . statistical analysis showed significant differences in escape latency between different training days ( f5,80=58.8 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.364 , p= 0.779 , n=84 ) , and no interaction between days and groups ( f15,80=1.12 , p= 0.338 , n=84 ) . traveled distance , average escape latency , and swimming speed during 6-day training in mwm average escape latency ( a ) , traveled distance ( b ) , and swimming speed ( c ) across all training days . as shown the mean latency and traveled distance to find the platform declined progressively in all animals . analysis showed distance traveled and escape latency on the 6 day of training were statistically different ( p < 0.001 ) from the first day of training . data is expressed as meansem of 84 animals . * * * p < 0.001 versus the first day of training in each group data related to the distance traveled to reach the platform followed similar to the latency pattern . all groups traveled shorter distances to reach the platform as training progressed ( figure 2b ) . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the analysis of swimming speed also showed no significant differences as training days progressed ( f5,80=0.449 , p=0.813 , n=84 ) among groups ( f3,80=1.067 , p= 0.373 , n=84 ) and no interaction between days and groups ( f15,80= 1.442 , p= 0.129 , n=84 ) ( figure 2c ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . exerted stress followed learning in mwm . the probe test was performed 22 days after last acquisition trials . in probe trials , time spent in target quadrant was used to evaluate long - term memory . a two - way analysis of variance ( anova ) , with zones as repeated measure , showed significant effects of group ( f3,50=3.02 , p= 0.043 , n=54 ) and zone ( f1,50=10.73 , p=0.007 , n=54 ) , and a significant interaction between group and zone ( f3,50=3.91 , p=0.017 , n=54 ) . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . data are expressed as meansem * p<0.05 , * * p<0.01 , * * * p<0.001 and p<0.05 , p<0.01 compared with controls # # # p < 0.001 within group comparisons figure 3b represents the average proximity to the platform . one - way anova showed a significant difference among the four groups ( f3,54=3.427 , p= 0.023 , n=58 ) . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . one - way anova on platform location latency data indicated a significant difference among the four groups ( f3,54=3.111 , p=0.034 , n=58 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . one - way anova on quadrant entries also displayed a significant difference between groups ( f3,54=3.281 , p=0.027 , n=58 ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . these findings show that stress could impair memory retrieval . to control the differences in the mwm per - formance , we recorded swimming speed of animals ( figure 4a ) . one - way anova showed no significant differences of swimming speed among the four groups ( f3,54=1.90 , p= 0.14 , n=58 ) . also , there were no significant differences in total distance traveled in the four groups ( f3,54=1.97 , p= 0.13 , n=58 , figure 4b ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . data is expressed as mean sem of 15 - 16 animals in each group there was a significant difference in cortico - sterone levels between control and chronically stressed groups after 21 days stress ( 8.373.3 ng / ml in control versus 103.98.9 ng / ml in the chronically stressed group , p<0.01 ) . also , one - way anova analysis indicated that there were significant differences in corticosterone levels between groups ( figure 5 , f3,36=10.67 , p=0.000 , n=40 ) . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . distance traveled , escape latency and swimming speed data of the animals during the 6 days of training in water maze are illustrated in figure 2 a , two - way anova ( group training days ) was used to analyze the escape latencies during training . all animals were able to improve their performance as shown by the reduction of escape latencies ( figure 2a ) . statistical analysis showed significant differences in escape latency between different training days ( f5,80=58.8 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.364 , p= 0.779 , n=84 ) , and no interaction between days and groups ( f15,80=1.12 , p= 0.338 , n=84 ) . traveled distance , average escape latency , and swimming speed during 6-day training in mwm average escape latency ( a ) , traveled distance ( b ) , and swimming speed ( c ) across all training days . as shown the mean latency and traveled distance to find the platform declined progressively in all animals . analysis showed distance traveled and escape latency on the 6 day of training were statistically different ( p < 0.001 ) from the first day of training . data is expressed as meansem of 84 animals . * * * p < 0.001 versus the first day of training in each group data related to the distance traveled to reach the platform followed similar to the latency pattern . all groups traveled shorter distances to reach the platform as training progressed ( figure 2b ) . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the analysis of swimming speed also showed no significant differences as training days progressed ( f5,80=0.449 , p=0.813 , n=84 ) among groups ( f3,80=1.067 , p= 0.373 , n=84 ) and no interaction between days and groups ( f15,80= 1.442 , p= 0.129 , n=84 ) ( figure 2c ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . exerted stress followed learning in mwm . the probe test was performed 22 days after last acquisition trials . in probe trials , time spent in target quadrant was used to evaluate long - term memory . a two - way analysis of variance ( anova ) , with zones as repeated measure , showed significant effects of group ( f3,50=3.02 , p= 0.043 , n=54 ) and zone ( f1,50=10.73 , p=0.007 , n=54 ) , and a significant interaction between group and zone ( f3,50=3.91 , p=0.017 , n=54 ) . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . data are expressed as meansem * p<0.05 , * * p<0.01 , * * * p<0.001 and p<0.05 , p<0.01 compared with controls # # # p < 0.001 within group comparisons figure 3b represents the average proximity to the platform . one - way anova showed a significant difference among the four groups ( f3,54=3.427 , p= 0.023 , n=58 ) . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . one - way anova on platform location latency data indicated a significant difference among the four groups ( f3,54=3.111 , p=0.034 , n=58 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . one - way anova on quadrant entries also displayed a significant difference between groups ( f3,54=3.281 , p=0.027 , n=58 ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . these findings show that stress could impair memory retrieval . to control the differences in the mwm per - formance , we recorded swimming speed of animals ( figure 4a ) . one - way anova showed no significant differences of swimming speed among the four groups ( f3,54=1.90 , p= 0.14 , n=58 ) . also , there were no significant differences in total distance traveled in the four groups ( f3,54=1.97 , p= 0.13 , n=58 , figure 4b ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . data is expressed as mean sem of 15 - 16 animals in each group there was a significant difference in cortico - sterone levels between control and chronically stressed groups after 21 days stress ( 8.373.3 ng / ml in control versus 103.98.9 ng / ml in the chronically stressed group , p<0.01 ) . also , one - way anova analysis indicated that there were significant differences in corticosterone levels between groups ( figure 5 , f3,36=10.67 , p=0.000 , n=40 ) . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . distance traveled , escape latency and swimming speed data of the animals during the 6 days of training in water maze are illustrated in figure 2 a , two - way anova ( group training days ) was used to analyze the escape latencies during training . all animals were able to improve their performance as shown by the reduction of escape latencies ( figure 2a ) . statistical analysis showed significant differences in escape latency between different training days ( f5,80=58.8 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.364 , p= 0.779 , n=84 ) , and no interaction between days and groups ( f15,80=1.12 , p= 0.338 , n=84 ) . traveled distance , average escape latency , and swimming speed during 6-day training in mwm average escape latency ( a ) , traveled distance ( b ) , and swimming speed ( c ) across all training days . as shown the mean latency and traveled distance to find the platform declined progressively in all animals . analysis showed distance traveled and escape latency on the 6 day of training were statistically different ( p < 0.001 ) from the first day of training . data is expressed as meansem of 84 animals . * * * p < 0.001 versus the first day of training in each group data related to the distance traveled to reach the platform followed similar to the latency pattern . all groups traveled shorter distances to reach the platform as training progressed ( figure 2b ) . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the analysis of swimming speed also showed no significant differences as training days progressed ( f5,80=0.449 , p=0.813 , n=84 ) among groups ( f3,80=1.067 , p= 0.373 , n=84 ) and no interaction between days and groups ( f15,80= 1.442 , p= 0.129 , n=84 ) ( figure 2c ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . the probe test was performed 22 days after last acquisition trials . in probe trials , a two - way analysis of variance ( anova ) , with zones as repeated measure , showed significant effects of group ( f3,50=3.02 , p= 0.043 , n=54 ) and zone ( f1,50=10.73 , p=0.007 , n=54 ) , and a significant interaction between group and zone ( f3,50=3.91 , p=0.017 , n=54 ) . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . data are expressed as meansem * p<0.05 , * * p<0.01 , * * * p<0.001 and p<0.05 , p<0.01 compared with controls # # # p < 0.001 within group comparisons figure 3b represents the average proximity to the platform . one - way anova showed a significant difference among the four groups ( f3,54=3.427 , p= 0.023 , n=58 ) . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . one - way anova on platform location latency data indicated a significant difference among the four groups ( f3,54=3.111 , p=0.034 , n=58 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . one - way anova on quadrant entries also displayed a significant difference between groups ( f3,54=3.281 , p=0.027 , n=58 ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . these findings show that stress could impair memory retrieval . to control the differences in the mwm per - formance , we recorded swimming speed of animals ( figure 4a ) . one - way anova showed no significant differences of swimming speed among the four groups ( f3,54=1.90 , p= 0.14 , n=58 ) . also , there were no significant differences in total distance traveled in the four groups ( f3,54=1.97 , p= 0.13 , n=58 , figure 4b ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . there was a significant difference in cortico - sterone levels between control and chronically stressed groups after 21 days stress ( 8.373.3 ng / ml in control versus 103.98.9 ng / ml in the chronically stressed group , p<0.01 ) . also , one - way anova analysis indicated that there were significant differences in corticosterone levels between groups ( figure 5 , f3,36=10.67 , p=0.000 , n=40 ) . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . extensive evidence from animal and human studies indicate that stress and glucocorticoids influence cognitive function ( 41 - 43 ) . previous studies have shown stress levels of glucocorticoids impair memory retrieval in animals as well as humans ( 39 , 44 - 47 ) . in this study moreover , the impairing effect of chronic stress was not affected by acute stress . during learning , all animals were able to improve their performance as shown by the reduction of escape latencies and traveled distance . statistical analysis showed no significant difference in swi - mming speed , distance traveled , and escape latency among the four groups during training days . mean plasma corticosterone levels for stressed groups , immediately following immobilization stress and immediately after probe test ( a ) : meansem corticosterone levels of 10 animals immediately following the immobilization stress on the last day of the stress treatment . ( b ) : meansem of corticosterone levels in stressed groups immediately following the probe test * * p < 0.01 , * * * p<0.001 versus controls thus , memory performance impairment in stress exposure groups was due to disruption of memory retrieval . findings indicated chronic stress impaired memory retention as the stressed animals spent significantly less time in target quadrant , had longer platform location latencies , and larger average of proximity than their non - stressed control group . also , corticosterone levels significantly increased in chronically stressed animals as measured immediately after the period of 21 days stress and after the probe test . mclaughlin et al ( 2007 ) reported the use of chronic restraint stress , with wire mesh , for 6 hr / day for 21 days as a reliable and efficient method to produce psychological stress and to cause ca3 dendritic retraction and spatial memory deficits in male sprague dawley rats ( 48 ) . in this study , we used the same method for chronic stress and found that corticosterone levels significantly increased and spatial memory retrieval reduced in the stressed rats as compared to the control groups . the results of previous studies are in agreement with our finding that chronic stress has an impairing effect on learning and memory ( 40 , 49 , 50 ) . most studies on the relationship between chronic stress and spatial memory have focused on the hippocampus because of its crucial role in spatial learning and its well - known susceptibility to stress ( 33 ) . the hippocampus plays a critical role in spatial memory s ability because damage to the hippo - campus corresponds with spatial memory impairments in both animals ( 30 , 50 , 51 ) and humans ( 52 , 54 ) . stress resulted in enhanced release of catecholamines and glucocorticoids due to the activation of sympathoadrenal and hypothalamic - pituitary - adrenal axes ( 55 ) . the impairing effects of chronic stress on learning and memory are mainly mediated via elevated levels of glucocorticoids . chronic stress may affect hippocampal function through such mechanisms as ca3 neuronal remodeling ( 56 ) , suppression of synaptic activity ( 45 , 57 ) , and altered neurogenesis ( 51 , 58 , 59 ) . these changes in the hippocampus following chronic stress or elevation of glucocorticoids have been related to changes in spatial learning and memory ( 60 ) . also , acute stress increased average proximity and escape location latency compared with the controls . the finding that plasma levels of corticosterone , from blood collected immediately after the probe trial , was elevated in the acutely stressed group compared with the control group , suggests that increased adrenocortical function induced by the stressor may have disrupted memory retrieval . there is evidence that glucocorticoids impair retention performance when rats or human subjects are tested shortly after training when circulating levels of glucocorticoids are still elevated ( 44 - 46 ) these effects on retention performance suggest that the retention impairment is directly related to increased adrenocortical function . glucocorticoids can affect retention performance by selectively influencing memory retrieval proce - sses and these effects appear to depend on gr activation . in one study ( 20 ) , rats that were given an aversive experience of footshock exposure 30 min before retention testing , failed to remember the platform location as indicated by equal swim times in both target and opposite quadrants . stress effects on memory retrieval are time - dependent and retention performance was not impaired when rats were tested either 2 min or 4 hr after footshock exposure . this time course on retention impairment correlated with plasma corticosterone levels that peak 30 min after stress exposure and return to baseline within 4 our result in this study was in agreement with findings of de quervain et al that indicated foot - shock exposure 30 min before retention testing impaired memory retention ( 20 ) . also , our study indicated chronic plus acute stress impaired memory retention as the stressed animals spent significantly less time in the target quadrant and had longer platform location latencies and average of proximity than their non - stressed control group . also , corticosterone levels increased signi - ficantly more in these animals than controls at immediately after the probe test . as mentioned before , animal studies ( 20 ) , as well as studies on healthy human volunteers ( 23 ) , have demonstrated that retrieval of learned information is susceptible to glucocorticoid - induced impairment . by administrating cortisone at different times to different groups of healthy volunteers ( one hour before word presentation to test consolidation ; and one hour before the delayed recall test to test retrieval ) , it was found that only declarative memory was affected by acute exposure to glucocorticoids ; there was no effect on acquisition or consolidation . in our study , there was no significant difference in platform location latency , average of proximity , time in target quadrant , and number of crossings between acute , chronic , and chronic + acute stress groups . furthermore , there was no significant difference in corticosterone levels between chronic stress and chronic + acute stress groups . wright et al reported novel findings that chronic stress impairs spatial memory through changes in the hpa axis and that attenuating corticosterone levels can restore spatial memory ( 61 ) . these findings are consistent with the hypothesis presented by roozendaal et al ( 2001 ) and roozendaal ( 2002 ) that elevated corticosterone levels at the time of memory assessment may mediate spatial memory impairment in rats with a compromised hippocampus ( 62 , 63 ) . our data may indicate that enhanced corticosterone secretion as a result of exposure to behavioral tasks ( probe test ) may mediate this effect . corticosterone levels in chronically stressed animals immediately after the probe test was significantly more than chronically stressed group immediately after termination of stress . one limitation of the results in this study that needs to be addressed is that corticosterone levels must be measured in all groups immediately after ending of stress and after the probe test , but we measured it only in the chronically stressed and the control groups . chronic stress and the subsequent corticosterone hypersecretion increase adrenal weight ( 64 ) , which can release additional corticosterone in response to a stressor . there is evidence that chronic stress increased total corticosterone levels in response to the y - maze procedure and down - regulated hippo - campal gr mrna expression , which may have functional consequences at the level of receptor capacity . gr reduction or changes in other brain areas may have been responsible for the enhanced corticosterone response to the y - maze procedure because gr are proposed to mediate corticosterone release during the stress response and diurnal rhythms ( 65 , 67 ) , and alterations in extrahippo - campal brain areas have been shown to be involved in enhanced corticosterone response to novel stressors after chronic stress ( 68 , 69 ) . the reduction in gr mrna and an enhanced corticosterone response to the y - maze procedure in chronically stressed rats is consistent with the hypothesis that stress - induced corticosterone elevations on the day of memory assessment impairs spatial memory in chronically stressed rats . in our study , probably alterations of rats hippocampus in both chronic and chronic plus acute stress groups and similar corticosterone levels after the probe test are related to similar memory retrieval impairment in the two groups . in this study , we expected that prior chronic stress enhances hippocampal vulnerability to acute stress , thereby further increasing spatial memory retrieval impairment induced by acute stress . however , there were no significant differences in memory retrieval impairment of three stress exposure groups , suggesting that memory impair - ment has reached a ceiling effect in the chronic plus acute stress group , and so acute stress could not further increase memory retrieval impairment . also , one possibility for interpretation of this finding might be that other adaptation systems are activated during chronic stress that can prevent acute stress effects , thus that we did not observe additive effects . on the other hand , brain regions other than the hippocampus may have been affected by chronic restraint stress paradigm and could have contributed to the memory impairment , since corticosteroid receptors are present ubiquitously in the brain and consequently , every region has the potential to be affected by chronic stress . it will be interesting to see whether chronic restraint stress also induces morphological changes in regions other than the hippocampus . in addition , the interaction of glucocorticoid with several neurotransmitter systems in the brain such as adrenergic ( 70 , 71 ) , dopaminergic ( 21 ) , and opioidergic ( 22 , 72 ) systems may influence memory retrieval . chronic + acute stress same as acute and chronic stress alone impair retrieval of spatial memory . a similar effect of three types of stressors on memory retrieval suggests that the extent of memory impairment by stress is not influenced by prior stress experience . future studies may provide a clearer indication of the exact areas of the memory process that are impaired by excess levels of glucocorticoids and the mechanisms by which it happens .

objective(s):due to the prevalence and pervasiveness of stress in modern life and exposure to both chronic and acute stresses , it is not clear whether prior exposure to chronic stress can influence the impairing effects of acute stress on memory retrieval . this issue was tested in this study.materials and methods : adult male wistar rats were randomly assigned to the following groups : control , acute , chronic , and chronic + acute stress groups . the rats were trained with six trials per day for 6 consecutive days in the water maze . following training , the rats were either kept in control conditions or exposed to chronic stress in a restrainer 6 hr / day for 21 days . on day 22 , a probe test was done to measure memory retention . time spent in target and opposite areas , platform location latency , and proximity were used as indices of memory retention . to induce acute stress , 30 min before the probe test , animals received a mild footshock.results:stressed animals spent significantly less time in the target quadrant and more time in the opposite quadrant than control animals . moreover , the stressed animals showed significantly increased platform location latency and proximity as compared with control animals . no significant differences were found in these measures among stress exposure groups . finally , both chronic and acute stress significantly increased corticosterone levels.conclusion:our results indicate that both chronic and acute stress impair memory retrieval similarly . additionally , the impairing effects of chronic stress on memory retrieval were not influenced by acute stress .

Introduction Materials and Methods None Animals Experimental groups and stress paradigm Morris water maze (MWM) task Corticosterone assay Statistical analysis Results None Spatial learning in the MWM Effects of stress on memory retrieval Corticosterone assay Discussion Conclusion

focusing on the effects of stress on the hippocampus , a large body of work has uncovered effects of chronic and short - term stress on learning and memory ( 6 - 8 ) . chronic restraint stress causes alterations in biochemistry , pharmacology , and morphology within the hippo - campus , especially in ca1 and ca3 ( 27 - 29 ) , and cognitive alterations have been systematically reported after repeated exposures to stress ( 29 - 34 ) we are exposed to various forms of stress daily , a common occurrence in the lives of most individuals , which have both positive and negative effects on brain function . in the current study , this hypothesis was tested on hippocampus - dependent learning and memory using the morris water maze tasks . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . the probe test was performed 22 days after last acquisition trials . in probe trials , time spent in target quadrant was used to evaluate long - term memory . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . in probe trials , time spent in target quadrant was used to evaluate long - term memory . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . in this study moreover , the impairing effect of chronic stress was not affected by acute stress . ( b ) : meansem of corticosterone levels in stressed groups immediately following the probe test * * p < 0.01 , * * * p<0.001 versus controls thus , memory performance impairment in stress exposure groups was due to disruption of memory retrieval . findings indicated chronic stress impaired memory retention as the stressed animals spent significantly less time in target quadrant , had longer platform location latencies , and larger average of proximity than their non - stressed control group . also , corticosterone levels significantly increased in chronically stressed animals as measured immediately after the period of 21 days stress and after the probe test . mclaughlin et al ( 2007 ) reported the use of chronic restraint stress , with wire mesh , for 6 hr / day for 21 days as a reliable and efficient method to produce psychological stress and to cause ca3 dendritic retraction and spatial memory deficits in male sprague dawley rats ( 48 ) . in this study , we used the same method for chronic stress and found that corticosterone levels significantly increased and spatial memory retrieval reduced in the stressed rats as compared to the control groups . the impairing effects of chronic stress on learning and memory are mainly mediated via elevated levels of glucocorticoids . also , acute stress increased average proximity and escape location latency compared with the controls . the finding that plasma levels of corticosterone , from blood collected immediately after the probe trial , was elevated in the acutely stressed group compared with the control group , suggests that increased adrenocortical function induced by the stressor may have disrupted memory retrieval . in one study ( 20 ) , rats that were given an aversive experience of footshock exposure 30 min before retention testing , failed to remember the platform location as indicated by equal swim times in both target and opposite quadrants . this time course on retention impairment correlated with plasma corticosterone levels that peak 30 min after stress exposure and return to baseline within 4 our result in this study was in agreement with findings of de quervain et al that indicated foot - shock exposure 30 min before retention testing impaired memory retention ( 20 ) . also , our study indicated chronic plus acute stress impaired memory retention as the stressed animals spent significantly less time in the target quadrant and had longer platform location latencies and average of proximity than their non - stressed control group . also , corticosterone levels increased signi - ficantly more in these animals than controls at immediately after the probe test . by administrating cortisone at different times to different groups of healthy volunteers ( one hour before word presentation to test consolidation ; and one hour before the delayed recall test to test retrieval ) , it was found that only declarative memory was affected by acute exposure to glucocorticoids ; there was no effect on acquisition or consolidation . in our study , there was no significant difference in platform location latency , average of proximity , time in target quadrant , and number of crossings between acute , chronic , and chronic + acute stress groups . furthermore , there was no significant difference in corticosterone levels between chronic stress and chronic + acute stress groups . wright et al reported novel findings that chronic stress impairs spatial memory through changes in the hpa axis and that attenuating corticosterone levels can restore spatial memory ( 61 ) . corticosterone levels in chronically stressed animals immediately after the probe test was significantly more than chronically stressed group immediately after termination of stress . one limitation of the results in this study that needs to be addressed is that corticosterone levels must be measured in all groups immediately after ending of stress and after the probe test , but we measured it only in the chronically stressed and the control groups . there is evidence that chronic stress increased total corticosterone levels in response to the y - maze procedure and down - regulated hippo - campal gr mrna expression , which may have functional consequences at the level of receptor capacity . gr reduction or changes in other brain areas may have been responsible for the enhanced corticosterone response to the y - maze procedure because gr are proposed to mediate corticosterone release during the stress response and diurnal rhythms ( 65 , 67 ) , and alterations in extrahippo - campal brain areas have been shown to be involved in enhanced corticosterone response to novel stressors after chronic stress ( 68 , 69 ) . in our study , probably alterations of rats hippocampus in both chronic and chronic plus acute stress groups and similar corticosterone levels after the probe test are related to similar memory retrieval impairment in the two groups . in this study , we expected that prior chronic stress enhances hippocampal vulnerability to acute stress , thereby further increasing spatial memory retrieval impairment induced by acute stress . however , there were no significant differences in memory retrieval impairment of three stress exposure groups , suggesting that memory impair - ment has reached a ceiling effect in the chronic plus acute stress group , and so acute stress could not further increase memory retrieval impairment . in addition , the interaction of glucocorticoid with several neurotransmitter systems in the brain such as adrenergic ( 70 , 71 ) , dopaminergic ( 21 ) , and opioidergic ( 22 , 72 ) systems may influence memory retrieval . chronic + acute stress same as acute and chronic stress alone impair retrieval of spatial memory . a similar effect of three types of stressors on memory retrieval suggests that the extent of memory impairment by stress is not influenced by prior stress experience .

in recent decades , the prevalence of obesity has increased worldwide in all age groups . increasing epidemiological evidence has demonstrated that obesity is associated with an increased risk of cancer , especially colon cancer [ 24 ] . several factors , such as insulin resistance , increased levels of leptin , plasminogen activator inhibitor-1 , endogenous sex steroids , decreased levels of adiponectin , and chronic inflammation , are involved in carcinogenesis and cancer progression . however , a few studies that have investigated the influence of bmi on the outcomes of colon cancer patients have reported inconsistent findings [ 611 ] . despite support for the importance of obesity and metabolic syndrome as risk factors for the development of colorectal cancer , data are equivocal for the effects on colorectal cancer progression and outcome [ 68 ] . several studies have reported decreased survival and increased recurrence in patients with insulin resistance or high bmi [ 79 ] , whereas other studies have not [ 6 , 10 , 11 ] . although high bmi was associated with better median os in the cairo study , cairo2 study did not show this association . consequently , testing the role of bmi in patients receiving targeted therapy is an actual subject of research . bevacizumab is a humanised monoclonal antibody ( mab ) against vascular endothelial growth factor ( vegf ) , which is the major mediator of angiogenesis . activation of the vegf / vegf receptor axis triggers multiple signalling networks responsible for endothelial cell survival , mitogenesis , migration , and differentiation . elevated serum vegf levels have been observed in overweight or obese patients [ 13 , 14 ] . . reported that high visceral fat area ( vfa ) measured by computed tomography independently predicted poorer outcome in a retrospective series of patients given first - line bevacizumab - based therapy for metastatic colon cancer . although not all patients diagnosed with metastatic crc benefit from anti - vegf antibody treatment , there are currently no biomarkers available to predict the efficacy of anti - angiogenic therapies . vegf plays an important role as an endothelial mitogen in tumour angiogenesis , and increased levels of vegf may contribute to poorer outcomes in cancer in obese subjects . thus , bmi may be a prognostic factor for poor outcomes in metastatic crc patients receiving bevacizumab - based treatment . we performed a study of the associations between bmi and clinical outcomes in consecutive patients administered bevacizumab - based treatments for metastatic crc . from april 2007 to june 2011 , 80 consecutive patients with metastatic colorectal adenocarcinoma treated with fluoropyrimidine - based combination chemotherapy plus bevacizumab at the institute of oncology , istanbul university ( istanbul , turkey ) , were included in the study . patients , age between 18 and 80 years old with histologically confirmed locally advanced or metastatic crc , were determined . those with one or more unidimensionally measurable lesions , who were not amenable to curative resection , with an eastern cooperative oncology group ( ecog ) performance status of 02 , no prior history of cancer ( except basal cell carcinoma or carcinoma in situ of the cervix ) , adequate bone marrow ( white absolute neutrophil count , 1,500/mm , platelet count 100,000/mm ) and liver function ( total bilirubin 1.5 mg / dl and alanine and aspartate transaminase levels < 5 x upper limit of normal ) , and a life expectancy of longer than 3 months were included . patients received one of the following treatment regimens : simplified lv5fu2 ( leucovorin 400 mg / m , followed by 5-fluorouracil as a 400 mg / m bolus and a 2,400 mg / m infusion over 46 h ) , modified folfox regimen ( simplified lv5fu2 regimen plus oxaliplatin 85 mg / m ) , folfiri ( simplified lv5fu2 regimen plus irinotecan 180 mg / m every 2 weeks ) , xelox ( capecitabine 1,000 mg / m , b.i.d . , p.o . mg / m every 3 weeks ) , or xeliri ( capecitabine 1,000 mg / m , b.i.d . , p.o . bevacizumab was administered at a dosage of 5 mg / kg every 2 weeks or 7.5 mg / kg every 3 weeks . the dosages of chemotherapeutic agents were determined according to the measured body surface area ( bsa ) ; however , for patients with bsa over 2 m , dosages were adjusted according to 2 m. for bevacizumab , no dosing reduction was accomplished , i.e. patients received the exact planned dose for their actual weight . all of the patients had unresectable liver metastases associated with primary tumours at the initial consultation . patients who underwent hepatic resection and local ablative therapies , chemo - embolisation , or radio - embolisation were excluded . other key exclusion criteria were as follows : clinically significant cardiovascular disease ; exclusive bone metastasis ; use of full - dose anti - coagulants or thrombolytics ; known cns metastases ; serious non - healing wound , ulcer , clinically significant bleeding diathesis , or coagulopathy . tumour response was evaluated using a computed tomography scan or magnetic resonance imaging depending on which imaging methods were used at baseline . tumour responses and progression were determined every 8 weeks using the response evaluation criteria in solid tumours by the investigators and classified as follows : complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , or progressive disease ( pd ) . patients with either cr or pr were classified as responders , and patients with sd or pd were considered non - responders . baseline demographic , clinical , and laboratory data , including age , gender , performance status , tumour marker levels , k - ras mutation status , and treatment details , were collected retrospectively for all patients using uniform database templates to ensure consistent data collection . the follow - up duration was calculated from the date of the first bevacizumab administration to the date of death or last follow - up visit . the time to progression ( ttp ) was calculated as the period from the initiation of treatment to the first observation of disease progression or to disease - specific death . the influence of putative prognostic factors on the progression of disease was evaluated by univariate analysis . fifty - four ( 67.5 % ) of the 80 patients with complete clinical and pathological data experienced disease progression during follow - up . descriptive , univariate , and multivariate analyses were performed for those 54 patients with progression . univariate cox proportional - hazards models of all the potential baseline predictors were built to compute the hazard ratios ( hr ) with 95 % confidence intervals ( ci ) . a multivariable binary logistic regression analysis was accomplished using a stepwise algorithm including age ( years ; < 60 vs. 60 ) , bmi ( < 25 vs. 25 ) , primary tumour status ( intact versus non - intact ) , type of chemotherapy regimens ( irinotecan based versus oxaliplatin based ) , k - ras mutational status ( wild type versus mutated ) , carcinoembryonic antigen ( cea ) level ( median , < 28 vs. 28 ) , and number of disease sites . for bmi , patients were categorised as underweight / normal weight ( bmi < 25 kg / m ) , or overweight / obese ( bmi 25 kg / m ) . from april 2007 to june 2011 , 80 consecutive patients with metastatic colorectal adenocarcinoma treated with fluoropyrimidine - based combination chemotherapy plus bevacizumab at the institute of oncology , istanbul university ( istanbul , turkey ) , were included in the study . patients , age between 18 and 80 years old with histologically confirmed locally advanced or metastatic crc , were determined . those with one or more unidimensionally measurable lesions , who were not amenable to curative resection , with an eastern cooperative oncology group ( ecog ) performance status of 02 , no prior history of cancer ( except basal cell carcinoma or carcinoma in situ of the cervix ) , adequate bone marrow ( white absolute neutrophil count , 1,500/mm , platelet count 100,000/mm ) and liver function ( total bilirubin 1.5 mg / dl and alanine and aspartate transaminase levels < 5 x upper limit of normal ) , and a life expectancy of longer than 3 months were included . patients received one of the following treatment regimens : simplified lv5fu2 ( leucovorin 400 mg / m , followed by 5-fluorouracil as a 400 mg / m bolus and a 2,400 mg / m infusion over 46 h ) , modified folfox regimen ( simplified lv5fu2 regimen plus oxaliplatin 85 mg / m ) , folfiri ( simplified lv5fu2 regimen plus irinotecan 180 mg / m every 2 weeks ) , xelox ( capecitabine 1,000 mg / m , b.i.d . , p.o . mg / m every 3 weeks ) , or xeliri ( capecitabine 1,000 mg / m , b.i.d . , p.o . bevacizumab was administered at a dosage of 5 mg / kg every 2 weeks or 7.5 mg / kg every 3 weeks . the dosages of chemotherapeutic agents were determined according to the measured body surface area ( bsa ) ; however , for patients with bsa over 2 m , dosages were adjusted according to 2 m. for bevacizumab , no dosing reduction was accomplished , i.e. patients received the exact planned dose for their actual weight . all of the patients had unresectable liver metastases associated with primary tumours at the initial consultation . patients who underwent hepatic resection and local ablative therapies , chemo - embolisation , or radio - embolisation were excluded . other key exclusion criteria were as follows : clinically significant cardiovascular disease ; exclusive bone metastasis ; use of full - dose anti - coagulants or thrombolytics ; known cns metastases ; serious non - healing wound , ulcer , clinically significant bleeding diathesis , or coagulopathy . tumour response was evaluated using a computed tomography scan or magnetic resonance imaging depending on which imaging methods were used at baseline . tumour responses and progression were determined every 8 weeks using the response evaluation criteria in solid tumours by the investigators and classified as follows : complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , or progressive disease ( pd ) . patients with either cr or pr were classified as responders , and patients with sd or pd were considered non - responders . baseline demographic , clinical , and laboratory data , including age , gender , performance status , tumour marker levels , k - ras mutation status , and treatment details , were collected retrospectively for all patients using uniform database templates to ensure consistent data collection . the follow - up duration was calculated from the date of the first bevacizumab administration to the date of death or last follow - up visit . the time to progression ( ttp ) was calculated as the period from the initiation of treatment to the first observation of disease progression or to disease - specific death . the influence of putative prognostic factors on the progression of disease was evaluated by univariate analysis . fifty - four ( 67.5 % ) of the 80 patients with complete clinical and pathological data experienced disease progression during follow - up . descriptive , univariate , and multivariate analyses were performed for those 54 patients with progression . univariate cox proportional - hazards models of all the potential baseline predictors were built to compute the hazard ratios ( hr ) with 95 % confidence intervals ( ci ) . a multivariable binary logistic regression analysis was accomplished using a stepwise algorithm including age ( years ; < 60 vs. 60 ) , bmi ( < 25 vs. 25 ) , primary tumour status ( intact versus non - intact ) , type of chemotherapy regimens ( irinotecan based versus oxaliplatin based ) , k - ras mutational status ( wild type versus mutated ) , carcinoembryonic antigen ( cea ) level ( median , < 28 vs. 28 ) , and number of disease sites . for bmi , patients were categorised as underweight / normal weight ( bmi < 25 kg / m ) , or overweight / obese ( bmi 25 kg / m ) . at the time of the final follow - up , 4 ( 5 % ) within 54 patients ( 67.5 % ) who exhibited tumour progression were deceased due to disease - related factors . remaining 26 ( 32.5 % ) patients had no evidence of progression during the last evaluation . the clinical and demographic data of 54 metastatic crc patients who had disease progression during chemotherapy with bevacizumab were summarised in table 1 and 2 . the median age for these patients was 60.5 years ( range 3478 ) ; 59.3 % ( n = 32 ) of the patients were male , and 40.7 % ( n = 22 ) were female . distant metastasis was present in 63 % of the patients at the time of diagnosis . the primary location of the malignancy was sigmoid colon and recto - sigmoid junction in 18 patients ( 33.4 % ) , rectum in 17 patients ( 31.5 % ) , right colon - caecum in 14 patients ( 25.8 % ) , left colon in 3 patients ( 5.6 % ) , and transverse colon in two patients ( 3.7 % ) . twenty - nine ( 53.7 % ) patients had k - ras mutations , and 25 ( 46.3 % ) patients had no mutations.table 1basal characteristics of metastatic crc patients who progressed during bevacizumab - containing chemotherapycharacteristicpatient number ( % ) characteristic bmi age bmi < 2521 ( 38.9)median ( years)60.5bmi 2533 ( 61.1)(range)(3478 ) sex cea male32 ( 59.3)median ( ng / ml)5female22 ( 40.7)(range)(0.820,300 ) kras analysis ca 199 mutant29 ( 53.7)median ( ng / ml)39.5wild25 ( 46.3)(range)(115,100 ) number of metastatic site 133 ( 61.1)>121 ( 38.9 ) bmi body mass index , cea carcinoembryonic antigen , ca 199 carbohydrate antigen 199table 2treatment details of patients with disease progressioncharacteristic n = 54 ( 100 % ) primary tumour resection yes45 ( 83.3 % ) no9 ( 16.7 % ) no . of cycles median10(range)(232 ) combined drug oxaliplatin19 ( 35.2 % ) irinotecan35 ( 64.8 % ) response to bevacizumab - based therapy cr + pr23 ( 42.6 % ) sd + pd31 ( 57.4 % ) cr complete response , pr partial response , sd stable disease , pd progressive disease basal characteristics of metastatic crc patients who progressed during bevacizumab - containing chemotherapy bmi body mass index , cea carcinoembryonic antigen , ca 199 carbohydrate antigen 199 treatment details of patients with disease progression cr complete response , pr partial response , sd stable disease , pd progressive disease all patients received bevacizumab - containing chemotherapy regimens as first - line therapy . oxaliplatin - based chemotherapy was administered to 19 patients ( 35.2 % ) , and irinotecan - based chemotherapy was administered to 35 patients ( 64.8 % ) in combination with bevacizumab . height and weight were recorded before initiation of bevacizumab and used to assign patients to group a ( bmi < 25 kg / m ) and group b ( bmi 25 kg / m ) . baseline characteristics ( age , gender , number of metastatic locations , cea , and ca 199 levels and k - ras mutation status ) and treatment details such as number of cycles , resection status of the primary tumour , type of chemotherapeutic agent combined with bevacizumab , and response to treatment of all patients are represented in tables 1 and 2 . twenty - one ( 56.3 % ) of 37 patients in group a and 33 ( 76.7 % ) of 43 patients in group b progressed during a median of 10-months follow - up ( range 357 months ) . for 54 patients who had disease progression and complete clinical data concerning the variables included in univariate analysis , the influence of the 7 variables on ttp was evaluated with mann the effect of these variables on ttp was analysed with binary logistic regression model . in univariate analysis , bmi 25 ( p = 0.004 ) and higher number of metastatic sites ( p = 0.032 ) were associated with poor ttp . the median ttp was 11.7 months in the group a and 6 months in the group b ( p = 0.004 ) . a multivariate logistic regression model for ttp was constructed that included age , bmi , serum cea level , k - ras mutational status , primary tumour status ( intact or non - intact ) , chemotherapy regimens , and number of disease sites . the results are displayed in table 3 . the only independent prognostic factor for ttp was bmi ( p = 0.01 ; or , 4.37 ; 95 % ci , 1.3414.78 ) for patients with metastatic crc treated with bevacizumab.table 3logistic regression model of clinical features for predicting time to tumour progression ( ttp)parameter n ttp ( months ) medianmin.max.univariate analysis * p valuebmi , kg / m 0.004 < 252111.705.6819.02 25336.001.3817.74cea median ( ng / ml)0.566 < 28349.491.3818.60 28207.263.6819.02age ( years)0.264 < 60279.493.2519.02 60277.001.3817.74primary tumour0.618 intact910.281.3818.60 non - intact457.522.0019.02k - ras mutation status0.683 wild type259.491.3817.74 mutated297.003.2519.02chemotherapy0.683 irinotecan - based358.713.2519.02 oxaliplatin - based197.001.3818.60no . of disease sites0.032 < 23310.282.0019.02 2215.721.3817.74 in multivariate analysis , the only independent prognostic factor for ttp was bmi ( p = 0.01 ; hr 4.37 ; 95 % ci 1.3414.78 ) for patients with mcrc treated with combination chemotherapy and bevacizumab * mann whitney u test logistic regression model of clinical features for predicting time to tumour progression ( ttp ) in multivariate analysis , the only independent prognostic factor for ttp was bmi ( p = 0.01 ; hr 4.37 ; 95 % ci 1.3414.78 ) for patients with mcrc treated with combination chemotherapy and bevacizumab * mann whitney u test recent phase iii trials have shown that adding bevacizumab to a first - line conventional chemotherapeutic regimen improved progression - free survival and overall survival in patients with metastatic crc [ 18 , 19 ] . despite extensive investigation , there are no validated predictive biomarkers of the efficacy of vegf - targeted therapies , such as bevacizumab . obese animal models have been shown to be resistant to anti - vegf treatment , which suggests that increased amounts of visceral fat may be associated with high vegf levels and resistance to bevacizumab - based regimens in patients with metastatic crc . in the present study , the median ttp was 11.7 months in the bmi < 25 group and 6 months in the bmi 25 group ( p = 0.004 ) . in addition , the multivariate analysis indicated that increased bmi was the most important predictor of progression in the patients receiving the bevacizumab treatment . evidence accumulated over the past decade has clearly established excess body fat as a risk factor for colorectal cancer . specifically , risk increases with increasing bmi in a sex-(greater risk for men ) and site - specific ( greater occurrence in the colon versus rectum ) manner . a number of plausible biological mechanisms are responsible for the observed associations , including increased insulin resistance , increased availability of insulin - like growth factor ( igf)-1 ( which is mitogenic , proapoptotic , and proangiogenic and increases cell motility ) , and altered adipokine metabolism . these alterations result in increased leptin , which is mitogenic , anti - apoptotic , and proangiogenic , and decreased adiponectin , which is also anti - angiogenic and anti - inflammatory [ 5 , 22 ] . obesity is a well - established risk factor for developing crc and has been associated with increased mortality from colon cancer [ 24 , 25 ] . obesity is also associated with a sedentary lifestyle and typical western diet , which are associated with increased rates of cancer recurrence and death among patients with a history of curative surgical resection for crc [ 7 , 26 , 27 ] . the patho - physiology is not completely understood but may involve adipose tissue production of adipokines and proangiogenic cytokines , such as vegf , which may promote cancer growth and dysregulated angiogenesis . all of the studies evaluating the association between body mass and mortality caused by crc have focused on patients at the time of diagnosis of localised diseases . to our knowledge , only one trial has analysed the influence of obesity on the prognosis of metastatic crc patients receiving vegf - targeted therapy . obesity is strongly associated with changes in the physiological function of adipose tissue and may cause insulin resistance , chronic inflammation , and altered secretion of adipokines . adipose tissue is now recognised to function as an endocrine and paracrine organ that releases cytokine - like polypeptides responsible for widespread biological effects [ 28 , 29 ] . in particular , adipocytes produce insulin - like growth factor and multiple angiogenic factors , including vegf and leptin . leptin exerts direct angiogenic effects [ 29 , 30 ] , upregulates vegf mrna expression , and induces vegf - mediated angiogenesis by colonic epithelial cells . inflammatory cells infiltrating the adipose tissue and adipose stromal cells also contribute to vegf production [ 30 , 33 ] . elevated serum vegf levels have been reported in overweight and obese patients [ 13 , 14 ] . visceral fat accumulation has been shown to be associated with colorectal adenoma formation , colon carcinogenesis , and increased risk for developing colon cancer . the amount of visceral fat independently predicts disease - free survival in patients with resectable crc . the data strongly suggest that visceral fat may induce the accumulation of protumorigenic factors and be associated with poorer outcomes in patients with crc . the role of bevacizumab - based therapy ( in combination with conventional chemotherapy ) has been established as an option for metastatic crc . biomarkers are available for other mechanism - based therapies in metastatic crc , including k - ras status and cetuximab therapy , in which measurement of the predictive biomarker clearly discriminates the subsequent treatment response . while using bmi as a lifestyle biomarker may appear to be unconventional ( compared with traditional gene or protein biomarkers ) , lifestyle is used as a predictive factor for treatment response of other cancer types ; smoking status is used for directing anti - epidermal growth factor receptor ( egfr ) therapy ( erlotinib ) in non - small cell lung cancer . the limitations of our study include a relatively small number of patients , limited follow - up duration , single - centre patient recruitment , and a retrospective design . in addition , the definition of obesity is controversial , and it is unclear whether bmi is the most appropriate measure of obesity . bmi and bsa are crude measures of obesity , and vfa ( visceral fat area ) or sfa ( subcutaneous fat area ) may be more appropriate obesity indicators . computed tomography can be used to accurately assess intra - abdominal fat via measurements of sfa and vfa at the level of the umbilicus . however , in clinical practice , sfa and vfa measurements may be difficult and expensive . moreover , there is still a controversion among different centres about dosing of chemotherapy for overweight patients due to the concerns of toxicity . in our study , dosage adjustment of chemotherapeutic agents for patients with bsa over 2 m might have confounded the evaluation of treatment efficacy . however , bevacizumab dosing was applied according to the actual patient weight ; thus , patients are supposed to receive ideal planned anti - vegf therapy . in conclusion , our results provide the evidence that the bmi of patients prior to initiating vegf - targeted therapy is a simple prognostic factor for patients with metastatic crc . further studies may help determine whether the predictive effects of high bmi are related to either a larger distribution volume of vegf - targeted therapies , the production of high levels of vegf by visceral fat , or both . consequently , patients with high bmis may not benefit from vegf - targeted therapy or may require higher dosages . if further validation studies with vegf - targeted therapies corroborate our results , bmi should be incorporated into clinical patient care and stratification schema for future clinical trials . vegf - targeted therapies should consider physiological parameters related to the patient in addition to pathologic parameters related to the tumour .

high bmi is a well - known risk factor for the development and recurrence of several solid tumours , including crc . obesity is associated with increased levels of vascular endothelial growth factor ( vegf ) . bevacizumab is the main targeted therapy for inhibiting tumour angiogenesis by blocking the vegf / vegf receptor pathway . elevated vegf in obese patients might provoke resistance to anti - vegf therapy . we evaluated the efficacy of bevacizumab on ttp among mcrc patients through stratifying them according to their bmi . patients with mcrc who had been treated with fluoropyrimidine - based combination chemotherapy with bevacizumab were included in the study . patients were assigned according to their bmi before initiation of therapy ( group a : bmi < 25 kg / m2 , group b : bmi 25 kg / m2 ) . multivariate analysis was performed to evaluate the risk of tumour progression . between april 2007 and june 2011 , 80 patients were treated with chemotherapy and bevacizumab as first - line therapy ( n = 37 for group a , n = 43 for group b ) . tumours in 56.3 % of the patients in group a ( n = 21 ) and 76.3 % of the patients in group b ( n = 33 ) progressed during a median 10-months ( 357 months ) follow - up . the median ttp was 11.7 months in the group a and 6 months in the group b ( p = 0.004 ) . in a multivariate analysis , high bmi ( 25 kg / m2 ) was associated with significantly shorter ttp ( p = 0.01 ; hr : 4.37 ) . high bmi among mcrc patients treated with bevacizumab is associated with shorter ttp . further study in larger databases is warranted for confirming the negative prognostic effect of obesity during treatment with anti - vegf agents .

Introduction Patients and methods Eligible patients Statistical analyses Results Discussion

increasing epidemiological evidence has demonstrated that obesity is associated with an increased risk of cancer , especially colon cancer [ 24 ] . several factors , such as insulin resistance , increased levels of leptin , plasminogen activator inhibitor-1 , endogenous sex steroids , decreased levels of adiponectin , and chronic inflammation , are involved in carcinogenesis and cancer progression . despite support for the importance of obesity and metabolic syndrome as risk factors for the development of colorectal cancer , data are equivocal for the effects on colorectal cancer progression and outcome [ 68 ] . several studies have reported decreased survival and increased recurrence in patients with insulin resistance or high bmi [ 79 ] , whereas other studies have not [ 6 , 10 , 11 ] . although high bmi was associated with better median os in the cairo study , cairo2 study did not show this association . bevacizumab is a humanised monoclonal antibody ( mab ) against vascular endothelial growth factor ( vegf ) , which is the major mediator of angiogenesis . activation of the vegf / vegf receptor axis triggers multiple signalling networks responsible for endothelial cell survival , mitogenesis , migration , and differentiation . reported that high visceral fat area ( vfa ) measured by computed tomography independently predicted poorer outcome in a retrospective series of patients given first - line bevacizumab - based therapy for metastatic colon cancer . although not all patients diagnosed with metastatic crc benefit from anti - vegf antibody treatment , there are currently no biomarkers available to predict the efficacy of anti - angiogenic therapies . vegf plays an important role as an endothelial mitogen in tumour angiogenesis , and increased levels of vegf may contribute to poorer outcomes in cancer in obese subjects . thus , bmi may be a prognostic factor for poor outcomes in metastatic crc patients receiving bevacizumab - based treatment . we performed a study of the associations between bmi and clinical outcomes in consecutive patients administered bevacizumab - based treatments for metastatic crc . from april 2007 to june 2011 , 80 consecutive patients with metastatic colorectal adenocarcinoma treated with fluoropyrimidine - based combination chemotherapy plus bevacizumab at the institute of oncology , istanbul university ( istanbul , turkey ) , were included in the study . those with one or more unidimensionally measurable lesions , who were not amenable to curative resection , with an eastern cooperative oncology group ( ecog ) performance status of 02 , no prior history of cancer ( except basal cell carcinoma or carcinoma in situ of the cervix ) , adequate bone marrow ( white absolute neutrophil count , 1,500/mm , platelet count 100,000/mm ) and liver function ( total bilirubin 1.5 mg / dl and alanine and aspartate transaminase levels < 5 x upper limit of normal ) , and a life expectancy of longer than 3 months were included . the dosages of chemotherapeutic agents were determined according to the measured body surface area ( bsa ) ; however , for patients with bsa over 2 m , dosages were adjusted according to 2 m. for bevacizumab , no dosing reduction was accomplished , i.e. all of the patients had unresectable liver metastases associated with primary tumours at the initial consultation . the follow - up duration was calculated from the date of the first bevacizumab administration to the date of death or last follow - up visit . the time to progression ( ttp ) was calculated as the period from the initiation of treatment to the first observation of disease progression or to disease - specific death . fifty - four ( 67.5 % ) of the 80 patients with complete clinical and pathological data experienced disease progression during follow - up . a multivariable binary logistic regression analysis was accomplished using a stepwise algorithm including age ( years ; < 60 vs. 60 ) , bmi ( < 25 vs. 25 ) , primary tumour status ( intact versus non - intact ) , type of chemotherapy regimens ( irinotecan based versus oxaliplatin based ) , k - ras mutational status ( wild type versus mutated ) , carcinoembryonic antigen ( cea ) level ( median , < 28 vs. 28 ) , and number of disease sites . for bmi , patients were categorised as underweight / normal weight ( bmi < 25 kg / m ) , or overweight / obese ( bmi 25 kg / m ) . from april 2007 to june 2011 , 80 consecutive patients with metastatic colorectal adenocarcinoma treated with fluoropyrimidine - based combination chemotherapy plus bevacizumab at the institute of oncology , istanbul university ( istanbul , turkey ) , were included in the study . those with one or more unidimensionally measurable lesions , who were not amenable to curative resection , with an eastern cooperative oncology group ( ecog ) performance status of 02 , no prior history of cancer ( except basal cell carcinoma or carcinoma in situ of the cervix ) , adequate bone marrow ( white absolute neutrophil count , 1,500/mm , platelet count 100,000/mm ) and liver function ( total bilirubin 1.5 mg / dl and alanine and aspartate transaminase levels < 5 x upper limit of normal ) , and a life expectancy of longer than 3 months were included . the dosages of chemotherapeutic agents were determined according to the measured body surface area ( bsa ) ; however , for patients with bsa over 2 m , dosages were adjusted according to 2 m. for bevacizumab , no dosing reduction was accomplished , i.e. all of the patients had unresectable liver metastases associated with primary tumours at the initial consultation . patients with either cr or pr were classified as responders , and patients with sd or pd were considered non - responders . the follow - up duration was calculated from the date of the first bevacizumab administration to the date of death or last follow - up visit . the time to progression ( ttp ) was calculated as the period from the initiation of treatment to the first observation of disease progression or to disease - specific death . fifty - four ( 67.5 % ) of the 80 patients with complete clinical and pathological data experienced disease progression during follow - up . a multivariable binary logistic regression analysis was accomplished using a stepwise algorithm including age ( years ; < 60 vs. 60 ) , bmi ( < 25 vs. 25 ) , primary tumour status ( intact versus non - intact ) , type of chemotherapy regimens ( irinotecan based versus oxaliplatin based ) , k - ras mutational status ( wild type versus mutated ) , carcinoembryonic antigen ( cea ) level ( median , < 28 vs. 28 ) , and number of disease sites . for bmi , patients were categorised as underweight / normal weight ( bmi < 25 kg / m ) , or overweight / obese ( bmi 25 kg / m ) . at the time of the final follow - up , 4 ( 5 % ) within 54 patients ( 67.5 % ) who exhibited tumour progression were deceased due to disease - related factors . the clinical and demographic data of 54 metastatic crc patients who had disease progression during chemotherapy with bevacizumab were summarised in table 1 and 2 . the median age for these patients was 60.5 years ( range 3478 ) ; 59.3 % ( n = 32 ) of the patients were male , and 40.7 % ( n = 22 ) were female . distant metastasis was present in 63 % of the patients at the time of diagnosis . the primary location of the malignancy was sigmoid colon and recto - sigmoid junction in 18 patients ( 33.4 % ) , rectum in 17 patients ( 31.5 % ) , right colon - caecum in 14 patients ( 25.8 % ) , left colon in 3 patients ( 5.6 % ) , and transverse colon in two patients ( 3.7 % ) . twenty - nine ( 53.7 % ) patients had k - ras mutations , and 25 ( 46.3 % ) patients had no mutations.table 1basal characteristics of metastatic crc patients who progressed during bevacizumab - containing chemotherapycharacteristicpatient number ( % ) characteristic bmi age bmi < 2521 ( 38.9)median ( years)60.5bmi 2533 ( 61.1)(range)(3478 ) sex cea male32 ( 59.3)median ( ng / ml)5female22 ( 40.7)(range)(0.820,300 ) kras analysis ca 199 mutant29 ( 53.7)median ( ng / ml)39.5wild25 ( 46.3)(range)(115,100 ) number of metastatic site 133 ( 61.1)>121 ( 38.9 ) bmi body mass index , cea carcinoembryonic antigen , ca 199 carbohydrate antigen 199table 2treatment details of patients with disease progressioncharacteristic n = 54 ( 100 % ) primary tumour resection yes45 ( 83.3 % ) no9 ( 16.7 % ) no . of cycles median10(range)(232 ) combined drug oxaliplatin19 ( 35.2 % ) irinotecan35 ( 64.8 % ) response to bevacizumab - based therapy cr + pr23 ( 42.6 % ) sd + pd31 ( 57.4 % ) cr complete response , pr partial response , sd stable disease , pd progressive disease basal characteristics of metastatic crc patients who progressed during bevacizumab - containing chemotherapy bmi body mass index , cea carcinoembryonic antigen , ca 199 carbohydrate antigen 199 treatment details of patients with disease progression cr complete response , pr partial response , sd stable disease , pd progressive disease all patients received bevacizumab - containing chemotherapy regimens as first - line therapy . oxaliplatin - based chemotherapy was administered to 19 patients ( 35.2 % ) , and irinotecan - based chemotherapy was administered to 35 patients ( 64.8 % ) in combination with bevacizumab . height and weight were recorded before initiation of bevacizumab and used to assign patients to group a ( bmi < 25 kg / m ) and group b ( bmi 25 kg / m ) . baseline characteristics ( age , gender , number of metastatic locations , cea , and ca 199 levels and k - ras mutation status ) and treatment details such as number of cycles , resection status of the primary tumour , type of chemotherapeutic agent combined with bevacizumab , and response to treatment of all patients are represented in tables 1 and 2 . twenty - one ( 56.3 % ) of 37 patients in group a and 33 ( 76.7 % ) of 43 patients in group b progressed during a median of 10-months follow - up ( range 357 months ) . for 54 patients who had disease progression and complete clinical data concerning the variables included in univariate analysis , the influence of the 7 variables on ttp was evaluated with mann the effect of these variables on ttp was analysed with binary logistic regression model . in univariate analysis , bmi 25 ( p = 0.004 ) and higher number of metastatic sites ( p = 0.032 ) were associated with poor ttp . the median ttp was 11.7 months in the group a and 6 months in the group b ( p = 0.004 ) . a multivariate logistic regression model for ttp was constructed that included age , bmi , serum cea level , k - ras mutational status , primary tumour status ( intact or non - intact ) , chemotherapy regimens , and number of disease sites . the only independent prognostic factor for ttp was bmi ( p = 0.01 ; or , 4.37 ; 95 % ci , 1.3414.78 ) for patients with metastatic crc treated with bevacizumab.table 3logistic regression model of clinical features for predicting time to tumour progression ( ttp)parameter n ttp ( months ) medianmin.max.univariate analysis * p valuebmi , kg / m 0.004 < 252111.705.6819.02 25336.001.3817.74cea median ( ng / ml)0.566 < 28349.491.3818.60 28207.263.6819.02age ( years)0.264 < 60279.493.2519.02 60277.001.3817.74primary tumour0.618 intact910.281.3818.60 non - intact457.522.0019.02k - ras mutation status0.683 wild type259.491.3817.74 mutated297.003.2519.02chemotherapy0.683 irinotecan - based358.713.2519.02 oxaliplatin - based197.001.3818.60no . of disease sites0.032 < 23310.282.0019.02 2215.721.3817.74 in multivariate analysis , the only independent prognostic factor for ttp was bmi ( p = 0.01 ; hr 4.37 ; 95 % ci 1.3414.78 ) for patients with mcrc treated with combination chemotherapy and bevacizumab * mann whitney u test logistic regression model of clinical features for predicting time to tumour progression ( ttp ) in multivariate analysis , the only independent prognostic factor for ttp was bmi ( p = 0.01 ; hr 4.37 ; 95 % ci 1.3414.78 ) for patients with mcrc treated with combination chemotherapy and bevacizumab * mann whitney u test recent phase iii trials have shown that adding bevacizumab to a first - line conventional chemotherapeutic regimen improved progression - free survival and overall survival in patients with metastatic crc [ 18 , 19 ] . despite extensive investigation , there are no validated predictive biomarkers of the efficacy of vegf - targeted therapies , such as bevacizumab . obese animal models have been shown to be resistant to anti - vegf treatment , which suggests that increased amounts of visceral fat may be associated with high vegf levels and resistance to bevacizumab - based regimens in patients with metastatic crc . in the present study , the median ttp was 11.7 months in the bmi < 25 group and 6 months in the bmi 25 group ( p = 0.004 ) . in addition , the multivariate analysis indicated that increased bmi was the most important predictor of progression in the patients receiving the bevacizumab treatment . evidence accumulated over the past decade has clearly established excess body fat as a risk factor for colorectal cancer . specifically , risk increases with increasing bmi in a sex-(greater risk for men ) and site - specific ( greater occurrence in the colon versus rectum ) manner . a number of plausible biological mechanisms are responsible for the observed associations , including increased insulin resistance , increased availability of insulin - like growth factor ( igf)-1 ( which is mitogenic , proapoptotic , and proangiogenic and increases cell motility ) , and altered adipokine metabolism . obesity is a well - established risk factor for developing crc and has been associated with increased mortality from colon cancer [ 24 , 25 ] . obesity is also associated with a sedentary lifestyle and typical western diet , which are associated with increased rates of cancer recurrence and death among patients with a history of curative surgical resection for crc [ 7 , 26 , 27 ] . to our knowledge , only one trial has analysed the influence of obesity on the prognosis of metastatic crc patients receiving vegf - targeted therapy . obesity is strongly associated with changes in the physiological function of adipose tissue and may cause insulin resistance , chronic inflammation , and altered secretion of adipokines . in particular , adipocytes produce insulin - like growth factor and multiple angiogenic factors , including vegf and leptin . elevated serum vegf levels have been reported in overweight and obese patients [ 13 , 14 ] . the data strongly suggest that visceral fat may induce the accumulation of protumorigenic factors and be associated with poorer outcomes in patients with crc . the role of bevacizumab - based therapy ( in combination with conventional chemotherapy ) has been established as an option for metastatic crc . biomarkers are available for other mechanism - based therapies in metastatic crc , including k - ras status and cetuximab therapy , in which measurement of the predictive biomarker clearly discriminates the subsequent treatment response . while using bmi as a lifestyle biomarker may appear to be unconventional ( compared with traditional gene or protein biomarkers ) , lifestyle is used as a predictive factor for treatment response of other cancer types ; smoking status is used for directing anti - epidermal growth factor receptor ( egfr ) therapy ( erlotinib ) in non - small cell lung cancer . the limitations of our study include a relatively small number of patients , limited follow - up duration , single - centre patient recruitment , and a retrospective design . in addition , the definition of obesity is controversial , and it is unclear whether bmi is the most appropriate measure of obesity . in our study , dosage adjustment of chemotherapeutic agents for patients with bsa over 2 m might have confounded the evaluation of treatment efficacy . however , bevacizumab dosing was applied according to the actual patient weight ; thus , patients are supposed to receive ideal planned anti - vegf therapy . in conclusion , our results provide the evidence that the bmi of patients prior to initiating vegf - targeted therapy is a simple prognostic factor for patients with metastatic crc . further studies may help determine whether the predictive effects of high bmi are related to either a larger distribution volume of vegf - targeted therapies , the production of high levels of vegf by visceral fat , or both . consequently , patients with high bmis may not benefit from vegf - targeted therapy or may require higher dosages .

cpv strain and genomic dna preparation : the culture supernatant of cpv strain c154 ( cpv-2b ) containing viral particles at 1 10 tcid50/ml was kindly provided by dr . c. k. chuang of the agricultural technology research institute , taiwan . the cpv suspension was serially diluted tenfold ( 1010 tcid50/ml ) , and the cpv genomic dna was prepared from 100 l of cpv supernatant using viral nucleic acid extraction kit ii ( genedirex , las vegas , nv , u.s.a . ) , according to the manufacturer s instructions . design and synthesis of the primers and probe : according to the published vp2 gene sequences of cpv in genbank ( accession numbers including ab054213 , ab054214 , u72695 , u72696 , u72698 , ab115504 , d78585 , m38245 , fj222821 and fj005236 ) ( including type 2a , 2b and 2c ) , sequences were aligned using the software program dnastar ( madison , wi , u.s.a . ) . the conserved sequence within the vp2 gene with high homology was selected as the target for designing the lamp primer set using the software primerexplorer v4 ( http://primerexplorer.jp/elamp4.0.0/index.html ) . the primer pair , cpv - f ( 5-biotin labeled ) and cpv - r , was used as the pcr primer to amplify a 319-bp fragment ( fig . 1.nucleotide sequences alignment of the vp2 gene from different cpv isolates including antigenic variants of type 2a , 2b and 2c . there were 5 ( ab054213 , ab054214 , u72695 , u72696 and u72698 ) , 2 ( ab115504 and d78585 ) , 2 ( m38245 and fj005236 ) and 1 ( fj222821 ) cpv isolates from taiwan , japan , united state america and italy , respectively . the designed nucleic acid sequences of labeled - cpv - probe and primers were indicated , boxed and/or bolded . ) . nucleotide sequences alignment of the vp2 gene from different cpv isolates including antigenic variants of type 2a , 2b and 2c . there were 5 ( ab054213 , ab054214 , u72695 , u72696 and u72698 ) , 2 ( ab115504 and d78585 ) , 2 ( m38245 and fj005236 ) and 1 ( fj222821 ) cpv isolates from taiwan , japan , united state america and italy , respectively . the designed nucleic acid sequences of labeled - cpv - probe and primers were indicated , boxed and/or bolded . the region selected as the internal capture probe was located inside the forward inner primer ( fip)/backward inner primer ( bip ) lamp - amplified fragment of vp2 and labeled with biotin at the 5 end ( mdbio inc . , the optimum hybridization temperatures for capture probes [ biotin - labeled probe for lamp elisa or pcr elisa and fluorescein isothiocyanate ( fitc)-labeled probe for lfd ] were also evaluated . pcr : pcr was performed in a 25-l volume containing 1 l of cpv template dna ; 4 m of each primer ; 200 m each of datp , dctp , dgtp and dttp ( promega , madison , wi , u.s.a . ) ; 5 l of 5 pcr buffer ( 100 mm tris - hcl , 9 mm mgcl2 , 110 mm nh4cl , 110 mm kcl , 0.3% igepal ca-630 and 0.25% tween 20 , ph 8.9 ) ; 0.5 u of onetaq dna polymerase ( new england biolabs , ipswich , ma , u.s.a . ) ; and h2o . the pcr conditions were as follows : denaturation at 95c for 5 min , followed by 35 cycles of denaturation at 95c for 30 sec , annealing at 58c for 45 sec , extension at 72c for 45 sec and final extension at 72c for a further 10 min . the amplified pcr products were analyzed by electrophoresis on a 2% agarose gel containing 0.5 g / ml ethidium bromide . lamp : lamp was performed in a 20-l volume containing 1.2 m each of fip and bip ; 0.3 m each of the f3 and b3 primers ; 10 l of the 2 reaction mixture ( 40 mm tris - hcl , 20 mm kcl , 16 mm mgso4 , 20 mm ( nh4)2so4 , 0.2% tween 20 , 1.6 m betaine and 2.8 mm dntps , ph 8.8 ) ; 1 l template dna ; and 1 l of bst dna polymerase ( dna amplification kit ; eiken chemical co. , tochigi , japan ) . the reaction temperature was optimized by incubating the lamp mixture at 59 , 61 , 63 or 65c for 60 min . the reaction time was optimized by incubating the mixture for 15 , 30 , 45 and 60 min at a predetermined temperature ( 65c ) . after heating at 80c for 5 min to terminate the lamp reaction , the lamp products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide . cpv detection by the lfd assay : a generic lfd strip ( milenia genline hybridetect ; milenia biotec gmbh , gieen , germany ) was used for the lfd assay . under the test condition , a specific dna probe was designed from the sequences between the f1p and b1p regions of lamp amplicons ( table 1table 1.the information of primers and probes used in this study for the sequence detection of vp2 gene of canine parvovirusprimers and probesnucleotide positionsequences ( 53)primer sets for lampcpv - f314091433gggataaagaatttgatactgacttcpv - b316271605gagaggctcttagtttagctttabiotin - cpv - fip ( f1c - f2)15121488/14401461biotin - aggcgcaacttttacaaataattgaaagacttcatgtaaatgcaccacpv - bip ( b1c - b2)15301554/15951573tgatcctgatgcatctgctaatatgtacctttccaccaaaaatctgaprimer pair for pcrbiotin - cpv - f13751398biotin - aatgtaccaccagtttatccaaat cpv - r16661693tacttggtacatagttaaattggttatcprobe for lamp - elisabiotin - cpv - probe15201546biotin - caaatgaatatgatcctgatgcatctgprobe for lamp - lfdfitc - cpv - probe15201546fitc - caaatgaatatgatcctgatgcatctg ) . according to the detection system established , the dna probe was labeled with biotin or fitc at the 5 end . for this assay , 20 pmol of the fitc - labeled cpv probe was added to the biotin - labeled lamp amplicons and hybridized at 58c for 15 min . after hybridization , 8 l of the reaction solution was mixed with 120 l assay buffer , and the lfd strip was dipped into it for 5 min . the detection results were determined by observing the control and test lines on the lfd strips . pcr and lamp amplicon detection combined with elisa : detection using pcr elisa and lamp elisa ( fig . the biotin - labeled specific oligonucleotide probes are applied onto the surface of streptavidin - coated well and used to hybridize with biotin - labeled lamp amplicons or biotin - labeled pcr products . after the nonspecific binding lamp amplicons or biotin - labeled pcr products were removed , the conjugates of streptavidin- horseradish peroxidase ( hrp ) were added to perform the elisa detection . ) each well of a 96-well microtiter plate ( nunc a / s , roskilde , denmark ) was coated with 100 l streptavidin ( 5 mg / ml ; sigma , st . louis , mo , u.s.a . ) in 10 mm phosphate - buffered saline ( pbs , ph 7 , sigma ) and refrigerated at 4c overnight . the streptavidin - unbound sites were blocked with blocking solution [ 1% ( w / v ) bovine serum albumin in pbs ] for 1 hr at room temperature . the plate was washed three times with pbst ( pbs containing 0.05% tween 20 ) . subsequently , each well received 100 l of 2.5 m biotin - labeled cpv probe diluted in pbst . the plates were incubated at 37c for 1 hr . after washing three times with pbst the biotin - labeled specific oligonucleotide probes are applied onto the surface of streptavidin - coated well and used to hybridize with biotin - labeled lamp amplicons or biotin - labeled pcr products . after the nonspecific binding lamp amplicons or biotin - labeled pcr products were removed , the conjugates of streptavidin- horseradish peroxidase ( hrp ) were added to perform the elisa detection . after amplification , 5 l of the pcr or lamp amplicons were diluted with 95 l hybridization solution ( 50 mm phosphate buffer and 2 mm edta , ph 7.2 ) and denatured at 95c for 5 min . after cooling on ice , the denatured biotin - labeled pcr or lamp amplicons were added to the capture probe after washing three times with pbst , 100 l of a 1:1,000 dilution of streptavidin horseradish peroxide ( perkinelmer , waltham , ma , u.s.a . ) in pbs was added to each well , and the plates were incubated at 37c for 45 min . the wells were then washed five times with pbst , and the 3,3,5,5-tetramethylbenzidine liquid substrate system for elisa ( 100 l , sigma ) was added to each well . after incubation in the dark at room temperature , the reaction was stopped by adding 50 l of 2 m h2so4 . absorbance was detected at 450 nm using the tecan / sunrise elisa reader ( advance biotechnology , taipei , taiwan ) . preparation of dna from fecal samples artificially contaminated with cpv : five grams of a cpv - free dog fecal sample in 50 ml of sterile pbs was completely mixed by vortexing and distributed into 50 vials . the aliquots were inoculated with 100 l cpv suspension ( 1010 tcid50/ml ) . the samples were centrifuged at 6,000 g for 15 min , and the supernatant was collected . the supernatant was used for dna extraction , and the samples were pretreated by rapid boiling and chilling as described previously . template dna was prepared from the cpv - contaminated fecal samples using an ultraclean faecal dna isolation kit ( mo bio laboratories inc . , establishment of the lamp assay for cpv detection : several taiwan isolates and strains from japan , italy and usa including antigenic types 2a , 2b and 2c were selected for developing effective visual detection methods for different cpv strains . given that the sequence of the vp2 gene of cpv is frequently used as the target for cpv detection , the pcr / lamp primers were designed on the basis of the alignment of vp2 gene sequences from the selected strains . according to alignment analysis , a highly conserved sequence was selected as a suitable target for designing the pcr / lamp primers ( fig . 1 ) . a set of four primers capable of recognizing six distinct regions on the target sequence was designed : two outer primers ( cpv - f3 and cpv - b3 ) and two inner primers ( cpv - fip and cpv - bip ) . for elisa and lfd analyses , locations and sequences of the primers and specific probes for cpv detection are shown in fig . 1 and table 1 , respectively . optimization of the lamp assay : when lamp was performed to determine the optimal temperature and time of reaction , a ladder - like pattern of the lamp products appeared on the 2% agarose gel at 59 , 61 , 63 and 65c ( fig . 3afig . 3.optimization of loop - mediated isothermal amplification ( lamp ) assay for the detection of vp2 sequence from cpv genomic dna . ( a ) for the test of optimal temperature , reaction temperature of 65 , 63 , 61 and 59c was tested using the genomic dna extracted from 100 l of cpv suspension ( 10 tcid50/ml ) as template . ( b ) for the interactive test of reaction time and template dna concentration , the cpv genomic dnas were extracted from 10 , 10 and 10 tcid50/ml , and then , each cpv genomic dna was used in the lamp reaction for 15 , 30 , 45 and 60 min . indicated the 100-bp ladder dna marker , and the molecular of partial dna ladders were also noted . ) . a slight difference in band clarity was observed with increasing reaction temperature . we selected 65c as the optimal working temperature for lamp , because of the intense signal and specificity observed at the higher temperature . the lamp product could be amplified as early as 30 min when the template dna concentration was high ( isolated from 10 tcid50/ml of cpv ) , whereas at a low template dna concentration ( 10 tcid50/ml of cpv ) , the amplified lamp was observed at 45 min at least ( fig . we selected the optimal reaction condition of 65c for 60 min to ensure positive detection at a low template dna concentration . optimization of loop - mediated isothermal amplification ( lamp ) assay for the detection of vp2 sequence from cpv genomic dna . ( a ) for the test of optimal temperature , reaction temperature of 65 , 63 , 61 and 59c was tested using the genomic dna extracted from 100 l of cpv suspension ( 10 tcid50/ml ) as template . ( b ) for the interactive test of reaction time and template dna concentration , the cpv genomic dnas were extracted from 10 , 10 and 10 tcid50/ml , and then , each cpv genomic dna was used in the lamp reaction for 15 , 30 , 45 and 60 min . indicated the 100-bp ladder dna marker , and the molecular of partial dna ladders were also noted . sensitivity of pcr , lamp and lamp combined with the lfd assay : to determine the sensitivity of cpv detection by lamp and lamp combined with lfd , the cpv stock ( 10 tcid50/ml ) was serially diluted tenfold , and the cpv genomic dna was prepared from 100 l of cpv supernatant . each viral dna ( 1 l ) was used as a template for conventional pcr or lamp . in pcr , the respective dilutions were subjected to thermal cycling using the primer pair of biotin - labeled cpv - f and cpv - r ( table 1 ) , which amplified a 319-bp fragment from the vp2 gene of cpv ( fig . 4.sensitivity of pcr ( a ) , loop - mediated isothermal amplification ( lamp ) ( b ) , lamp combined with enzyme - linked immunosorbent assay ( elisa ) ( c ) and lamp combined with lateral flow dipstick ( lfd ) ( d ) assays for the detection of vp2 sequence from cpv genomic dna . the templates of cpv genomic dna were extracted from cpv suspension ranging from 10 to 10 tcid50/ml . a 20-l reaction mixture was the same as for conventional lamp ; however , a 5-biotinylated fip was used to replace the fip primer . the reaction was performed at 65c for 60 min , and the products were analyzed by electrophoresis on a 2% agarose gel and the lfd assay . the results showed that pcr and lamp could detect cpv at concentrations of 10 tcid50/ml and 10 tcid50/ml , respectively ( fig . 4a and 4b ) and that lamp lfd was also able to detect cpv at concentrations as low as 10 tcid50/ml ( fig . sensitivity of pcr ( a ) , loop - mediated isothermal amplification ( lamp ) ( b ) , lamp combined with enzyme - linked immunosorbent assay ( elisa ) ( c ) and lamp combined with lateral flow dipstick ( lfd ) ( d ) assays for the detection of vp2 sequence from cpv genomic dna . the templates of cpv genomic dna were extracted from cpv suspension ranging from 10 to 10 tcid50/ml . sensitivity of pcr and lamp combined with elisa : the genomic dnas extracted from serially tenfold diluted cpv suspensions were used as templates for biotin - labeled pcr and lamp . as described in materials and methods , the labeled pcr products and lamp amplicons the overall scheme of pcr elisa and lamp elisa is shown in fig . the pcr elisa and lamp elisa results were spectrophotometrically obtained using a microplate reader that provided an absorbance value corresponding to the amount of labeled pcr products or labeled lamp amplicons attached to the surface of microtiter plate wells . in pcr elisa , a linear relationship ( y1=0.4064x + 2.127 , r=0.9833 ) was found at higher cpv titers , from 10 to 10 tcid50/ml . in lamp elisa , cpv dilutions corresponding to 1010 tcid50/ml gave a plateau absorbance value of od450 ranging from 1.78 to 1.66 , and a linear relationship ( y2=0.165x + 1.1216 , r=0.9869 ) was observed with cpv titers decreasing from 10 to 10 tcid50/ml ( fig . 5fig . 5.semi-quantification and limitation of lamp - elisa and pcr - elisa for the detection of vp2 sequence from cpv genomic dna . different titers of cpv ( 10 10 tcid50/ml ) were applied to prepare the genomic dna and then applied for pcr or lamp amplification . the pcr products and lamp amplicoms were used for the elisa assays as the demonstration in fig . each value was derived from three independent detections , and the error bars mean standard deviation ( sd ) . ) . it is indicated the detection limit of the developed pcr - elisa and lamp - elisa was 10 and 10 tcid50/ml , respectively . semi - quantification and limitation of lamp - elisa and pcr - elisa for the detection of vp2 sequence from cpv genomic dna . different titers of cpv ( 10 10 tcid50/ml ) were applied to prepare the genomic dna and then applied for pcr or lamp amplification . the pcr products and lamp amplicoms were used for the elisa assays as the demonstration in fig . each value was derived from three independent detections , and the error bars mean standard deviation ( sd ) . specificity of lamp detection by gel electrophoresis and lfd : in order to evaluate the specificity of established lamp , potential cross - reactions were performed using dna / rna extracted from different pathogens including cdv , infectious canine hepatitis virus ( ichv ) , leptospira canicola and bordetella bronchiseptica . biotin - labeled lamp amplicons were analyzed by 2% agarose gel electrophoresis with ethidium bromide and by the lfd assay . 6.specificity of loop - mediated isothermal amplification ( lamp ) ( a ) combined with lateral flow dipstick ( lfd ) ( b ) for the cpv detection . cpv genomic dna , canine distemper virus ( cdv ) rna , canine hepatitis virus ( ichv ) genomic dna , b. bronchiseptica genomic dna and l. canicola genomic dna were applied in the lamp detections . , cross - amplification tests using templates from cdv , ichv , l. canicola and b. bronchiseptica showed that no amplicons were detected , whereas the reaction using the cpv template gave a positive result . the similar results were also observed in the lfd assay ; the test band appeared only for cpv detection ( fig . these results indicated that the lamp - based assay methods developed in this study were specific for cpv . specificity of loop - mediated isothermal amplification ( lamp ) ( a ) combined with lateral flow dipstick ( lfd ) ( b ) for the cpv detection . cpv genomic dna , canine distemper virus ( cdv ) rna , canine hepatitis virus ( ichv ) genomic dna , b. bronchiseptica genomic dna and l. canicola genomic dna were applied in the lamp detections . cpv detection in artificially contaminated dog fecal samples : cpv was artificially inoculated into dog fecal samples and subjected to cpv detection by pcr according to the results shown in table 2table 2.sensitivity of pcr - elisa , lamp - elisa and lamp - lfd assays for the cpv detection in artificially contaminated fecal sample of dogmethodspbs(negative control)cpv inoculation ( tcid50/ml ) 101101010pcr - elisa++lamp - elisa++++lamp - lfd++++a ) positive was determined by the value of od450>0.261 determined by a spectrophotometry ( as shown in fig . b ) positive was determined by the value of od450>0.447 determined by a spectrophotometry ( as shown in fig . d ) each cpv inoculation detected by different assays was performed from three independent fecal samples and got the same detection result . , the fecal samples with cpv inoculated at 10 tcid50/ml gave positive results by pcr elisa , and both lamp elisa and lamp lfd provided positive results at 1 tcid50/ml of cpv . importantly , the positive signals produced by all of the detection methods including pcr elisa , lamp elisa and lamp lfd could be easily read with the naked eye . a ) positive was determined by the value of od450>0.261 determined by a spectrophotometry ( as shown in fig . b ) positive was determined by the value of od450>0.447 determined by a spectrophotometry ( as shown in fig . d ) each cpv inoculation detected by different assays was performed from three independent fecal samples and got the same detection result . several detection methods have been developed to detect cpv proteins and nucleic acids , and many of these tests are effective and accurate in detecting the viral infection in laboratory . however , they require expensive equipment and are often laborious and time - consuming . early and rapid diagnosis is necessary so that cpv - infected dogs can be isolated to prevent the spread of the disease and to administer supportive treatment for reducing morbidity and mortality . therefore , a novel nucleic acid amplification method , termed lamp , which amplifies specific dna sequences under isothermal conditions within a few hours , was developed as a simple , rapid , specific and cost - effective alternative . lamp is an excellent technology for the detection of nucleic acids present at very low levels in biological and environmental samples with its remarkable sensitivity . therefore , lamp could be applied to gene analysis and study of genetic traits and mostly to detect etiological cause of infections . in the present study , the cpv detection limit by pcr , pcr - elisa , lamp , lamp elisa and lamp lfd was 10 , 10 , 10 , 10 and 10 tcid50/ml , respectively . the results indicated that the sensitivity of lamp elisa and lamp lfd for cpv detection is higher than that of conventional molecular methods . in addition to reducing assay time and elevating the sensitivity , combination of lamp with elisa or with lfd confirmed amplicon identity by hybridization and eliminates the need to handle , such as ethidium bromide . our results confirmed the previous report of cpv detection from cpv - suspected fecal samples by lamp having a detection limit of 10 tcid50/ml and to show that lamp is more sensitive than pcr - based tests . elisa , while lamp elisa provided a positive signal at 10 tcid50/ml . furthermore , the performance of lamp and pcr diagnostic systems has been extensively compared by several groups . according to the report of cho et al . , the detection rates of cpv - suspected fecal samples by lamp and pcr were 80% and 74% , respectively . mukhopadhyay et al . also compared the detection rates of cpv by lamp and pcr from clinical samples to be 74.28% and 57.85% . in general , lamp has been found to have sensitivity similar or superior to that of pcr [ 18 , 25 , 32 ] . we have demonstrated this consistency of the 12 clinical samples tested and 10 ( 83% ) and 8 ( 67% ) were detected positive for cpv by lamp and pcr , respectively ( data not shown ) . the positive results of cpv detection could be presented fully when lamp combined with elisa and lfd . however , immunization with modified - live cpv vaccine may result in shedding of the virus for a period of 3 to 14 days post vaccination . therefore , it is possible that there is a positive result that can be produced by a recent cpv vaccination with our developed lamp elisa and lamp lfd assays . in the present report , we described the development of lamp elisa and lamp lfd diagnostic systems with an assay time of <3 hr for cpv levels of clinical concern with a pretty high sensitivity . we have known the most commercial test kits for cpv diagnosis are fabricated by the immunochromatographic assay or elisa technology . the detection by the kits can be completed within mins , but the detection limit of the kits is approximate 10 tcid50/ml of cpv in canine feces . the purpose of our study was to develop a highly sensitive assay used to detect the canine cpv as possible as early after the virus infection . although the overall detection time is about 3 hr including sample preparation and detection , the test procedure of the developed lamp elisa and lamp lfd assays is easy to be performed and can be used in field test . our system comprised the amplification of a part of vp2 sequences that is unique to cpv , followed by hybridization to a specific probe for exact identification of cpv . our results confirmed the results of recent reports that indicated lamp elisa or lamp lfd to be highly - sensitive methods that can be easily applied for the visual detection of clinical pathogens [ 2 , 32 ] . the high sensitivity of lamp elisa and lamp lfd may allow the identification of dogs shedding cpv at low titers in their feces , helping veterinarians to adopt adequate measures of prophylaxis to prevent cpv infection . our data also showed that the cpv detection limit for artificially contaminated fecal samples using lamp elisa and lamp lfd was 1 tcid50/ml . this sensitivity was lower than the results shown in figs . 4 , 5 and table 2 . we suggest that the lower sensitivity of cpv detection in fecal samples than in pbs may be related to loss of the virus during isolation of cpv from the fecal samples . it may also be related to the presence of intestinal cells and bacteria along with cpv in the prepared dna , which might have reduced the efficiency of lamp in amplifying the target cpv dna from the samples . however , our results indicated that both lamp elisa and lamp lfd developed in the present study are applicable to cpv detection in naturally contaminated fecal samples . in conclusion , when lamp was combined with elisa or with lfd , the detection signal of lamp amplicons could be spectrophotometrically obtained and easily read with the naked eye and without agarose gel electrophoresis . the assays could be completed within 3 hr . to our knowledge , this is the first report employing lamp combined with elisa or lfd for detection of cpv . these results indicated that a simple and cost - effective lamp - based technique can be developed into a rapid and reliable molecular diagnostic method with potential for routine use in the clinical detection of cpv and other veterinary clinical pathogens .

abstractloop - mediated isothermal amplification ( lamp ) combined with enzyme - linked immunosorbent assay ( lamp elisa ) and with lateral flow dipstick ( lamp lfd ) are rapid , sensitive and specific methods for the visual detection of clinical pathogens . in this study , lamp elisa and lamp lfd were developed for the visual detection of canine parvovirus ( cpv ) . for lamp , a set of four primers ( biotin - labeled forward inner primers ) was designed to specifically amplify a region of the vp2 gene of cpv . the optimum time and temperature for lamp were 60 min and 65c , respectively . the specific capture oligonucleotide probes , biotin - labeled cpv probe for lamp elisa and fluorescein isothiocyanate - labeled cpv probe for lamp lfd were also designed for hybridization with lamp amplicons on streptavidin - coated wells and lfd strips , respectively . for the comparison of detection sensitivity , conventional pcr and lamp for cpv detection were also performed . the cpv detection limits by pcr , pcr elisa , lamp , lamp elisa and lamp lfd were 102 , 102 , 101 , 101 and 101 tcid50/ml , respectively . in tests using artificially contaminated dog fecal samples , the samples with cpv inoculation levels of 1 tcid50/ml gave positive results by both lamp elisa and lamp lfd . our data indicated that both lamp elisa and lamp lfd are promising as rapid , sensitive and specific methods for an efficient diagnosis of cpv infection .

MATERIALS AND METHODS RESULTS DISCUSSION

design and synthesis of the primers and probe : according to the published vp2 gene sequences of cpv in genbank ( accession numbers including ab054213 , ab054214 , u72695 , u72696 , u72698 , ab115504 , d78585 , m38245 , fj222821 and fj005236 ) ( including type 2a , 2b and 2c ) , sequences were aligned using the software program dnastar ( madison , wi , u.s.a . ) 1.nucleotide sequences alignment of the vp2 gene from different cpv isolates including antigenic variants of type 2a , 2b and 2c . there were 5 ( ab054213 , ab054214 , u72695 , u72696 and u72698 ) , 2 ( ab115504 and d78585 ) , 2 ( m38245 and fj005236 ) and 1 ( fj222821 ) cpv isolates from taiwan , japan , united state america and italy , respectively . nucleotide sequences alignment of the vp2 gene from different cpv isolates including antigenic variants of type 2a , 2b and 2c . there were 5 ( ab054213 , ab054214 , u72695 , u72696 and u72698 ) , 2 ( ab115504 and d78585 ) , 2 ( m38245 and fj005236 ) and 1 ( fj222821 ) cpv isolates from taiwan , japan , united state america and italy , respectively . , the optimum hybridization temperatures for capture probes [ biotin - labeled probe for lamp elisa or pcr elisa and fluorescein isothiocyanate ( fitc)-labeled probe for lfd ] were also evaluated . cpv detection by the lfd assay : a generic lfd strip ( milenia genline hybridetect ; milenia biotec gmbh , gieen , germany ) was used for the lfd assay . under the test condition , a specific dna probe was designed from the sequences between the f1p and b1p regions of lamp amplicons ( table 1table 1.the information of primers and probes used in this study for the sequence detection of vp2 gene of canine parvovirusprimers and probesnucleotide positionsequences ( 53)primer sets for lampcpv - f314091433gggataaagaatttgatactgacttcpv - b316271605gagaggctcttagtttagctttabiotin - cpv - fip ( f1c - f2)15121488/14401461biotin - aggcgcaacttttacaaataattgaaagacttcatgtaaatgcaccacpv - bip ( b1c - b2)15301554/15951573tgatcctgatgcatctgctaatatgtacctttccaccaaaaatctgaprimer pair for pcrbiotin - cpv - f13751398biotin - aatgtaccaccagtttatccaaat cpv - r16661693tacttggtacatagttaaattggttatcprobe for lamp - elisabiotin - cpv - probe15201546biotin - caaatgaatatgatcctgatgcatctgprobe for lamp - lfdfitc - cpv - probe15201546fitc - caaatgaatatgatcctgatgcatctg ) . for this assay , 20 pmol of the fitc - labeled cpv probe was added to the biotin - labeled lamp amplicons and hybridized at 58c for 15 min . pcr and lamp amplicon detection combined with elisa : detection using pcr elisa and lamp elisa ( fig . the biotin - labeled specific oligonucleotide probes are applied onto the surface of streptavidin - coated well and used to hybridize with biotin - labeled lamp amplicons or biotin - labeled pcr products . after the nonspecific binding lamp amplicons or biotin - labeled pcr products were removed , the conjugates of streptavidin- horseradish peroxidase ( hrp ) were added to perform the elisa detection . ) subsequently , each well received 100 l of 2.5 m biotin - labeled cpv probe diluted in pbst . after washing three times with pbst the biotin - labeled specific oligonucleotide probes are applied onto the surface of streptavidin - coated well and used to hybridize with biotin - labeled lamp amplicons or biotin - labeled pcr products . after the nonspecific binding lamp amplicons or biotin - labeled pcr products were removed , the conjugates of streptavidin- horseradish peroxidase ( hrp ) were added to perform the elisa detection . after amplification , 5 l of the pcr or lamp amplicons were diluted with 95 l hybridization solution ( 50 mm phosphate buffer and 2 mm edta , ph 7.2 ) and denatured at 95c for 5 min . after cooling on ice , the denatured biotin - labeled pcr or lamp amplicons were added to the capture probe after washing three times with pbst , 100 l of a 1:1,000 dilution of streptavidin horseradish peroxide ( perkinelmer , waltham , ma , u.s.a . ) preparation of dna from fecal samples artificially contaminated with cpv : five grams of a cpv - free dog fecal sample in 50 ml of sterile pbs was completely mixed by vortexing and distributed into 50 vials . , establishment of the lamp assay for cpv detection : several taiwan isolates and strains from japan , italy and usa including antigenic types 2a , 2b and 2c were selected for developing effective visual detection methods for different cpv strains . given that the sequence of the vp2 gene of cpv is frequently used as the target for cpv detection , the pcr / lamp primers were designed on the basis of the alignment of vp2 gene sequences from the selected strains . a set of four primers capable of recognizing six distinct regions on the target sequence was designed : two outer primers ( cpv - f3 and cpv - b3 ) and two inner primers ( cpv - fip and cpv - bip ) . for elisa and lfd analyses , locations and sequences of the primers and specific probes for cpv detection are shown in fig . optimization of the lamp assay : when lamp was performed to determine the optimal temperature and time of reaction , a ladder - like pattern of the lamp products appeared on the 2% agarose gel at 59 , 61 , 63 and 65c ( fig . 3.optimization of loop - mediated isothermal amplification ( lamp ) assay for the detection of vp2 sequence from cpv genomic dna . ( a ) for the test of optimal temperature , reaction temperature of 65 , 63 , 61 and 59c was tested using the genomic dna extracted from 100 l of cpv suspension ( 10 tcid50/ml ) as template . ( b ) for the interactive test of reaction time and template dna concentration , the cpv genomic dnas were extracted from 10 , 10 and 10 tcid50/ml , and then , each cpv genomic dna was used in the lamp reaction for 15 , 30 , 45 and 60 min . we selected 65c as the optimal working temperature for lamp , because of the intense signal and specificity observed at the higher temperature . the lamp product could be amplified as early as 30 min when the template dna concentration was high ( isolated from 10 tcid50/ml of cpv ) , whereas at a low template dna concentration ( 10 tcid50/ml of cpv ) , the amplified lamp was observed at 45 min at least ( fig . optimization of loop - mediated isothermal amplification ( lamp ) assay for the detection of vp2 sequence from cpv genomic dna . ( a ) for the test of optimal temperature , reaction temperature of 65 , 63 , 61 and 59c was tested using the genomic dna extracted from 100 l of cpv suspension ( 10 tcid50/ml ) as template . ( b ) for the interactive test of reaction time and template dna concentration , the cpv genomic dnas were extracted from 10 , 10 and 10 tcid50/ml , and then , each cpv genomic dna was used in the lamp reaction for 15 , 30 , 45 and 60 min . sensitivity of pcr , lamp and lamp combined with the lfd assay : to determine the sensitivity of cpv detection by lamp and lamp combined with lfd , the cpv stock ( 10 tcid50/ml ) was serially diluted tenfold , and the cpv genomic dna was prepared from 100 l of cpv supernatant . in pcr , the respective dilutions were subjected to thermal cycling using the primer pair of biotin - labeled cpv - f and cpv - r ( table 1 ) , which amplified a 319-bp fragment from the vp2 gene of cpv ( fig . 4.sensitivity of pcr ( a ) , loop - mediated isothermal amplification ( lamp ) ( b ) , lamp combined with enzyme - linked immunosorbent assay ( elisa ) ( c ) and lamp combined with lateral flow dipstick ( lfd ) ( d ) assays for the detection of vp2 sequence from cpv genomic dna . the results showed that pcr and lamp could detect cpv at concentrations of 10 tcid50/ml and 10 tcid50/ml , respectively ( fig . sensitivity of pcr ( a ) , loop - mediated isothermal amplification ( lamp ) ( b ) , lamp combined with enzyme - linked immunosorbent assay ( elisa ) ( c ) and lamp combined with lateral flow dipstick ( lfd ) ( d ) assays for the detection of vp2 sequence from cpv genomic dna . sensitivity of pcr and lamp combined with elisa : the genomic dnas extracted from serially tenfold diluted cpv suspensions were used as templates for biotin - labeled pcr and lamp . as described in materials and methods , the labeled pcr products and lamp amplicons the overall scheme of pcr elisa and lamp elisa is shown in fig . the pcr elisa and lamp elisa results were spectrophotometrically obtained using a microplate reader that provided an absorbance value corresponding to the amount of labeled pcr products or labeled lamp amplicons attached to the surface of microtiter plate wells . in pcr elisa , a linear relationship ( y1=0.4064x + 2.127 , r=0.9833 ) was found at higher cpv titers , from 10 to 10 tcid50/ml . in lamp elisa , cpv dilutions corresponding to 1010 tcid50/ml gave a plateau absorbance value of od450 ranging from 1.78 to 1.66 , and a linear relationship ( y2=0.165x + 1.1216 , r=0.9869 ) was observed with cpv titers decreasing from 10 to 10 tcid50/ml ( fig . 5.semi-quantification and limitation of lamp - elisa and pcr - elisa for the detection of vp2 sequence from cpv genomic dna . it is indicated the detection limit of the developed pcr - elisa and lamp - elisa was 10 and 10 tcid50/ml , respectively . semi - quantification and limitation of lamp - elisa and pcr - elisa for the detection of vp2 sequence from cpv genomic dna . the pcr products and lamp amplicoms were used for the elisa assays as the demonstration in fig . biotin - labeled lamp amplicons were analyzed by 2% agarose gel electrophoresis with ethidium bromide and by the lfd assay . 6.specificity of loop - mediated isothermal amplification ( lamp ) ( a ) combined with lateral flow dipstick ( lfd ) ( b ) for the cpv detection . , cross - amplification tests using templates from cdv , ichv , l. canicola and b. bronchiseptica showed that no amplicons were detected , whereas the reaction using the cpv template gave a positive result . the similar results were also observed in the lfd assay ; the test band appeared only for cpv detection ( fig . these results indicated that the lamp - based assay methods developed in this study were specific for cpv . specificity of loop - mediated isothermal amplification ( lamp ) ( a ) combined with lateral flow dipstick ( lfd ) ( b ) for the cpv detection . cpv detection in artificially contaminated dog fecal samples : cpv was artificially inoculated into dog fecal samples and subjected to cpv detection by pcr according to the results shown in table 2table 2.sensitivity of pcr - elisa , lamp - elisa and lamp - lfd assays for the cpv detection in artificially contaminated fecal sample of dogmethodspbs(negative control)cpv inoculation ( tcid50/ml ) 101101010pcr - elisa++lamp - elisa++++lamp - lfd++++a ) positive was determined by the value of od450>0.261 determined by a spectrophotometry ( as shown in fig . , the fecal samples with cpv inoculated at 10 tcid50/ml gave positive results by pcr elisa , and both lamp elisa and lamp lfd provided positive results at 1 tcid50/ml of cpv . importantly , the positive signals produced by all of the detection methods including pcr elisa , lamp elisa and lamp lfd could be easily read with the naked eye . therefore , a novel nucleic acid amplification method , termed lamp , which amplifies specific dna sequences under isothermal conditions within a few hours , was developed as a simple , rapid , specific and cost - effective alternative . lamp is an excellent technology for the detection of nucleic acids present at very low levels in biological and environmental samples with its remarkable sensitivity . in the present study , the cpv detection limit by pcr , pcr - elisa , lamp , lamp elisa and lamp lfd was 10 , 10 , 10 , 10 and 10 tcid50/ml , respectively . the results indicated that the sensitivity of lamp elisa and lamp lfd for cpv detection is higher than that of conventional molecular methods . in addition to reducing assay time and elevating the sensitivity , combination of lamp with elisa or with lfd confirmed amplicon identity by hybridization and eliminates the need to handle , such as ethidium bromide . our results confirmed the previous report of cpv detection from cpv - suspected fecal samples by lamp having a detection limit of 10 tcid50/ml and to show that lamp is more sensitive than pcr - based tests . , the detection rates of cpv - suspected fecal samples by lamp and pcr were 80% and 74% , respectively . we have demonstrated this consistency of the 12 clinical samples tested and 10 ( 83% ) and 8 ( 67% ) were detected positive for cpv by lamp and pcr , respectively ( data not shown ) . the positive results of cpv detection could be presented fully when lamp combined with elisa and lfd . therefore , it is possible that there is a positive result that can be produced by a recent cpv vaccination with our developed lamp elisa and lamp lfd assays . in the present report , we described the development of lamp elisa and lamp lfd diagnostic systems with an assay time of <3 hr for cpv levels of clinical concern with a pretty high sensitivity . although the overall detection time is about 3 hr including sample preparation and detection , the test procedure of the developed lamp elisa and lamp lfd assays is easy to be performed and can be used in field test . our results confirmed the results of recent reports that indicated lamp elisa or lamp lfd to be highly - sensitive methods that can be easily applied for the visual detection of clinical pathogens [ 2 , 32 ] . the high sensitivity of lamp elisa and lamp lfd may allow the identification of dogs shedding cpv at low titers in their feces , helping veterinarians to adopt adequate measures of prophylaxis to prevent cpv infection . our data also showed that the cpv detection limit for artificially contaminated fecal samples using lamp elisa and lamp lfd was 1 tcid50/ml . we suggest that the lower sensitivity of cpv detection in fecal samples than in pbs may be related to loss of the virus during isolation of cpv from the fecal samples . it may also be related to the presence of intestinal cells and bacteria along with cpv in the prepared dna , which might have reduced the efficiency of lamp in amplifying the target cpv dna from the samples . however , our results indicated that both lamp elisa and lamp lfd developed in the present study are applicable to cpv detection in naturally contaminated fecal samples . in conclusion , when lamp was combined with elisa or with lfd , the detection signal of lamp amplicons could be spectrophotometrically obtained and easily read with the naked eye and without agarose gel electrophoresis . to our knowledge , this is the first report employing lamp combined with elisa or lfd for detection of cpv . these results indicated that a simple and cost - effective lamp - based technique can be developed into a rapid and reliable molecular diagnostic method with potential for routine use in the clinical detection of cpv and other veterinary clinical pathogens .

first , stressors are not isolated occurrences but can have lifelong effects on health through setting up different patterns of vulnerability and resilience [ 1 , 2 ] . the second premise is that there are increasing individual differences in stress and health trajectories with age . however , there are surprisingly few longitudinal studies of changes in stress , especially in later life . inconsistent results in the literature on the effects of stress on health and mortality may reflect the aggregation of individuals who may have had very different experiences over their life . first , using 18 years of data in a large sample of middle - aged and older men , we sought to identify patterns of change in stressful life events across the lifespan . the second was to examine the effects of different types of stress trajectories on mortality . specifically , we contrasted an allostatic load model which assumes that higher levels of chronic stress will have the greatest adverse effects on health , with a hormesis model [ 57 ] , which suggests that moderate amount of stress may have protective effects on health . early correlational studies found that the number of life events is negatively associated with age ( for reviews see [ 8 , 9 ] ) . however , most life events inventories sample events more common in young adulthood , such as graduations , new jobs , marriages , having children , and divorces . newer life event measures that targeted middle - aged and older adults show little or slightly negative correlations with age although health - related events show increased frequency in late life , as do loss events [ 10 , 11 ] . a few longitudinal studies have examined age - related changes in the number and type of stressful life events . chiriboga found that young adults reported more life events ( both negative and positive ) than older adults , but there were no differences between individuals in mid - life and late life . however , the longitudinal analyses spanning a 12-year period did not find a linear decrease with age . rather , the number of events fluctuated over time , suggesting both cohort and period effects . in a preliminary six - year longitudinal study , yancura et al . found suggestions of nonlinear change over time , with the number of self - reported life events increasing until about age 65 and then decreasing into later life . ensel and lin [ 14 , 15 ] used data spanning 15 years , but the 12-year gap between the last two assessments did not permit an examination of trajectories over time , although they did find that the more distal events predicted the more recent ones a few studies examined growth curve stress trajectories in adults [ 10 , 11 , 16 ] but over short periods of time ( e.g. , three to seven years ) . the results varied by type of stressor , the context , and respondent characteristics . for example , there was little change in stress over a three - year period among married women in their thirties , but recently divorced women had higher levels of stress that decreased over three years . divorced women with antisocial behavior patterns decreased less , however . in older adults , especially in african americans , loss - related events tended to increase [ 10 , 11 ] . the adverse effects of stress on both mental and physical health are now well documented [ 1720 ] . however , there are inconsistent results in the literature on the relationship between stress and mortality . while some studies have found that high levels of stressful life events do predict mortality [ 21 , 22 ] , this may be restricted primarily to health - related stressful life events . other studies have found no relationships between life events and mortality , and one found an inverse association . others have suggested that chronic stress may be a better predictor of health outcomes than the simple occurrence of a life event [ 26 , 27 ] . for example , while schulz and beach showed an increased risk of mortality among caregivers , fredman et al . found a decreased risk perhaps because those who are healthier are more likely to take on the arduous task of caregiving . allostatic load ( al ; ) is a construct assessed across multiple biosystems , including cardiovascular , metabolic , neuroendocrine , and inflammatory markers that are presumed to reflect chronic psychosocial stress . a number of studies have shown that al is associated with higher mortality levels ( for a review , see ) . however , few studies have examined psychosocial stress measures in the context of allostatic load , and the ones that have generally find little or no relationship between various components of al and psychosocial stress . . found that stress was unrelated to a similar measure , the metabolic syndrome , although positive coping strategies were protective . there are a number of possible reasons for the difficulties in relating stress to mortality . in addition to individual differences in vulnerability to stress , such as personality , coping , and differential access to social resources , very few studies actually examine stress over a long period of time . given that the types of chronic illnesses related to mortality often take decades to develop , it would be surprising if a single assessment of life events were strongly associated with mortality . thus , examining long - term stress patterns may be a more promising way to link stress with mortality . while most stress theories implicitly assume that there is a linear relationship between stress and adverse outcomes , there is some evidence to suggest that there is a nonlinear relationship . biogerontologists have recently become interested in hormesis , which is defined as the phenomenon in which adaptive responses to low doses of otherwise harmful conditions improve the functional ability of cells and organisms most researchers assume that there is a linear , dose - response relationship between stress and adverse health outcomes , but hormesis suggests that there is a nonlinear or inverted u relationship , such that low or even moderate doses of an otherwise toxic substance provide protective benefits against future exposure to stresses . although still controversial , there is a growing body of evidence for hormesis in organisms such as flatworms , plants , and mice [ 6 , 7 , 35 ] , as well as in cardiac and neuronal cells . presumably , the mechanism is the activation of stress repair mechanisms at the cellular level , such as heat shock proteins . while hormesis has been studied most frequently in plant and animal models , it has been linked to longevity in humans [ 35 , 37 ] . however , the study of hormesis typically involves physical stressors such as radiation , and little work has been done with psychosocial stressors . dienstbier 's construct of physiological tougheningin which intermittent exposure to stress results in better functioning is consistent with the hormesis model . the field of stress - related growth has also challenged the assumption of linear effects of stress on health psychological outcomes . applying the models to human stress and health leads to some interesting and somewhat counterintuitive hypotheses , namely , individuals who experience intermittent or moderate amounts of stress may develop better coping resources which allow them to be more resilient to stress in later life . for example , in a longitudinal study , schnurr et al . found that individuals who experienced moderate combat exposure had improved mmpi profiles 20 years later , but individuals with no or high combat exposure had worsened mmpi profiles . people who have chronically high stress levels may experience resource depletion , leading to impaired ability to cope with stress . however , individuals who , for whatever reasons , avoid stressors , that is , who are extremely risk averse , may not develop the resources which allow them to adequately cope with problems that arise in late life . in a qualitative study , vaillant , described one such person who managed to adopt an extremely self - limiting lifestyle in that she managed to avoid employment , marriage , and even friends , but was poorly equipped to deal with problems in later life . a possible explanation for the inconsistent relations between stress and mortality is that hormesis may apply to psychosocial as well as physiological stressors . thus , the possibility exists that there is a nonlinear relationship between stress and mortality that moderate stress levels may be associated with better longevity , while both chronic high and low levels may be associated with poorer longevity . the primary aim of this study was to compare longitudinal change in life events and its consequences on mortality , using 18 years of data from the va normative aging study ( nas ) , a longitudinal panel study which has followed a large sample of men since the mid-1960s . the nas has collected up to nine assessments of stressful life events . to our knowledge , this constitutes the longest extant longitudinal study of stress and coping using quantitative measures and , as such , provides a unique resource to model changes in stress patterns from mid- to late life and their relationships to mortality . only a few studies have examined trajectories of life events in adulthood [ 10 , 11 , 16 ] . further , we hypothesized that by using latent class growth analyses ( lcgas ; ) , we would identify at least three different patterns of trajectories , with some individuals exhibiting chronically elevated levels of life events , others showing stable low levels , and others showing moderate or intermittent levels of life events . we estimated two sets of lcga models , one for stressful life events measure including health items , and one for a measure excluding health items . the purpose of the first set of analyses was to examine how stressful life events , including health problems , changed with age ; the second omitted health problems in order to avoid a potential confound between stress and health when predicting mortality . the second aim was to understand how these developmental trajectories of stress related to all - cause mortality , contrasting an allostatic load model with a hormetic one . if the allostatic load model is correct , there should be a linear relationship between stress and mortality . however , if the hormesis model is correct , then the relationship should be nonlinear ( u - shaped , with higher mortality at either end of stress distribution ) . we hypothesized that individuals in the chronic high- or low - stress categories would be more likely to have higher rates of mortality than those in the intermediate stress categories . the va normative aging study ( nas ) screened over six thousand men between 1961 and 1968 and enrolled 2,280 , aged 2181 , who had good health , defined as the absence of chronic illness and blood pressure below 140/90 as well as likely geographic stability , which was indexed by extensive ties in the boston area ( see ) . the men are equally divided between blue and white collar workers , and reflected the racial profile of boston in the late 1950s , that is , primarily white . as of august 2010 , 852 ( 37.4% ) were participating , 1,305 ( 57.2% ) were deceased , 89 ( 3.9% ) were lost or dropped out , and 34 ( 1.5% ) were too sick to participate . the current mean age is 79 ( sd = 6.4 , range = 6398 ) . in this study , we began with 1,565 men who completed the initial stress inventory in 1985 . we computed stress trajectories from 1985 to 2002 ( an 18-year period ) on the 1443 men who had at least three assessments of stressful life events . mean age of this sample in 1985 was 60.30 ( sd = 7.73 , range = 4187 ) . we then predicted subsequent mortality , using these stress trajectories as a predictor , and including covariates from various surveys and medical examinations , which decreased the n to 977 . this subsample did not differ from the larger sample in health ratings , smoking , alcohol consumption , or education although they were more likely to be married , x(1 ; n = 1222 ) = 4.30 , p < .05 . nas men complete biomedical examinations every three years as well as periodic mail surveys assessing psychosocial variables , which typically have response rates exceeding 80% . the nas began collecting stressful life events in 1985 , using three mail surveys over six years , which also included information on mental and physical health . , the health and social behavior ( hsb ) survey was developed to obtain psychosocial information at the time of the biomedical exam . the hsb was included in a packet of materials sent to the men before they reported for their triennial physical exam . the hsb included measures assessing stress and mental and physical health ; response rates typically exceed 95% . data collected between 1985 and 2002 were selected to develop the stressful life event trajectories . data from the triennial medical exams and several surveys were used for the covariates . in the nas , the elders life stress inventory ( elsi ; ) was included in both the social support and the health and social behavior ( hsb ) surveys . the elsi is a 30 item measure that assesses events likely to occur in middle - aged and older adults during the past year . the elsi uses a nested construction technique ; that is , where possible , there are several items in each domain such that the more items that are reported , the more serious are the problems in that domain . for example , the disruptions in marital relationships domain contains three items ( e.g. , marital problems , separation , and divorce ) . for those analyses with health outcomes , scoring for the elsi excludes the items related to worsening health . the elsi has good criterion validity , correlating about .2 to .3 with physical and psychological health outcomes , comparable to other stressful life event measures . although this life events measure is correlated with other types of stress measures , such as hassles and perceived stress , as well as personality measures such as neuroticism , it contributes independent variance to health outcomes . we scored the elsi in two ways : a total sum which includes all 30 items and one which omits the two items which tap health events . we used a total of 7,634 observations for 1443 men ( m = 5.29 , sd = 1.58 , range = 39 ) . in the models predicting mortality as a function of stress trajectory , we included a number of covariates that are known to affect mortality , including a self - reported health rating , demographics , and health behavior habits . educational attainment was derived from an item in the original social screening survey which was collected at study enrollment . less than high school ( 9.31% ) , high school ( 24.97% ) least some college ( 51.18% ) , and graduate or professional education ( 14.53% ) . marital status was derived from a survey in 1986 and dichotomized into married ( 88.46% ) versus not married ( 11.54% ) . for self - reported health , we used a single item assessing general health from the first social survey in 1985 taken from the sf-36 . health was rated from 15 , where 1 = poor and 5 = excellent ( m = 4.2 , sd = 0.72 ) . drinking status was derived from a survey item administered in 1986 and was coded to identify nondrinker ( 12.93% ) , light or moderate ( 71.69% ) , and heavy drinkers ( 15.38% ) . current smokers ( 13.03% ) were identified based on an interview at the physical exam closest to 1985 . vital status of nas participants was monitored by periodic mailings , and when notified of a death , official death certificates were obtained . death certificates were reviewed by a physician , and cause of death coded by an experienced rn using icd-9 . to identify different trajectory classes of stressful life events , we used a semi - parametric mixture - model approach for nagin and jones et al . . the method is implemented in the traj procedure in sas and uses a group - modeling approach that employs a mixture of probability distributions that are specified to match the data being analyzed . the mixture models used deal with heterogeneity in the population by modeling subpopulations which differ in regard to their parameter values . the marginal density for the outcome y is ( 1)f(y)=k=1kpr ( c = k)pr ( y = y c = k)=k=1kpkf(y,k ) , where pk the probability of belonging to class k corresponding to parameters k , which depend on time , thus creating longitudinally based latent trajectories . ( y = y | c = k ) in a number of ways , including a standard poisson model appropriate for count data . however , because approximately 30% of nas men did not report stressful life events ( see figure 1 ) , it was necessary to use a zero - inflated poisson ( zip ) model to account for the fact that there are more zeros than would be expect under the poisson assumption . as with standard poisson models , covariates such as age are related linearly to the log of the poisson mean . we used the voung test to compare the zero - inflated model with an ordinary poisson regression model for both the sle sum and the sle sum omitting the health items . in both instances , the vuong non - nested hypothesis test statistics suggested the zip model was superior to the standard poisson model ( p < .001 for both outcomes ) . in addition , we tested a quadratic term for age , based on preliminary analyses , which suggested that there might be nonlinear trajectories . the number of classes present in the data is determined by comparing models with the same class structure but varying numbers of classes . in order to identify the correct number of classes , we used the change in bayesian information criterion ( bic , ) between models as an approximation to the log of bayes factor , 2log e ( b10 ) 2(bic ) , where bic is the bic of the more complex ( alternative ) model minus the bic of the simpler ( null ) model . finally , we considered the model 's interpretability . in order to graph the predicted trajectories for each group , c , we used the following model , written with no covariates adjustments , which is based on the growth mixture - model described earlier in this paper : ( 2)cj = p0cj+(1p0cj)p1cj , p0cj = exp ( 0c+1cagej)1+exp ( 0c+1cagej),p1cj = exp ( 0c+1cagej + 2cagej2 ) , where cj indicates the predicted probability for the group c at time j and cj was composed using both predicted probabilities p0cj and p1cj which were obtained from the zero - inflation and the poisson parts of the model . note that parameters 0c and 1c are associated with the zero - inflation part of the model , while 0c , 1c , and 2c are coefficients associated with the poisson part of the model . here , the estimate 2c will be zero for groups where the nonlinear pattern is not best described with the group trajectory . a cox proportional hazard model was used to investigate the effect of stressful life event trajectory classes on mortality . the general equation is ( 3)(ti ci , xi)=0(ti)e1ci+2xi , where 0(ti ) is individual i 's risk of dying with baseline hazard rate ( 0 ) at time t , c is the class indicator estimate obtained from the growth mixture model in the first stage , and x represents the vectors of demographics and health behaviors . parameter 1 represents the effect of the class of stressful life event trajectories on risk of dying , and parameter 2 represents the effect of demographics ( i.e. , education ) and health behaviors ( i.e , drinking ) on risk of dying . following mroczek et al . , we used two different proportional hazard models , where the first model used only class assignments , and the second model utilized both class estimates and covariates to determine whether any of the effects of life events class on mortality were affected by the covariates . an iterative procedure was used to determine the number of classes , with the number of classes determined by change in the model fit criterion , bic . we first examined the total life events measure , which included the two health items . as can be seen in table 1 , the fit improved as classes were added to the model , starting with a single - class model and moving up to a model with four classes . however , beyond four classes , the fit became worse . thus , we determined that the four - class model provided the best estimate of the true number of classes . table 2 provides the intercept and linear slope estimates for the four classes . while we tested the quadratic term for all of the classes , it was only significant for the first class . we compared a simpler model in which only the first class had a quadratic term to a more complex model which estimated quadratic terms for all classes . the model comparison test for the more complex model did not significantly improve over the simpler model , 2bic = 14.23 , so we retained the simpler model . three of the classes showed decreasing slopes , with low , medium , and high intercept values . class 1 ( low stress ) , class 2 ( moderate stress ) and class 3 ( high stress ) contained 28.5% , 52.3% , and 18.2% of the sample . however , class 4 ( nonlinear ) shows an inverted u - shaped relationship between life events and age , with life events increasing from age 50 to about 70 , and decreasing thereafter . we repeated the procedure for the life events measure that omitted the two health items . as can be seen in table 3 , only three classes were found for this scoring of the life events measure . further , none of the quadratic terms were significant , so the final model included only linear terms for the slopes . as can be seen in figure 3 and table 4 , all of the groups had negative slopes , indicating decreases over time , with low , medium , and high intercepts ( classes 1 , 2 , and 3 had 32.36% , 55.09% , and 12.54% of the sample , resp . ) . these class assignments were used in the subsequent analyses predicting mortality , as they omitted the potential confounds of the health items . by 2010 , 693 ( 48.02% ) of the sample was deceased . the deaths were not equally distributed across the three trajectory classes of stressful life events ( no health items ) , x(2 , n = 1443 ) = 15.10 , p < .01 . only 40.69% of the low - stress group was deceased , compared to 51.19% and 53.04% of the moderate- and high - stress groups , respectively . in the first model , we investigated the effect of classes of stress trajectories on mortality . here , we were using the low - stress group as the comparison group . in the second model , we included covariates assessing marital status , education , self - rated health and alcohol and tobacco use . as can be seen in table 5 , both the moderate- and high - stress groups were almost 50% more likely to die than the low - stress group ( hazard ratios ( 95% ci ) = 1.43 ( 1.16 , 1.76 ) and 1.49 ( 1.10 , 2.02 ) , ps < .001 , and .01 , resp . ) . adding the covariates reduced these slightly , but they remained significant ( hazard ratios = 1.42 ( 1.14 , 1.76 ) and 1.37 ( 1.01 , 1.87 ) , p = 001 and p < .05 , resp . ) . being married and having better self - reported health were protective factors , while being a nondrinker and being a smoker were risk factors for mortality . first , we examined patterns of change in two measures of stressful life events over 18 years , one a summary score which included health events , and a second that excluded the two health events . using growth mixture models , we found four patterns for the stressful life event measure which included the health events . three showed linear decreases over time and were distinguished primarily by the initial number of events ( low , moderate , and high stress ) . the fourth pattern , however , demonstrated a nonlinear inverted u , which peaked at about age 70 . in the analysis of the life events measure which excluded health events , the first three patterns were replicated but the fourth pattern was not found , suggesting that the health events accounted for the nonlinearity . to our knowledge , this study is unique in two respects : first in examining longitudinal change in self - reported stressors over nearly two decades and second in identifying different patterns of change and relating these patterns to mortality . earlier studies either spanned much shorter periods of time [ 10 , 11 , 16 ] or used stressful life events coded from interviews . while this latter study found cross - sectional differences between young , middle - aged , and older adults , with younger adults having higher levels of stressful life events , they did not find longitudinal decreases over time . however , our study focused primarily on middle - aged and older men and did find decreases even using a measure which assesses events more likely to occur to older adults . it is possible that a different pattern of results might have occurred had we included young adults or women in the sample . second , we considered whether different patterns of stressful life events were associated with mortality . as reviewed earlier , research has shown a highly variable pattern of results , with different studies showing positive , negative , and no effects on mortality [ 21 , 22 , 24 , 25 ] . we hypothesized that multiple assessments of life events to tap stress chronicity might be presumed to show greater effects on health outcomes than a single measure . however , studies of allostatic load have been limited by omitting psychosocial measures of chronic stress in favor of clinical measures . further , some more recent studies failed to find associations between chronic stress and some of the allostatic load biomarkers ( see , for a review ) . thus , we sought to conduct a more stringent test of the impact of stress on mortality by examining different patterns of psychosocial stress , which included specific indicators of chronic stress over longer periods of time . further , we excluded health events , given that some have suggested that the relationship between stress and mortality is limited to health - related events . thus , we utilized the stress classes generated from the growth mixture models of stressful life events , excluding the ones related to health . moreover , while most theories of stress and health implicitly assume a linear relationship , we hypothesized that the pattern of results would support a nonlinear model . specifically , we tested the hormesis model currently being advocated by some biogerontologists , in which moderate stress levels are thought to be protective for health outcomes [ 6 , 7 , 34 ] . using a proportional hazards model , in which the different stress patterns were entered first , and then covariates added ( see ) , we found that both the moderate- and high - stress groups showed significantly higher risk of mortality than the low - stress group . this relationship was only slightly attenuated by the addition of standard health behavior risk factors ( e.g. , smoking and drinking ) as covariates . however , contrary to the prediction from hormesis theory , those with moderate - stress levels did not show enhanced longevity . rather , the low - stress group showed the lowest levels of mortality . however , it is noteworthy that both the moderate- and high - stress groups showed similar mortality risks , which is also not predicted by the allostatic load model . the first is that the allostatic load model may be largely correct but needs to acknowledge an asymptotic relationship in which higher levels of stress do not confer much additional risk over moderate levels ( see ) . the second possibility reflects the relative youth of the field of hormesis there are currently few guidelines for what level of stress might be considered hormetic . the low - stress group did have some stress averaging nearly 2 sles during their 50s , dropping down to about 1 in their 80s . it is possible that this low level was actually hormetic and provided protection over the higher levels . in other words , low ( but not zero ) stress , rather than moderate stress , is protective . one or two stressful life events a year may be manageable , allowing individuals to cope in a fashion which increases mastery , theoretically increasing hormesis . however , anything over two stressful life events might prove overwhelming . to adequately test this , we would need to find populations which experienced no stress , which would be difficult . nonetheless , this nonlinear relationship is quite interesting , and might account for the inconsistency in the findings in the literature . much as the relationship between age and mental health is muddied by its nonlinear ( j - shaped ) relationship [ 5658 ] , so too the nonlinear , asymptotic relationship between stress and mortality might have created similar inconsistencies . those with better self - rated health showed lower levels of mortality , supporting many other studies . likewise , married men had lower mortality , as previously found . while education had no significant effect , men who were teetotalers showed higher risk of mortality . smoking was only marginally related to mortality , undoubtedly because so few of the nas men have continued to smoke . nonetheless , stressful life event levels clearly had independent effects on mortality . a major limitation of this study was that the sample consisted primarily of white , middle - class men , and other patterns of results would likely be found in more diverse samples which include women and minorities . it is also possible that other types of stress measures , such as daily stressors , would show a different pattern of results . it is surprising that the effects of life events on mortality did not appear to be mediated through health behavior habits such as drinking and smoking but rather had independent effects . future studies should examine whether the types of biomarkers used to measure allostatic load mediate the effects of psychosocial stress on mortality . also , it is possible that the use of longitudinal trajectories may have obscured any hormesis - like relationships . growth mixture models smooth out the curve , and it is possible that an analytical technique which better addresses the intermittent nature of stress might show physiological toughening effects . nonetheless , this study shows that longitudinal assessments of stressful life events significantly predict mortality independent of standard risk factors and that high stressful life event levels did not convey appreciably greater risk than moderate ones . future studies should investigate both possible nonlinear relationships between stress and health as well as potential mediating pathways .

we examined long - term patterns of stressful life events ( sle ) and their impact on mortality contrasting two theoretical models : allostatic load ( linear relationship ) and hormesis ( inverted u relationship ) in 1443 nas men ( aged 4187 in 1985 ; m = 60.30 , sd = 7.3 ) with at least two reports of sles over 18 years ( total observations = 7,634 ) . using a zero - inflated poisson growth mixture model , we identified four patterns of sle trajectories , three showing linear decreases over time with low , medium , and high intercepts , respectively , and one an inverted u , peaking at age 70 . repeating the analysis omitting two health - related sles yielded only the first three linear patterns . compared to the low - stress group , both the moderate and the high - stress groups showed excess mortality , controlling for demographics and health behavior habits , hrs = 1.42 and 1.37 , ps < .01 and < .05 . the relationship between stress trajectories and mortality was complex and not easily explained by either theoretical model .

1. Introduction 2. Methods 3. Results 4. Discussion

first , stressors are not isolated occurrences but can have lifelong effects on health through setting up different patterns of vulnerability and resilience [ 1 , 2 ] . inconsistent results in the literature on the effects of stress on health and mortality may reflect the aggregation of individuals who may have had very different experiences over their life . first , using 18 years of data in a large sample of middle - aged and older men , we sought to identify patterns of change in stressful life events across the lifespan . the second was to examine the effects of different types of stress trajectories on mortality . specifically , we contrasted an allostatic load model which assumes that higher levels of chronic stress will have the greatest adverse effects on health , with a hormesis model [ 57 ] , which suggests that moderate amount of stress may have protective effects on health . however , most life events inventories sample events more common in young adulthood , such as graduations , new jobs , marriages , having children , and divorces . newer life event measures that targeted middle - aged and older adults show little or slightly negative correlations with age although health - related events show increased frequency in late life , as do loss events [ 10 , 11 ] . a few longitudinal studies have examined age - related changes in the number and type of stressful life events . chiriboga found that young adults reported more life events ( both negative and positive ) than older adults , but there were no differences between individuals in mid - life and late life . found suggestions of nonlinear change over time , with the number of self - reported life events increasing until about age 65 and then decreasing into later life . ensel and lin [ 14 , 15 ] used data spanning 15 years , but the 12-year gap between the last two assessments did not permit an examination of trajectories over time , although they did find that the more distal events predicted the more recent ones a few studies examined growth curve stress trajectories in adults [ 10 , 11 , 16 ] but over short periods of time ( e.g. , three to seven years ) . however , there are inconsistent results in the literature on the relationship between stress and mortality . while some studies have found that high levels of stressful life events do predict mortality [ 21 , 22 ] , this may be restricted primarily to health - related stressful life events . other studies have found no relationships between life events and mortality , and one found an inverse association . allostatic load ( al ; ) is a construct assessed across multiple biosystems , including cardiovascular , metabolic , neuroendocrine , and inflammatory markers that are presumed to reflect chronic psychosocial stress . however , few studies have examined psychosocial stress measures in the context of allostatic load , and the ones that have generally find little or no relationship between various components of al and psychosocial stress . thus , examining long - term stress patterns may be a more promising way to link stress with mortality . while most stress theories implicitly assume that there is a linear relationship between stress and adverse outcomes , there is some evidence to suggest that there is a nonlinear relationship . biogerontologists have recently become interested in hormesis , which is defined as the phenomenon in which adaptive responses to low doses of otherwise harmful conditions improve the functional ability of cells and organisms most researchers assume that there is a linear , dose - response relationship between stress and adverse health outcomes , but hormesis suggests that there is a nonlinear or inverted u relationship , such that low or even moderate doses of an otherwise toxic substance provide protective benefits against future exposure to stresses . applying the models to human stress and health leads to some interesting and somewhat counterintuitive hypotheses , namely , individuals who experience intermittent or moderate amounts of stress may develop better coping resources which allow them to be more resilient to stress in later life . in a qualitative study , vaillant , described one such person who managed to adopt an extremely self - limiting lifestyle in that she managed to avoid employment , marriage , and even friends , but was poorly equipped to deal with problems in later life . a possible explanation for the inconsistent relations between stress and mortality is that hormesis may apply to psychosocial as well as physiological stressors . thus , the possibility exists that there is a nonlinear relationship between stress and mortality that moderate stress levels may be associated with better longevity , while both chronic high and low levels may be associated with poorer longevity . the primary aim of this study was to compare longitudinal change in life events and its consequences on mortality , using 18 years of data from the va normative aging study ( nas ) , a longitudinal panel study which has followed a large sample of men since the mid-1960s . the nas has collected up to nine assessments of stressful life events . to our knowledge , this constitutes the longest extant longitudinal study of stress and coping using quantitative measures and , as such , provides a unique resource to model changes in stress patterns from mid- to late life and their relationships to mortality . only a few studies have examined trajectories of life events in adulthood [ 10 , 11 , 16 ] . further , we hypothesized that by using latent class growth analyses ( lcgas ; ) , we would identify at least three different patterns of trajectories , with some individuals exhibiting chronically elevated levels of life events , others showing stable low levels , and others showing moderate or intermittent levels of life events . we estimated two sets of lcga models , one for stressful life events measure including health items , and one for a measure excluding health items . the purpose of the first set of analyses was to examine how stressful life events , including health problems , changed with age ; the second omitted health problems in order to avoid a potential confound between stress and health when predicting mortality . the second aim was to understand how these developmental trajectories of stress related to all - cause mortality , contrasting an allostatic load model with a hormetic one . if the allostatic load model is correct , there should be a linear relationship between stress and mortality . we hypothesized that individuals in the chronic high- or low - stress categories would be more likely to have higher rates of mortality than those in the intermediate stress categories . the men are equally divided between blue and white collar workers , and reflected the racial profile of boston in the late 1950s , that is , primarily white . as of august 2010 , 852 ( 37.4% ) were participating , 1,305 ( 57.2% ) were deceased , 89 ( 3.9% ) were lost or dropped out , and 34 ( 1.5% ) were too sick to participate . in this study , we began with 1,565 men who completed the initial stress inventory in 1985 . we computed stress trajectories from 1985 to 2002 ( an 18-year period ) on the 1443 men who had at least three assessments of stressful life events . mean age of this sample in 1985 was 60.30 ( sd = 7.73 , range = 4187 ) . we then predicted subsequent mortality , using these stress trajectories as a predictor , and including covariates from various surveys and medical examinations , which decreased the n to 977 . this subsample did not differ from the larger sample in health ratings , smoking , alcohol consumption , or education although they were more likely to be married , x(1 ; n = 1222 ) = 4.30 , p < .05 . nas men complete biomedical examinations every three years as well as periodic mail surveys assessing psychosocial variables , which typically have response rates exceeding 80% . the nas began collecting stressful life events in 1985 , using three mail surveys over six years , which also included information on mental and physical health . the hsb was included in a packet of materials sent to the men before they reported for their triennial physical exam . in the nas , the elders life stress inventory ( elsi ; ) was included in both the social support and the health and social behavior ( hsb ) surveys . although this life events measure is correlated with other types of stress measures , such as hassles and perceived stress , as well as personality measures such as neuroticism , it contributes independent variance to health outcomes . we scored the elsi in two ways : a total sum which includes all 30 items and one which omits the two items which tap health events . we used a total of 7,634 observations for 1443 men ( m = 5.29 , sd = 1.58 , range = 39 ) . in the models predicting mortality as a function of stress trajectory , we included a number of covariates that are known to affect mortality , including a self - reported health rating , demographics , and health behavior habits . for self - reported health , we used a single item assessing general health from the first social survey in 1985 taken from the sf-36 . health was rated from 15 , where 1 = poor and 5 = excellent ( m = 4.2 , sd = 0.72 ) . vital status of nas participants was monitored by periodic mailings , and when notified of a death , official death certificates were obtained . to identify different trajectory classes of stressful life events , we used a semi - parametric mixture - model approach for nagin and jones et al . however , because approximately 30% of nas men did not report stressful life events ( see figure 1 ) , it was necessary to use a zero - inflated poisson ( zip ) model to account for the fact that there are more zeros than would be expect under the poisson assumption . we used the voung test to compare the zero - inflated model with an ordinary poisson regression model for both the sle sum and the sle sum omitting the health items . in both instances , the vuong non - nested hypothesis test statistics suggested the zip model was superior to the standard poisson model ( p < .001 for both outcomes ) . in addition , we tested a quadratic term for age , based on preliminary analyses , which suggested that there might be nonlinear trajectories . in order to identify the correct number of classes , we used the change in bayesian information criterion ( bic , ) between models as an approximation to the log of bayes factor , 2log e ( b10 ) 2(bic ) , where bic is the bic of the more complex ( alternative ) model minus the bic of the simpler ( null ) model . finally , we considered the model 's interpretability . in order to graph the predicted trajectories for each group , c , we used the following model , written with no covariates adjustments , which is based on the growth mixture - model described earlier in this paper : ( 2)cj = p0cj+(1p0cj)p1cj , p0cj = exp ( 0c+1cagej)1+exp ( 0c+1cagej),p1cj = exp ( 0c+1cagej + 2cagej2 ) , where cj indicates the predicted probability for the group c at time j and cj was composed using both predicted probabilities p0cj and p1cj which were obtained from the zero - inflation and the poisson parts of the model . note that parameters 0c and 1c are associated with the zero - inflation part of the model , while 0c , 1c , and 2c are coefficients associated with the poisson part of the model . a cox proportional hazard model was used to investigate the effect of stressful life event trajectory classes on mortality . the general equation is ( 3)(ti ci , xi)=0(ti)e1ci+2xi , where 0(ti ) is individual i 's risk of dying with baseline hazard rate ( 0 ) at time t , c is the class indicator estimate obtained from the growth mixture model in the first stage , and x represents the vectors of demographics and health behaviors . parameter 1 represents the effect of the class of stressful life event trajectories on risk of dying , and parameter 2 represents the effect of demographics ( i.e. , education ) and health behaviors ( i.e , drinking ) on risk of dying . , we used two different proportional hazard models , where the first model used only class assignments , and the second model utilized both class estimates and covariates to determine whether any of the effects of life events class on mortality were affected by the covariates . we first examined the total life events measure , which included the two health items . as can be seen in table 1 , the fit improved as classes were added to the model , starting with a single - class model and moving up to a model with four classes . while we tested the quadratic term for all of the classes , it was only significant for the first class . we compared a simpler model in which only the first class had a quadratic term to a more complex model which estimated quadratic terms for all classes . three of the classes showed decreasing slopes , with low , medium , and high intercept values . class 1 ( low stress ) , class 2 ( moderate stress ) and class 3 ( high stress ) contained 28.5% , 52.3% , and 18.2% of the sample . however , class 4 ( nonlinear ) shows an inverted u - shaped relationship between life events and age , with life events increasing from age 50 to about 70 , and decreasing thereafter . we repeated the procedure for the life events measure that omitted the two health items . as can be seen in figure 3 and table 4 , all of the groups had negative slopes , indicating decreases over time , with low , medium , and high intercepts ( classes 1 , 2 , and 3 had 32.36% , 55.09% , and 12.54% of the sample , resp . ) these class assignments were used in the subsequent analyses predicting mortality , as they omitted the potential confounds of the health items . the deaths were not equally distributed across the three trajectory classes of stressful life events ( no health items ) , x(2 , n = 1443 ) = 15.10 , p < .01 . only 40.69% of the low - stress group was deceased , compared to 51.19% and 53.04% of the moderate- and high - stress groups , respectively . in the first model , we investigated the effect of classes of stress trajectories on mortality . here , we were using the low - stress group as the comparison group . in the second model , we included covariates assessing marital status , education , self - rated health and alcohol and tobacco use . as can be seen in table 5 , both the moderate- and high - stress groups were almost 50% more likely to die than the low - stress group ( hazard ratios ( 95% ci ) = 1.43 ( 1.16 , 1.76 ) and 1.49 ( 1.10 , 2.02 ) , ps < .001 , and .01 , resp . ) adding the covariates reduced these slightly , but they remained significant ( hazard ratios = 1.42 ( 1.14 , 1.76 ) and 1.37 ( 1.01 , 1.87 ) , p = 001 and p < .05 , resp . ) first , we examined patterns of change in two measures of stressful life events over 18 years , one a summary score which included health events , and a second that excluded the two health events . using growth mixture models , we found four patterns for the stressful life event measure which included the health events . three showed linear decreases over time and were distinguished primarily by the initial number of events ( low , moderate , and high stress ) . the fourth pattern , however , demonstrated a nonlinear inverted u , which peaked at about age 70 . in the analysis of the life events measure which excluded health events , the first three patterns were replicated but the fourth pattern was not found , suggesting that the health events accounted for the nonlinearity . earlier studies either spanned much shorter periods of time [ 10 , 11 , 16 ] or used stressful life events coded from interviews . while this latter study found cross - sectional differences between young , middle - aged , and older adults , with younger adults having higher levels of stressful life events , they did not find longitudinal decreases over time . however , our study focused primarily on middle - aged and older men and did find decreases even using a measure which assesses events more likely to occur to older adults . second , we considered whether different patterns of stressful life events were associated with mortality . as reviewed earlier , research has shown a highly variable pattern of results , with different studies showing positive , negative , and no effects on mortality [ 21 , 22 , 24 , 25 ] . thus , we sought to conduct a more stringent test of the impact of stress on mortality by examining different patterns of psychosocial stress , which included specific indicators of chronic stress over longer periods of time . further , we excluded health events , given that some have suggested that the relationship between stress and mortality is limited to health - related events . thus , we utilized the stress classes generated from the growth mixture models of stressful life events , excluding the ones related to health . moreover , while most theories of stress and health implicitly assume a linear relationship , we hypothesized that the pattern of results would support a nonlinear model . using a proportional hazards model , in which the different stress patterns were entered first , and then covariates added ( see ) , we found that both the moderate- and high - stress groups showed significantly higher risk of mortality than the low - stress group . however , contrary to the prediction from hormesis theory , those with moderate - stress levels did not show enhanced longevity . rather , the low - stress group showed the lowest levels of mortality . however , it is noteworthy that both the moderate- and high - stress groups showed similar mortality risks , which is also not predicted by the allostatic load model . the first is that the allostatic load model may be largely correct but needs to acknowledge an asymptotic relationship in which higher levels of stress do not confer much additional risk over moderate levels ( see ) . the low - stress group did have some stress averaging nearly 2 sles during their 50s , dropping down to about 1 in their 80s . one or two stressful life events a year may be manageable , allowing individuals to cope in a fashion which increases mastery , theoretically increasing hormesis . however , anything over two stressful life events might prove overwhelming . much as the relationship between age and mental health is muddied by its nonlinear ( j - shaped ) relationship [ 5658 ] , so too the nonlinear , asymptotic relationship between stress and mortality might have created similar inconsistencies . smoking was only marginally related to mortality , undoubtedly because so few of the nas men have continued to smoke . nonetheless , stressful life event levels clearly had independent effects on mortality . a major limitation of this study was that the sample consisted primarily of white , middle - class men , and other patterns of results would likely be found in more diverse samples which include women and minorities . it is surprising that the effects of life events on mortality did not appear to be mediated through health behavior habits such as drinking and smoking but rather had independent effects . future studies should examine whether the types of biomarkers used to measure allostatic load mediate the effects of psychosocial stress on mortality . growth mixture models smooth out the curve , and it is possible that an analytical technique which better addresses the intermittent nature of stress might show physiological toughening effects . nonetheless , this study shows that longitudinal assessments of stressful life events significantly predict mortality independent of standard risk factors and that high stressful life event levels did not convey appreciably greater risk than moderate ones . future studies should investigate both possible nonlinear relationships between stress and health as well as potential mediating pathways .

protein interaction network around known and candidate components of the tnf/nfb pathway ( bouwmeester et al . , 2004 ) . using an integrated approach comprising tandem affinity purification and liquidchromatography mass spectrometry , traf7 was identified with taptagged mekk3 , a mapkkk required for the tnfinduced activation of nfb ( yang et al . , 2001 ) . independently , traf7 was also identified using bioinformatics tools in a search for sequences containing a traflike ring finger domain ( xu et al . , 2004 ) . as other traf family members , traf7 contains an aminoterminal ring finger domain ( aa 125160 ) , followed by an adjacent zinc finger domain ( aa 221287 ) . however , instead of the classical traf domain , the carboxy terminus of traf7 contains seven wd40 repeats ( bouwmeester et al . , 2004 ; xu et al . , 2004 ) . due to the lack of a canonical carboxyterminal traf domain , some authors question whether traf7 is a proper traf protein . as mentioned before , traf7 has been identified for its association with mekk3 . however , while other traf family members interact with various signaling molecules , including protein kinases , through the traf domain , the interaction of traf7 with mekk3 is mediated by the wd40 repeatscontaining region of the protein ( bouwmeester et al . , 2004 ) . functionally , traf7 and mekk3 cooperate in the signal transduction pathways that lead to activation of jnk and p38 map kinases following tnf stimulation ( bouwmeester et al . in addition to ap1 , the main transcription factors activated by jnk , traf7 also positively regulates the transcriptional activity of chop / gadd153 , a transcription factor activated following exposure to different cellular stresses , such as uv light , genotoxic agents , and protein misfolding in the endoplasmic reticulum ( xu et al . , 2004 ) . similarly to traf2 , traf3 , and traf6 ( karin and gallagher , 2009 ; hcker et al . , 2011 ; xie , 2013 ) , the ring domain of traf7 possesses an e3 ubiquitin ligase activity ( bouwmeester et al . , 2004 ) . in addition to autoubiquitination ( bouwmeester et al . , 2004 ) , traf7 also promotes ubiquitination of several cellular targets , including the adapter molecule nfb essential modulator ( nemo ) ( zotti et al . , 2011 ) , the p65 subunit of nfb transcription factor ( tsikitis et al . , 2010 ; zotti et al . , 2011 ) , the antiapoptotic protein cflip ( scudiero et al . , 2012 ) and the tumor suppressor protein p53 ( wang et al . , 2013 ) . ubiquitin possesses seven lysines ( lys6 , lys11 , lys27 , lys29 , lys33 , lys48 , and lys63 ) and the consequence of an ubiquitination reaction depends on the lystype of ubiquitin linkage ( kulathu and komander , 2012 ) . indeed , traf7 is able to catalyze different lystypes of ubiquitinations . in fact , traf7 promotes predominantly lys29linked polyubiquitination of nemo , p65 , and cflip ( zotti et al . , 2011 ; scudiero et al . , 2012 ) , and this type of ubiquitination is associated with lysosomal degradation of the target proteins ( chastagner et al . , 2006 ; zotti et al . , 2011 ; scudiero et al . , , traf7 promotes lys48linked ubiquitination of p53 ( wang et al . , 2013 ) , which is usually followed by the proteosomal degradation of the ubiquitinated protein . along with ubiquitination reactions , traf7 also binds to and stimulates sumoylation of the protooncogene product cmyb ( morita et al . , 2005 ) , a transcription factor that regulates proliferation and differentiation of hematopoietic cells ( mucenski et al . , 1991 ) . having a primary role in the regulation of the acquired and innate immune responses , it is not surprising that the first evidence for an involvement of traf proteins in tumors came from the molecular analysis of specific b cells malignancies . demchenko et al . , 2010 ) and biallelic or monoallelic deletion of traf3 , often associated with epigenetic alteration , homozygous deletions of the traf3 locus have also been observed in cases of chronic lymphocytic leukemias ( nagel et al . , 2009 ) , waldenstrm 's macroglobulinemia a distinct bcell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow and igm paraprotein production ( braggio et al . , 2009 ) , and classical hodgkin lymphoma ( otto et al . , 2012 ) . in addition to the above mentioned inactivating mutations of traf2 found in b cell malignancies , a significant fraction of diffuse large bcell lymphoma ( dlbl ) carry somatic activating mutations in traf2 ( compagno et al . , 2009 ) . these mutations produce traf2 molecules with considerably enhanced ability to activate nfb , which catalyzes transcription of cytoprotective and antiapoptotic genes . thus , in the same cell type , the b lymphocytes , traf2 can function as both oncogene and tumor suppressor gene . in the same study by compagno et al . ( 2009 ) , mutations of traf5 were identified even with higher frequency compared to traf2 ( 5% traf5 vs. 3% traf2 ) . however , the effect of these traf5 mutations on nfb activation , or other signaling pathways , has not been investigated yet . while traf2 and traf3 have been involved in several lymphoid tumors , traf4 and traf6 are involved in a broader spectrum of cancers . indeed , traf4 cdna was initially isolated from metastatic breast cancer ( rgnier et al . , 1995 ; tomasetto et al . , 1995 ) , where the traf4 gene was amplified . in a subsequent larger study including epithelial tumors , traf4 was found overexpressed in approximately 60% of lung , breast and ovarian , 44% of bladder , and 28% of colon carcinomas ( camilleribrot et al . , 2007 ) . moreover , traf4 overexpression was not only most commonly observed in epithelial tumors ( 48% ) , but also in 31% of melanomas , 21% of neurogenic tumors , and 17% of lymphomas ( camilleribrot et al . , 2007 ) . thus , traf4 protein overexpression appears to be a common feature of several human cancers . frequent amplification of the traf6 locus , with concomitant mrna overexpression , has been reported in lung cancers ( starczynowski et al . , 2011 ) , colon cancer ( sun et al . , 2014 ) , and osteosarcoma samples ( meng et al . , 2012 ) . expression of traf6 was found upregulated also in pancreatic cancer tissues , resulting in deregulated expression of multiple genes involved in cell growth , apoptosis , and migration ( rong et al . , 2014 ) . meningiomas are tumors originating from the meningeal coverings of the brain and the spinal cord ( mawrin and perry , 2010 ) . they represent one of the most frequent primary brain tumor , accounting for about one third of all cerebral nervous system tumors . the who currently recognizes 15 different variants of meningioma ( louis et al . , 2007 ) , classified in three grades of malignancy . the secretory subtype of meningioma , which represents approximately 3% of all meningiomas , follows a more aggressive clinical course due to increased brain swelling ( probstcousin et al . , 1997 ; mawrin and perry , 2010 ; mawrin et al . , 2015 ) . a large fraction of all sporadic meningiomas ( 4060% ) display recurrent genetic alterations resulting in inactivating mutations in the nf2 gene locus located on chromosome 22 ( rouleau et al . , 1993 ; , 1993 ; ruttledge et al . , 1994 ; peyre and kalamarides , 2014 ; domingues et al . , 2015 ) . the rate of nf2 mutations is similar in menigiomas of different who grades , suggesting that nf2 inactivation is critical for tumor initiation , rather than progression ( mawrin and perry , 2010 ; choy et al . , 2011 ) . the nf2 locus encodes for merlin , a pleiotropic protein involved in cytoskeleton organization and cell contact inhibition ( pecinaslaus , 2013 ) , which also regulates transmembrane receptor accumulation and receptor signaling , including erb receptors , plateletderived growth factor receptor , insulinlike growth factor 1 receptor and vascular endothelial growth factor receptors ( lallemand et al . , 2009 ) . besides these roles in cytoskeleton organization and receptor signaling , merlin also regulates ubiquitination events , by inhibiting the e3 ubiquitin ligase crl4(dcaf1 ) in the nucleus ( li et al . , 2010 ) . , 2013 ; clark et al . , 2013 ; reuss et al . , 2013 ; abedalthagafi et al . , 2016 ; clark et al . , 2016 ) have led to the discovery of driver traf7 mutations implicated in meningioma tumorigenesis . overall , these studies show that mutations in traf7 are observed in nearly onefourth of the meningiomas studied , and in all meningiomas of the secretory subtype . interestingly , traf7 mutations are exclusive of nf2 mutations suggesting that the two proteins act along the same pathway and often cooccur with mutations in klf4 or in akt1 . klf4 is a transcription factor that regulates differentiation in a variety of different cell types and its expression is essential to reprogram adult cells in pluripotent adult stem cells ( takahashi et al . , 2007 ) , whereas akt1 is serinethreonine kinase involved in proliferation , and is a well known oncogene ( vivanco and sawyers , 2013 ) . also , whereas the cooccurring mutations in klf4 and akt1 are always the same ( k409q for klf4 and e17k for akt1 ) , the traf7 mutations found in the various samples are distributed along the whole protein , with a marked predilection for the wd40 repeats ( fig . , the akt1e17k mutation found in meningiomas often occurs in various other human cancers , including breast , colon , and ovarian cancers ( carpten et al . , 2007 ) . traf7 mutations in human cancers . in the domain structure of traf7 , on the top are indicated the mutations found in meningiomas , on the bottom the mutations found in mesothelioma . specifically , combination of klf4 and traf7 mutations was only found in virtually 100% of secretory meningiomas moreover , meningiomas arising in specific anatomical positions are characterized by mutations in the same genes . in fact , tumors with traf7/akt1/klf4 mutations predominantly localize medially in the skull base , whereas meningiomas with nf2 mutations predominantly localize in the hemispheres or laterally in the skull base ( clark et al . , 2013 ) . malignant mesothelioma ( mm ) is an aggressive cancer that results from unregulated proliferation of the mesothelial cells lining the pleural , peritoneal , and pericardial cavities ( yang et al . , 2008 ) . the vast majority of mms is associated to exposure to mineral fibers , particularly asbestos and erionite . mms are histologically classified in the three subtypes epithelial , biphasic ( or mixed ) , and sarcomatoid . malignant pleural mesothelioma ( mpm ) is the most common type of mm , accounting for approximately 70% of all cases . unfortunately , mpm is commonly diagnosed at advanced stages and its survival rate is lower than 12 months ( yang et al . , 2008 ) . the genes whose role has been well established in mpm are cdkn2a , nf2 ( i.e. , the same gene mutated in meningiomas ) , and bap1 ( cheng et al . , 1994 ; bianchi et al . , 1995 ; the cdkn2a locus encodes for several proteins , the most wellstudied are the p16(ink4a ) and the p14(arf ) proteins , which both function as tumor suppressors ( zhao et al . , 2016 ) . bap1 ( brca1associated protein 1 ) also is a tumor suppressor gene , and it encodes for a deubiquitinating enzyme that regulates key cellular pathways , including cell cycle , cellular differentiation , transcription , and dna damage response ( wang et al . , 2016 ) . in a recent sequencing work of transcriptomes and exomes from 216 mpms , traf7 was found among the significantly mutated genes ( 5/216 ; 2,3% ) ( bueno et al . , 2016 ) . as in the case of meningiomas , most of the mms mutations observed in traf7 ( 4/5 ) were localized at the carboxyterminal wd40 repeats ( fig . mutations in traf7 often cooccurred with mutations of p53 , but , similarly to what observed in meningiomas , rarely with mutations of nf2 , which was found mutated in 19% of the samples . from the histological point of view , traf7 was found mutated in the epithelial and biphasic subtypes of mpm , but not in the sarcomatoid subtype . in addition to mpm , a somatic mutation of traf7 has been reported in a case of papillary peritoneal mesothelioma , where nf2 was not mutated as well ( yu et al . , 2011 ) . having a primary role in the regulation of the acquired and innate immune responses , it is not surprising that the first evidence for an involvement of traf proteins in tumors came from the molecular analysis of specific b cells malignancies . 2010 ) and biallelic or monoallelic deletion of traf3 , often associated with epigenetic alteration , have been detected in multiple myeloma samples ( annunziata et al . homozygous deletions of the traf3 locus have also been observed in cases of chronic lymphocytic leukemias ( nagel et al . , 2009 ) , waldenstrm 's macroglobulinemia a distinct bcell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow and igm paraprotein production ( braggio et al . , 2009 ) , and classical hodgkin lymphoma ( otto et al . , 2012 ) . in addition to the above mentioned inactivating mutations of traf2 found in b cell malignancies , a significant fraction of diffuse large bcell lymphoma ( dlbl ) carry somatic activating mutations in traf2 ( compagno et al . , 2009 ) . these mutations produce traf2 molecules with considerably enhanced ability to activate nfb , which catalyzes transcription of cytoprotective and antiapoptotic genes . thus , in the same cell type , the b lymphocytes , traf2 can function as both oncogene and tumor suppressor gene . in the same study by compagno et al . ( 2009 ) , mutations of traf5 were identified even with higher frequency compared to traf2 ( 5% traf5 vs. 3% traf2 ) . however , the effect of these traf5 mutations on nfb activation , or other signaling pathways , has not been investigated yet . while traf2 and traf3 have been involved in several lymphoid tumors , traf4 and traf6 indeed , traf4 cdna was initially isolated from metastatic breast cancer ( rgnier et al . , 1995 1995 ) , where the traf4 gene was amplified . in a subsequent larger study including epithelial tumors , traf4 was found overexpressed in approximately 60% of lung , breast and ovarian , 44% of bladder , and 28% of colon carcinomas moreover , traf4 overexpression was not only most commonly observed in epithelial tumors ( 48% ) , but also in 31% of melanomas , 21% of neurogenic tumors , and 17% of lymphomas ( camilleribrot et al . , 2007 ) . thus , traf4 protein overexpression appears to be a common feature of several human cancers . frequent amplification of the traf6 locus , with concomitant mrna overexpression , has been reported in lung cancers ( starczynowski et al . , 2011 ) , , 2014 ) , and osteosarcoma samples ( meng et al . , 2012 ) . expression of traf6 was found upregulated also in pancreatic cancer tissues , resulting in deregulated expression of multiple genes involved in cell growth , apoptosis , and migration ( rong et al . , 2014 ) . meningiomas are tumors originating from the meningeal coverings of the brain and the spinal cord ( mawrin and perry , 2010 ) . they represent one of the most frequent primary brain tumor , accounting for about one third of all cerebral nervous system tumors . the who currently recognizes 15 different variants of meningioma ( louis et al . , 2007 ) , classified in three grades of malignancy . the secretory subtype of meningioma , which represents approximately 3% of all meningiomas , follows a more aggressive clinical course due to increased brain swelling ( probstcousin et al . , 1997 ; mawrin and perry , 2010 ; mawrin et al . , 2015 ) . a large fraction of all sporadic meningiomas ( 4060% ) display recurrent genetic alterations resulting in inactivating mutations in the nf2 gene locus located on chromosome 22 ( rouleau et al . , 1993 ; , 1994 ; peyre and kalamarides , 2014 ; domingues et al . , 2015 ) . the rate of nf2 mutations is similar in menigiomas of different who grades , suggesting that nf2 inactivation is critical for tumor initiation , rather than progression ( mawrin and perry , 2010 ; choy et al . , 2011 ) . the nf2 locus encodes for merlin , a pleiotropic protein involved in cytoskeleton organization and cell contact inhibition ( pecinaslaus , 2013 ) , which also regulates transmembrane receptor accumulation and receptor signaling , including erb receptors , plateletderived growth factor receptor , insulinlike growth factor 1 receptor and vascular endothelial growth factor receptors ( lallemand et al . , 2009 ) . besides these roles in cytoskeleton organization and receptor signaling , merlin also regulates ubiquitination events , by inhibiting the e3 ubiquitin ligase crl4(dcaf1 ) in the nucleus ( li et al . , 2010 ) . several recent genomewide studies of meningiomas ( brastianos et al . , 2013 ; clark et al . , 2013 ; reuss et al . , 2013 ; 2016 ) have led to the discovery of driver traf7 mutations implicated in meningioma tumorigenesis . overall , these studies show that mutations in traf7 are observed in nearly onefourth of the meningiomas studied , and in all meningiomas of the secretory subtype . interestingly , traf7 mutations are exclusive of nf2 mutations suggesting that the two proteins act along the same pathway and often cooccur with mutations in klf4 or in akt1 . klf4 is a transcription factor that regulates differentiation in a variety of different cell types and its expression is essential to reprogram adult cells in pluripotent adult stem cells ( takahashi et al . , 2007 ) , whereas akt1 is serinethreonine kinase involved in proliferation , and is a well known oncogene ( vivanco and sawyers , 2013 ) . also , whereas the cooccurring mutations in klf4 and akt1 are always the same ( k409q for klf4 and e17k for akt1 ) , the traf7 mutations found in the various samples are distributed along the whole protein , with a marked predilection for the wd40 repeats ( fig . , the akt1e17k mutation found in meningiomas often occurs in various other human cancers , including breast , colon , and ovarian cancers ( carpten et al . , 2007 ) . traf7 mutations in human cancers . in the domain structure of traf7 , on the top are indicated the mutations found in meningiomas , on the bottom the mutations found in mesothelioma . specifically , combination of klf4 and traf7 mutations was only found in virtually 100% of secretory meningiomas ( clark et al . , 2013 ) . moreover , meningiomas arising in specific anatomical positions are characterized by mutations in the same genes . in fact , tumors with traf7/akt1/klf4 mutations predominantly localize medially in the skull base , whereas meningiomas with nf2 mutations predominantly localize in the hemispheres or laterally in the skull base ( clark et al . , 2013 ) . malignant mesothelioma ( mm ) is an aggressive cancer that results from unregulated proliferation of the mesothelial cells lining the pleural , peritoneal , and pericardial cavities ( yang et al . , 2008 ) . the vast majority of mms is associated to exposure to mineral fibers , particularly asbestos and erionite . mms are histologically classified in the three subtypes epithelial , biphasic ( or mixed ) , and sarcomatoid . malignant pleural mesothelioma ( mpm ) is the most common type of mm , accounting for approximately 70% of all cases . unfortunately , mpm is commonly diagnosed at advanced stages and its survival rate is lower than 12 months ( yang et al . , 2008 ) . the genes whose role has been well established in mpm are cdkn2a , nf2 ( i.e. , the same gene mutated in meningiomas ) , and bap1 ( cheng et al . , 1994 ; bianchi et al . , 1995 ; bott et al . , 2011 ; the cdkn2a locus encodes for several proteins , the most wellstudied are the p16(ink4a ) and the p14(arf ) proteins , which both function as tumor suppressors ( zhao et al . , 2016 ) . bap1 ( brca1associated protein 1 ) also is a tumor suppressor gene , and it encodes for a deubiquitinating enzyme that regulates key cellular pathways , including cell cycle , cellular differentiation , transcription , and dna damage response ( wang et al . , 2016 ) . in a recent sequencing work of transcriptomes and exomes from 216 mpms , traf7 was found among the significantly mutated genes ( 5/216 ; 2,3% ) ( bueno et al . , 2016 ) . as in the case of meningiomas , most of the mms mutations observed in traf7 ( 4/5 ) were localized at the carboxyterminal wd40 repeats ( fig . mutations in traf7 often cooccurred with mutations of p53 , but , similarly to what observed in meningiomas , rarely with mutations of nf2 , which was found mutated in 19% of the samples . from the histological point of view , traf7 was found mutated in the epithelial and biphasic subtypes of mpm , but not in the sarcomatoid subtype . in addition to mpm , a somatic mutation of traf7 has been reported in a case of papillary peritoneal mesothelioma , where nf2 was not mutated as well ( yu et al . , 2011 ) . recent data generated through deep sequencing of primary tumors have clearly indicated the involvement of traf7 in the genesis of various human cancers . specifically , traf7 is mutated in about 25% of meningiomas and significantly , although at a lower rate , in human mesotheliomas . an inactivating mutation of traf7 has also been reported in a case of merkel cell carcinoma , a rare and aggressive cutaneous neuroendocrine carcinoma ( goh et al . , 2015 ) . it is certainly interesting the fact that mutations in traf7 in human meningioma and mesothelioma cancers are alternative to mutations in nf2 , a gene which , strikingly , also shows a high rate of mutation in these two types of tumors . however , merlin , the product of the nf2 gene , is a multifaceted protein , functioning as a scaffold protein that connects cytoskeleton dynamics to signaling transduction pathways that control cell death and proliferation , including those triggered by tyrosine kinase receptors , small gtpases , mammalian target of rapamycin ( mtor ) , pi3k / akt , and hippo pathways ( petrilli and fernndezvalle , 2016 ) . thus , in this variety of different functions it is not obvious to distinguish which of them is / are shared by traf7 and nf2 in promoting cancer development . most of the traf7 mutations identified in human tumors localize at the wd40 repeats region and at the neighboring coiled coil domain , that is , the regions through which traf7 establishes interactions with several molecular partners ( zotti et al . , for example , the coiled coil domain of traf7 is involved in the interaction with nemo ( zotti et al . , 2011 ) , an adapter molecule essential for activation of the canonical pathway of nfb , a transcription factor that by promoting expression of antiapoptotic and proliferative genes is known to be generally hyperactive in the vast majority of human cancers ( karin , 2006 ; wu et al . , 2015 ) . generally , traf7 exerts a negative control on nfb activity , by promoting ubiquitination and lysosomal degradation of nemo and that of the p65 subunit of nfb ( zotti et al . , 2011 ) . traf7 also promotes ubiquitination and lysosomal degradation of cflip ( scudiero et al . , 2012 ) , a very well known inhibitor of apoptosis by interfering with caspases activation in tumor cells ( safa and pollok , 2011 ; shirley and micheau , 2013 ) whose stability is known to be controlled by traf proteins ( guiet et al . , 2002 ) . consistent with its role as a crucial negative regulator of the apoptotic pathway , cflip is found overexpressed in many different types of cancer , including mpm ( rippo et al . , 2004 ) . interestingly , the tumor suppressor protein p53 is another target of the ubiquitinating activity of traf7 , and traf7mediated ubiquitination of p53 plays a critical role in breast cancer development and progression ( wang et al . , 2013 ) . the wd40 repeats seem to be the main hotspot for traf7 mutations found in human cancers . repeatscontaining proteins are generally abundantly present in eukaryote genomes the human genome encodes for 349 predicted wd40 repeatcontaining proteins and are characterized by the presence of repeating units with 4060 variable residues that ended with tryptophan ( w ) and aspartate ( d ) dipeptides ( zhang and zhang , 2015 ) . for instance , the copresence of a ring finger domain and wd40 repeats allows to classify traf7 as a member of a smaller family of e3 ubiquitin ligases named rfwd proteins , which includes cop1 and rfwd3 ( marine , 2012 ) . rfwd proteins target transcriptional regulators such as p53 , etv , and ets , and are known to be involved in tumorigenesis as cop1 is a tumor suppressor and rfwd3 is a susceptibility locus in myeloma and testicular cancer ( marine , 2012 ; chung et al . , 2013 ; mitchell et al . , the basic function of wd40 proteins is to serve as scaffold platforms for protein protein and protein dna interactions . in fact , some functionally relevant molecular interactions are established by traf7 through its carboxyterminal wd40 repeats , including association with mekk3 . indeed , coexpression of mekk3 with traf7 results in activation of the jnk and p38 map kinases pathways ( bouwmeester et al . , 2004 ; xu et al . , 2004 ) , which constitute a pivotal signaling pathway in cytokine and stressinduced apoptosis coherently with its positive role in activation of p38 and jnk pathways , and its negative role in regulating nfb activation and cflip expression level , traf7 promotes apoptotic pathways . however , which of the multiple functions performed by traf7 is compromised as a result of mutations found in different human tumors remains still to be defined . in this regard , the next coming years will definitely witness profound advances in our understanding of the biology of traf7 , and will clarify whether traf7 can be considered as a potential molecular target for the therapeutic approach of the diverse human cancers in which this protein is involved .

the seven members of the tumor necrosis factor receptor ( tnfr)associated factor ( traf ) family of intracellular proteins were originally discovered and characterized as signaling adaptor molecules coupled to the cytoplasmic regions of receptors of the tnfr superfamily . functionally , trafs act both as a scaffold and/or enzymatic proteins to regulate activation of mitogenactivated protein kinases ( mapks ) and transcription factors of nuclear factorb family ( nfb ) . given the wide variety of stimuli intracellularly conveyed by traf proteins , they are physiologically involved in multiple biological processes , including embryonic development , tissue homeostasis , and regulation of innate and adaptive immune responses . in the last few years , it has become increasingly evident the involvement of traf7 , the last member of the traf family to be discovered , in the genesis and progression of several human cancers , placing traf7 in the spotlight as a novel tumor suppressor protein . in this paper , we review and discuss the literature recently produced on this subject . j. cell . physiol . 232 : 12331238 , 2017 . 2016 the authors . journal of cellular physiology published by wiley periodicals , inc .

TRAF7 TRAF proteins in tumors TRAF7 in meningioma TRAF7 in mesothelioma Conclusion

as other traf family members , traf7 contains an aminoterminal ring finger domain ( aa 125160 ) , followed by an adjacent zinc finger domain ( aa 221287 ) . however , instead of the classical traf domain , the carboxy terminus of traf7 contains seven wd40 repeats ( bouwmeester et al . however , while other traf family members interact with various signaling molecules , including protein kinases , through the traf domain , the interaction of traf7 with mekk3 is mediated by the wd40 repeatscontaining region of the protein ( bouwmeester et al . functionally , traf7 and mekk3 cooperate in the signal transduction pathways that lead to activation of jnk and p38 map kinases following tnf stimulation ( bouwmeester et al . in addition to ap1 , the main transcription factors activated by jnk , traf7 also positively regulates the transcriptional activity of chop / gadd153 , a transcription factor activated following exposure to different cellular stresses , such as uv light , genotoxic agents , and protein misfolding in the endoplasmic reticulum ( xu et al . similarly to traf2 , traf3 , and traf6 ( karin and gallagher , 2009 ; hcker et al . , 2011 ; xie , 2013 ) , the ring domain of traf7 possesses an e3 ubiquitin ligase activity ( bouwmeester et al . , 2004 ) , traf7 also promotes ubiquitination of several cellular targets , including the adapter molecule nfb essential modulator ( nemo ) ( zotti et al . , 2011 ) , the p65 subunit of nfb transcription factor ( tsikitis et al . , 2011 ) , the antiapoptotic protein cflip ( scudiero et al . , 2012 ) and the tumor suppressor protein p53 ( wang et al . ubiquitin possesses seven lysines ( lys6 , lys11 , lys27 , lys29 , lys33 , lys48 , and lys63 ) and the consequence of an ubiquitination reaction depends on the lystype of ubiquitin linkage ( kulathu and komander , 2012 ) . in fact , traf7 promotes predominantly lys29linked polyubiquitination of nemo , p65 , and cflip ( zotti et al . , 2012 ) , and this type of ubiquitination is associated with lysosomal degradation of the target proteins ( chastagner et al . , 2013 ) , which is usually followed by the proteosomal degradation of the ubiquitinated protein . along with ubiquitination reactions , traf7 also binds to and stimulates sumoylation of the protooncogene product cmyb ( morita et al . having a primary role in the regulation of the acquired and innate immune responses , it is not surprising that the first evidence for an involvement of traf proteins in tumors came from the molecular analysis of specific b cells malignancies . , 2010 ) and biallelic or monoallelic deletion of traf3 , often associated with epigenetic alteration , homozygous deletions of the traf3 locus have also been observed in cases of chronic lymphocytic leukemias ( nagel et al . , 2009 ) , and classical hodgkin lymphoma ( otto et al . in addition to the above mentioned inactivating mutations of traf2 found in b cell malignancies , a significant fraction of diffuse large bcell lymphoma ( dlbl ) carry somatic activating mutations in traf2 ( compagno et al . thus , in the same cell type , the b lymphocytes , traf2 can function as both oncogene and tumor suppressor gene . in the same study by compagno et al . while traf2 and traf3 have been involved in several lymphoid tumors , traf4 and traf6 are involved in a broader spectrum of cancers . thus , traf4 protein overexpression appears to be a common feature of several human cancers . frequent amplification of the traf6 locus , with concomitant mrna overexpression , has been reported in lung cancers ( starczynowski et al . expression of traf6 was found upregulated also in pancreatic cancer tissues , resulting in deregulated expression of multiple genes involved in cell growth , apoptosis , and migration ( rong et al . meningiomas are tumors originating from the meningeal coverings of the brain and the spinal cord ( mawrin and perry , 2010 ) . they represent one of the most frequent primary brain tumor , accounting for about one third of all cerebral nervous system tumors . a large fraction of all sporadic meningiomas ( 4060% ) display recurrent genetic alterations resulting in inactivating mutations in the nf2 gene locus located on chromosome 22 ( rouleau et al . the nf2 locus encodes for merlin , a pleiotropic protein involved in cytoskeleton organization and cell contact inhibition ( pecinaslaus , 2013 ) , which also regulates transmembrane receptor accumulation and receptor signaling , including erb receptors , plateletderived growth factor receptor , insulinlike growth factor 1 receptor and vascular endothelial growth factor receptors ( lallemand et al . overall , these studies show that mutations in traf7 are observed in nearly onefourth of the meningiomas studied , and in all meningiomas of the secretory subtype . klf4 is a transcription factor that regulates differentiation in a variety of different cell types and its expression is essential to reprogram adult cells in pluripotent adult stem cells ( takahashi et al . , 2007 ) , whereas akt1 is serinethreonine kinase involved in proliferation , and is a well known oncogene ( vivanco and sawyers , 2013 ) . also , whereas the cooccurring mutations in klf4 and akt1 are always the same ( k409q for klf4 and e17k for akt1 ) , the traf7 mutations found in the various samples are distributed along the whole protein , with a marked predilection for the wd40 repeats ( fig . , the akt1e17k mutation found in meningiomas often occurs in various other human cancers , including breast , colon , and ovarian cancers ( carpten et al . traf7 mutations in human cancers . in the domain structure of traf7 , on the top are indicated the mutations found in meningiomas , on the bottom the mutations found in mesothelioma . malignant mesothelioma ( mm ) is an aggressive cancer that results from unregulated proliferation of the mesothelial cells lining the pleural , peritoneal , and pericardial cavities ( yang et al . mms are histologically classified in the three subtypes epithelial , biphasic ( or mixed ) , and sarcomatoid . , the same gene mutated in meningiomas ) , and bap1 ( cheng et al . , 1995 ; the cdkn2a locus encodes for several proteins , the most wellstudied are the p16(ink4a ) and the p14(arf ) proteins , which both function as tumor suppressors ( zhao et al . bap1 ( brca1associated protein 1 ) also is a tumor suppressor gene , and it encodes for a deubiquitinating enzyme that regulates key cellular pathways , including cell cycle , cellular differentiation , transcription , and dna damage response ( wang et al . as in the case of meningiomas , most of the mms mutations observed in traf7 ( 4/5 ) were localized at the carboxyterminal wd40 repeats ( fig . mutations in traf7 often cooccurred with mutations of p53 , but , similarly to what observed in meningiomas , rarely with mutations of nf2 , which was found mutated in 19% of the samples . in addition to mpm , a somatic mutation of traf7 has been reported in a case of papillary peritoneal mesothelioma , where nf2 was not mutated as well ( yu et al . having a primary role in the regulation of the acquired and innate immune responses , it is not surprising that the first evidence for an involvement of traf proteins in tumors came from the molecular analysis of specific b cells malignancies . 2010 ) and biallelic or monoallelic deletion of traf3 , often associated with epigenetic alteration , have been detected in multiple myeloma samples ( annunziata et al . homozygous deletions of the traf3 locus have also been observed in cases of chronic lymphocytic leukemias ( nagel et al . , 2009 ) , and classical hodgkin lymphoma ( otto et al . in addition to the above mentioned inactivating mutations of traf2 found in b cell malignancies , a significant fraction of diffuse large bcell lymphoma ( dlbl ) carry somatic activating mutations in traf2 ( compagno et al . thus , in the same cell type , the b lymphocytes , traf2 can function as both oncogene and tumor suppressor gene . in the same study by compagno et al . while traf2 and traf3 have been involved in several lymphoid tumors , traf4 and traf6 indeed , traf4 cdna was initially isolated from metastatic breast cancer ( rgnier et al . in a subsequent larger study including epithelial tumors , traf4 was found overexpressed in approximately 60% of lung , breast and ovarian , 44% of bladder , and 28% of colon carcinomas moreover , traf4 overexpression was not only most commonly observed in epithelial tumors ( 48% ) , but also in 31% of melanomas , 21% of neurogenic tumors , and 17% of lymphomas ( camilleribrot et al . thus , traf4 protein overexpression appears to be a common feature of several human cancers . , 2011 ) , , 2014 ) , and osteosarcoma samples ( meng et al . expression of traf6 was found upregulated also in pancreatic cancer tissues , resulting in deregulated expression of multiple genes involved in cell growth , apoptosis , and migration ( rong et al . meningiomas are tumors originating from the meningeal coverings of the brain and the spinal cord ( mawrin and perry , 2010 ) . a large fraction of all sporadic meningiomas ( 4060% ) display recurrent genetic alterations resulting in inactivating mutations in the nf2 gene locus located on chromosome 22 ( rouleau et al . the nf2 locus encodes for merlin , a pleiotropic protein involved in cytoskeleton organization and cell contact inhibition ( pecinaslaus , 2013 ) , which also regulates transmembrane receptor accumulation and receptor signaling , including erb receptors , plateletderived growth factor receptor , insulinlike growth factor 1 receptor and vascular endothelial growth factor receptors ( lallemand et al . besides these roles in cytoskeleton organization and receptor signaling , merlin also regulates ubiquitination events , by inhibiting the e3 ubiquitin ligase crl4(dcaf1 ) in the nucleus ( li et al . overall , these studies show that mutations in traf7 are observed in nearly onefourth of the meningiomas studied , and in all meningiomas of the secretory subtype . klf4 is a transcription factor that regulates differentiation in a variety of different cell types and its expression is essential to reprogram adult cells in pluripotent adult stem cells ( takahashi et al . , 2007 ) , whereas akt1 is serinethreonine kinase involved in proliferation , and is a well known oncogene ( vivanco and sawyers , 2013 ) . also , whereas the cooccurring mutations in klf4 and akt1 are always the same ( k409q for klf4 and e17k for akt1 ) , the traf7 mutations found in the various samples are distributed along the whole protein , with a marked predilection for the wd40 repeats ( fig . , the akt1e17k mutation found in meningiomas often occurs in various other human cancers , including breast , colon , and ovarian cancers ( carpten et al . traf7 mutations in human cancers . in the domain structure of traf7 , on the top are indicated the mutations found in meningiomas , on the bottom the mutations found in mesothelioma . in fact , tumors with traf7/akt1/klf4 mutations predominantly localize medially in the skull base , whereas meningiomas with nf2 mutations predominantly localize in the hemispheres or laterally in the skull base ( clark et al . malignant mesothelioma ( mm ) is an aggressive cancer that results from unregulated proliferation of the mesothelial cells lining the pleural , peritoneal , and pericardial cavities ( yang et al . mms are histologically classified in the three subtypes epithelial , biphasic ( or mixed ) , and sarcomatoid . , the same gene mutated in meningiomas ) , and bap1 ( cheng et al . , 2011 ; the cdkn2a locus encodes for several proteins , the most wellstudied are the p16(ink4a ) and the p14(arf ) proteins , which both function as tumor suppressors ( zhao et al . bap1 ( brca1associated protein 1 ) also is a tumor suppressor gene , and it encodes for a deubiquitinating enzyme that regulates key cellular pathways , including cell cycle , cellular differentiation , transcription , and dna damage response ( wang et al . as in the case of meningiomas , most of the mms mutations observed in traf7 ( 4/5 ) were localized at the carboxyterminal wd40 repeats ( fig . mutations in traf7 often cooccurred with mutations of p53 , but , similarly to what observed in meningiomas , rarely with mutations of nf2 , which was found mutated in 19% of the samples . recent data generated through deep sequencing of primary tumors have clearly indicated the involvement of traf7 in the genesis of various human cancers . specifically , traf7 is mutated in about 25% of meningiomas and significantly , although at a lower rate , in human mesotheliomas . an inactivating mutation of traf7 has also been reported in a case of merkel cell carcinoma , a rare and aggressive cutaneous neuroendocrine carcinoma ( goh et al . it is certainly interesting the fact that mutations in traf7 in human meningioma and mesothelioma cancers are alternative to mutations in nf2 , a gene which , strikingly , also shows a high rate of mutation in these two types of tumors . however , merlin , the product of the nf2 gene , is a multifaceted protein , functioning as a scaffold protein that connects cytoskeleton dynamics to signaling transduction pathways that control cell death and proliferation , including those triggered by tyrosine kinase receptors , small gtpases , mammalian target of rapamycin ( mtor ) , pi3k / akt , and hippo pathways ( petrilli and fernndezvalle , 2016 ) . thus , in this variety of different functions it is not obvious to distinguish which of them is / are shared by traf7 and nf2 in promoting cancer development . most of the traf7 mutations identified in human tumors localize at the wd40 repeats region and at the neighboring coiled coil domain , that is , the regions through which traf7 establishes interactions with several molecular partners ( zotti et al . , for example , the coiled coil domain of traf7 is involved in the interaction with nemo ( zotti et al . , 2011 ) , an adapter molecule essential for activation of the canonical pathway of nfb , a transcription factor that by promoting expression of antiapoptotic and proliferative genes is known to be generally hyperactive in the vast majority of human cancers ( karin , 2006 ; wu et al . generally , traf7 exerts a negative control on nfb activity , by promoting ubiquitination and lysosomal degradation of nemo and that of the p65 subunit of nfb ( zotti et al . , 2012 ) , a very well known inhibitor of apoptosis by interfering with caspases activation in tumor cells ( safa and pollok , 2011 ; shirley and micheau , 2013 ) whose stability is known to be controlled by traf proteins ( guiet et al . consistent with its role as a crucial negative regulator of the apoptotic pathway , cflip is found overexpressed in many different types of cancer , including mpm ( rippo et al . interestingly , the tumor suppressor protein p53 is another target of the ubiquitinating activity of traf7 , and traf7mediated ubiquitination of p53 plays a critical role in breast cancer development and progression ( wang et al . the wd40 repeats seem to be the main hotspot for traf7 mutations found in human cancers . repeatscontaining proteins are generally abundantly present in eukaryote genomes the human genome encodes for 349 predicted wd40 repeatcontaining proteins and are characterized by the presence of repeating units with 4060 variable residues that ended with tryptophan ( w ) and aspartate ( d ) dipeptides ( zhang and zhang , 2015 ) . for instance , the copresence of a ring finger domain and wd40 repeats allows to classify traf7 as a member of a smaller family of e3 ubiquitin ligases named rfwd proteins , which includes cop1 and rfwd3 ( marine , 2012 ) . rfwd proteins target transcriptional regulators such as p53 , etv , and ets , and are known to be involved in tumorigenesis as cop1 is a tumor suppressor and rfwd3 is a susceptibility locus in myeloma and testicular cancer ( marine , 2012 ; chung et al . , the basic function of wd40 proteins is to serve as scaffold platforms for protein protein and protein dna interactions . in fact , some functionally relevant molecular interactions are established by traf7 through its carboxyterminal wd40 repeats , including association with mekk3 . indeed , coexpression of mekk3 with traf7 results in activation of the jnk and p38 map kinases pathways ( bouwmeester et al . , 2004 ) , which constitute a pivotal signaling pathway in cytokine and stressinduced apoptosis coherently with its positive role in activation of p38 and jnk pathways , and its negative role in regulating nfb activation and cflip expression level , traf7 promotes apoptotic pathways . however , which of the multiple functions performed by traf7 is compromised as a result of mutations found in different human tumors remains still to be defined . in this regard , the next coming years will definitely witness profound advances in our understanding of the biology of traf7 , and will clarify whether traf7 can be considered as a potential molecular target for the therapeutic approach of the diverse human cancers in which this protein is involved .

the ability of viruses to self - assemble into nanoscale particles of discrete size and definite geometry gives them potential utility in a variety of nano- and biotechnology applications . efforts to adapt icosahedral virus particles for use as templates for materials synthesis , as platforms for the multivalent presentation of ligands , and even as possible molecular electronic components have been described recently [ 1 - 7 ] . work reported to date has made use of cowpea chlorotic mottle virus [ 1 - 4 ] and cowpea mosaic virus [ 5 - 7 ] . experiments that explore the utility of the rna bacteriophage ms2 for similar purposes are presented here . rna bacteriophages represent attractive systems for engineering new properties into viruses and virus - like particles . each rna phage particle is comprised of 180 copies of a single coat protein polypeptide about 130 amino acids in length , one copy of the maturase protein , and one molecule of viral genome rna . the coat protein itself possesses all the information needed for assembly into an icosahedron with a diameter of about 25 nm . this means that virus capsids can be produced by expression of the coat gene from a plasmid in e. coli without the need for other viral components . the coat protein dimer , the structural unit from which capsids are assembled , possesses a high - affinity binding site for a specific rna hairpin . since this hairpin can function as a packaging signal , it is straight - forward to engineer the encapsidation of an arbitrarily chosen rna by fusing it to this so - called pac site and expressing it in an e. coli strain that also produces coat protein . it is , of course , a simple matter to introduce any desired amino acid substitution by site - directed mutagenesis of the coat protein cdna clone , but systems also exist that facilitate random mutagenesis and selection of coat mutants having altered rna binding and particle assembly properties . a simple assay for correct particle assembly makes it easy to screen out those mutants that acquire undesired defects in protein folding or assembly . moreover , because coat proteins produced from a plasmid in e. coli are fully competent for particle assembly , changes in coat protein structure that are incompatible with the normal virus life cycle can be easily introduced and propagated . moreover , cdna clones of viral rna are infectious , making it easy to produce viable recombinant viruses that incorporate any mutation that does not interfere with virus viability . both virus and virus - like particles are readily produced in large quantities and high purity . high resolution x - ray structures are available for a number of rna phages , including ms2 [ 12 - 18 ] , so that desirable sites for modification can be identified easily . here i describe the production of a bacteriophage ms2 coat protein mutant that displays a reactive thiol on the surface of the virus - like particle . it can bind a variety of metals and reacts with a large collection of organic reagents , thus making cysteines obvious targets for protein modification reactions . wild - type ms2 coat protein contains two cysteines , but they are sequestered in the interior of the protein where they should be relatively unreactive . the introduction of an accessible cysteine on the surface of the ms2 capsid therefore should create the opportunity for multivalent display of a large number of different potential ligands on its surface . based on their accessibility on the surface of the viral capsid , five different amino acids of ms2 coat protein were selected initially for cysteine substitution ( figure 1 ) . three of the five ( glycine13 , glycine14 , and threonine15 ) are located in the so - called ab - loop , a short -turn that connects the a and b -strands of coat protein . the other two ( aspartic acid114 and glycine115 ) reside in a loop connecting the two coat protein -helices . each of these five amino acids was converted to cysteine by site - directed mutagenesis and the mutant genes were cloned in the plasmid called pet3d and introduced into e. coli strain bl21(de3/plyss for over - expression . each mutant was screened by sds gel electrophoresis for the ability to produce more or less normal amounts of coat protein in the soluble fraction of cell lysates , and by agarose gel electrophoresis under native conditions for correct assembly of a virus - like particle . four of the five mutants , g13c , g14c , d114c and g115c , failed these tests ( figure 2 ) . in these cases no virus - like particles were detected and the coat proteins were found predominantly in the insoluble fraction of cell lysates . a view of the ms2 coat protein dimer with its two polypeptide chains shown as blue and red ribbons . the positions of amino acids altered in this study by site - directed mutagenesis are shown as yellow ( glycine13 ) , green ( glycine14 ) , magenta ( threonine15 ) , cyan ( glycine113 ) and white ( aspartic acid114 ) . for details of the structure of ms2 coat protein a. agarose gel electrophoresis of the soluble fractions of lysates of e. coli cells producing the wild - type ( wt ) and each of the mutant coat proteins ( lanes 16 ) . since the particles contain host cell - derived rna , they can be stained with ethidium bromide and visualized under uv illumination . lane 1 wild - type , lane 2 g13c , lane 3 g14c ; lane 4 t15c , lane 5 g113c , lane 6 d114c . b. sds gel electrophoresis of protein extracted from the same cells . here are shown the contents of both the soluble ( s ) and pellet ( p ) fractions of crude cell lysates . samples are labeled as in a , except for the addition of lane 0 , which is a control that produces no coat protein . in past work it has frequently been possible to suppress the effects of mutations on ms2 coat protein folding / stability by incorporating them into so - called single - chain dimers . because of the proximity of the n - terminus of one subunit of the coat protein dimer to the c - terminus of the other subunit , it is possible to genetically fuse them into a single polypeptide chain . covalently linking the two monomers in this manner makes the dimer relatively resistant to the destabilizing effects of many amino acid substitutions and even of peptide insertions [ 20 - 23 ] . in an effort to revert their effects on coat protein structure , the g13c , g14c , d114c and g115c mutations were incorporated into single - chain dimer constructs . however , in none of these cases was the ability to produce active coat protein restored ( results not shown ) . in contrast to the destabilizing substitutions , the t15c mutant ( where threonine15 is replaced by cysteine ) produced significant quantities of soluble coat protein that assembled into particles with the same electrophoretic mobility as wild - type virus . assembly into a virus - sized particle was verified by the behavior of the t15c mutant upon chromatography in sepharose cl-4b . as seen in figure 3 , wild - type ms2 and the t15c mutant particles both eluted in a discrete , symmetric peak at the same position . figure 4 shows the structure of a portion of the viral capsid with the location of residue 15 indicated in red . it illustrates how the existence of the t15c mutant should make it possible to attach chemically a variety of substances in a defined geometric array on outside of the particle . introduction of cysteine at other sites would allow variations in this pattern , each of them adhering to the constraints of icosahedral geometry , but allowing different relative spacings of the functional group . elution profiles of wild - type and t15c virus - like particles from a sepharose cl-4b column . the presence of coat protein in individual fractions was determined by sds polyacrylamide gel electrophoresis followed by staining with coomassie blue and densitometry . a protein roughly the size of the coat protein monomer ( lysozyme , mw about 14,000 ) elutes at position 33 . two views of a portion of the surface of the viral particle showing the exposure of threonine15 and the pattern of its display . note that the structure shown here ( downloadable as 1gav.pdb from , the protein data bank website ) is actually that of ga , a close ms2 relative with a highly similar structure t15c virus - like particles were purified from e. coli , using methods that included gel filtration chromatography on sepharose cl-4b and that were described previously for the wild - type virus - like particle . note that although the reducing agent dithiothreitol ( dtt ) was present in the cell lysis solution , it was absent from the chromatography buffer . therefore , when column - purified capsids were concentrated by ultracentrifugation , it was under conditions that allow the formation of disulfide bonds . upon attempting to redissolve the pelleted t15c particles it was immediately apparent that their behavior was different from wild - type . whereas wild - type particles dissolve readily in water , the mutant capsids were insoluble . agarose gel electrophoresis also indicated the formation of large aggregates , because mutant particles failed to enter the gel ( figure 5 ) . treatment with 10 mm dtt led to the immediate dissolution ( within a few minutes ) of the aggregate and to the restoration of wild - type electrophoretic behavior . thus , concentration of the capsids under non - reducing conditions allowed efficient inter - particle disulfide cross - linking . at intermediate dtt concentrations , gel electrophoresis produced a ladder of species representing intermediately aggregated states , i.e. capsid dimers , trimers , tetramers and so forth . when the aggregates were subjected to sds gel electrophoresis in the absence of reducing agent ( with nem included to prevent thiol - disulfide interchange during sample preparation ) about 3% of the coat protein was present in the form of a disulfide linked dimer , consistent with the idea that each capsid in the aggregate is cross - linked on average to about 5 others ( data not shown ) . agarose gel electrophoresis of ms2-t15c virus - like particles treated with dtt at the indicated concentrations . material on the right is fully reduced and possesses the electrophoretic behavior characteristic of ms2 itself ( see figures 2 and 6 ) . the accessibility of the new cysteine is further illustrated by its reaction with thiol - specific chemical reagents . for simplicity only the results obtained when capsids are reacted with fluorescein-5-maleimide are shown here , but similar results were obtained from reaction with 5,5'-dithio - bis(2-nitrobenzoic acid ) ( dtnb ) to form the 5-thio-2-nitrobenzoyl derivative , by reaction with na2so3 in the presence of dtnb to produce the thiosulfonate derivative , and when reacted with iodoacetic acid to form the carboxymethyl derivative . wild - type and t15c capsids were reacted with fluoroscein-5-maleimide under conditions described in materials and methods and the products were subjected to electrophoresis in agarose gels and photographed under uv illumination both before and after staining with ethidium bromide , which gives an orange fluorescence to all the capsids because of the rna each contains . in addition , its electrophoretic mobility increases , consistent with the addition of negative charges to the capsid ( fluorescein has a carboxyl group ) . the modification is specific for the t15c mutant wild - type ms2 remains unmodified and is abolished when the reagent is inactivated by prior addition of dtt to the reaction . when subjected to electrophoresis in sds - polyacrylamide gels a single fluorescent product staining of the gel with coomassie blue shows that attachment of fluorescein alters the mobility of coat protein , allowing an estimation of the extent of its modification . clearly , the great majority ( about 8090% ) of the t15c coat protein undergoes reaction under these conditions . longer reaction times ( up to 1.5 hours ) at a higher temperature ( 37c ) did not alter this pattern . failure to modify the wild - type coat protein indicates that the other cysteines ( residues 46 and 101 ) are not detectably accessible for reaction under these conditions . a. agarose gel electrophoresis of capsids unstained ( on the left ) and stained with ethidium bromide and photographed under uv illumination . lane 1 is unreacted ms2 , lane 2 is ms2 modified by reaction with fluorescein-5-maleimide , lane 3 is unreacted t15c , lane 4 is t15c reacted with fluorescein-5-maleimide . b. sds gel electrophoresis of the same samples shown in a. on the left is the gel stained with coomassie brilliant blue and at right it is illuminated in the uv . based on their accessibility on the surface of the viral capsid , five different amino acids of ms2 coat protein were selected initially for cysteine substitution ( figure 1 ) . three of the five ( glycine13 , glycine14 , and threonine15 ) are located in the so - called ab - loop , a short -turn that connects the a and b -strands of coat protein . the other two ( aspartic acid114 and glycine115 ) reside in a loop connecting the two coat protein -helices . each of these five amino acids was converted to cysteine by site - directed mutagenesis and the mutant genes were cloned in the plasmid called pet3d and introduced into e. coli strain bl21(de3/plyss for over - expression . each mutant was screened by sds gel electrophoresis for the ability to produce more or less normal amounts of coat protein in the soluble fraction of cell lysates , and by agarose gel electrophoresis under native conditions for correct assembly of a virus - like particle . four of the five mutants , g13c , g14c , d114c and g115c , failed these tests ( figure 2 ) . in these cases no virus - like particles were detected and the coat proteins were found predominantly in the insoluble fraction of cell lysates . a view of the ms2 coat protein dimer with its two polypeptide chains shown as blue and red ribbons . the positions of amino acids altered in this study by site - directed mutagenesis are shown as yellow ( glycine13 ) , green ( glycine14 ) , magenta ( threonine15 ) , cyan ( glycine113 ) and white ( aspartic acid114 ) . for details of the structure of ms2 coat protein a. agarose gel electrophoresis of the soluble fractions of lysates of e. coli cells producing the wild - type ( wt ) and each of the mutant coat proteins ( lanes 16 ) . since the particles contain host cell - derived rna , they can be stained with ethidium bromide and visualized under uv illumination . lane 1 wild - type , lane 2 g13c , lane 3 g14c ; lane 4 t15c , lane 5 g113c , lane 6 d114c . b. sds gel electrophoresis of protein extracted from the same cells . here are shown the contents of both the soluble ( s ) and pellet ( p ) fractions of crude cell lysates . samples are labeled as in a , except for the addition of lane 0 , which is a control that produces no coat protein . in past work it has frequently been possible to suppress the effects of mutations on ms2 coat protein folding / stability by incorporating them into so - called single - chain dimers . because of the proximity of the n - terminus of one subunit of the coat protein dimer to the c - terminus of the other subunit , it is possible to genetically fuse them into a single polypeptide chain . covalently linking the two monomers in this manner makes the dimer relatively resistant to the destabilizing effects of many amino acid substitutions and even of peptide insertions [ 20 - 23 ] . in an effort to revert their effects on coat protein structure , the g13c , g14c , d114c and g115c mutations were incorporated into single - chain dimer constructs . however , in none of these cases was the ability to produce active coat protein restored ( results not shown ) . in contrast to the destabilizing substitutions , the t15c mutant ( where threonine15 is replaced by cysteine ) produced significant quantities of soluble coat protein that assembled into particles with the same electrophoretic mobility as wild - type virus . assembly into a virus - sized particle was verified by the behavior of the t15c mutant upon chromatography in sepharose cl-4b . as seen in figure 3 , wild - type ms2 and the t15c mutant particles both eluted in a discrete , symmetric peak at the same position . figure 4 shows the structure of a portion of the viral capsid with the location of residue 15 indicated in red . it illustrates how the existence of the t15c mutant should make it possible to attach chemically a variety of substances in a defined geometric array on outside of the particle . introduction of cysteine at other sites would allow variations in this pattern , each of them adhering to the constraints of icosahedral geometry , but allowing different relative spacings of the functional group . elution profiles of wild - type and t15c virus - like particles from a sepharose cl-4b column . the presence of coat protein in individual fractions was determined by sds polyacrylamide gel electrophoresis followed by staining with coomassie blue and densitometry . a protein roughly the size of the coat protein monomer ( lysozyme , mw about 14,000 ) elutes at position 33 . two views of a portion of the surface of the viral particle showing the exposure of threonine15 and the pattern of its display . note that the structure shown here ( downloadable as 1gav.pdb from , the protein data bank website ) is actually that of ga , a close ms2 relative with a highly similar structure t15c virus - like particles were purified from e. coli , using methods that included gel filtration chromatography on sepharose cl-4b and that were described previously for the wild - type virus - like particle . note that although the reducing agent dithiothreitol ( dtt ) was present in the cell lysis solution , it was absent from the chromatography buffer . therefore , when column - purified capsids were concentrated by ultracentrifugation , it was under conditions that allow the formation of disulfide bonds . upon attempting to redissolve the pelleted t15c particles it was immediately apparent that their behavior was different from wild - type . whereas wild - type particles dissolve readily in water , the mutant capsids were insoluble . agarose gel electrophoresis also indicated the formation of large aggregates , because mutant particles failed to enter the gel ( figure 5 ) . treatment with 10 mm dtt led to the immediate dissolution ( within a few minutes ) of the aggregate and to the restoration of wild - type electrophoretic behavior . thus , concentration of the capsids under non - reducing conditions allowed efficient inter - particle disulfide cross - linking . at intermediate dtt concentrations , gel electrophoresis produced a ladder of species representing intermediately aggregated states , i.e. capsid dimers , trimers , tetramers and so forth . when the aggregates were subjected to sds gel electrophoresis in the absence of reducing agent ( with nem included to prevent thiol - disulfide interchange during sample preparation ) about 3% of the coat protein was present in the form of a disulfide linked dimer , consistent with the idea that each capsid in the aggregate is cross - linked on average to about 5 others ( data not shown ) . agarose gel electrophoresis of ms2-t15c virus - like particles treated with dtt at the indicated concentrations . material on the right is fully reduced and possesses the electrophoretic behavior characteristic of ms2 itself ( see figures 2 and 6 ) . the accessibility of the new cysteine is further illustrated by its reaction with thiol - specific chemical reagents . for simplicity only the results obtained when capsids are reacted with fluorescein-5-maleimide are shown here , but similar results were obtained from reaction with 5,5'-dithio - bis(2-nitrobenzoic acid ) ( dtnb ) to form the 5-thio-2-nitrobenzoyl derivative , by reaction with na2so3 in the presence of dtnb to produce the thiosulfonate derivative , and when reacted with iodoacetic acid to form the carboxymethyl derivative . wild - type and t15c capsids were reacted with fluoroscein-5-maleimide under conditions described in materials and methods and the products were subjected to electrophoresis in agarose gels and photographed under uv illumination both before and after staining with ethidium bromide , which gives an orange fluorescence to all the capsids because of the rna each contains . in addition , its electrophoretic mobility increases , consistent with the addition of negative charges to the capsid ( fluorescein has a carboxyl group ) . the modification is specific for the t15c mutant wild - type ms2 remains unmodified and is abolished when the reagent is inactivated by prior addition of dtt to the reaction . when subjected to electrophoresis in sds - polyacrylamide gels a single fluorescent product staining of the gel with coomassie blue shows that attachment of fluorescein alters the mobility of coat protein , allowing an estimation of the extent of its modification . clearly , the great majority ( about 8090% ) of the t15c coat protein undergoes reaction under these conditions . longer reaction times ( up to 1.5 hours ) at a higher temperature ( 37c ) did not alter this pattern . failure to modify the wild - type coat protein indicates that the other cysteines ( residues 46 and 101 ) are not detectably accessible for reaction under these conditions . a. agarose gel electrophoresis of capsids unstained ( on the left ) and stained with ethidium bromide and photographed under uv illumination . lane 1 is unreacted ms2 , lane 2 is ms2 modified by reaction with fluorescein-5-maleimide , lane 3 is unreacted t15c , lane 4 is t15c reacted with fluorescein-5-maleimide . b. sds gel electrophoresis of the same samples shown in a. on the left is the gel stained with coomassie brilliant blue and at right it is illuminated in the uv . considering their locations in the coat protein structure it is not surprising that some substitutions of ab loop residues disrupted folding . the loop makes a tight turn and the glycines present at positions 13 and 14 are probably needed to prevent the crowding that results when amino acids with bulkier side chains are introduced here . moreover , the defects caused by the g13c and g14c substitutions must be fairly severe , since they are not reverted by their incorporation into single - chain coat protein dimers . genetic fusion of the subunits of the dimer was shown previously to revert the destabilizing effects of variety of mutations , including a wide range of amino acid substitutions at different locations on the -sheet , temperature - sensitive mutations occurring at numerous sites through - out the structure ( unpublished observations ) , and even insertions into the ab - loop sequence itself . cysteine is a slightly smaller amino acid than the threonine it replaces and so would not be expected to introduce stereochemical difficulties of the sort that likely explain the g13c and g14c defects . it is less obvious why the substitutions at residues aspartic acid114 and glycine115 lead to folding - defects , but these residues also are involved in a turn of the polypeptide , this one connecting the two coat protein alpha - helices . the severity of the defects conferred by the cysteines introduced here is also indicated by the failure to revert them in single - chain dimers . as these results illustrate , amino acid substitutions can disrupt protein folding and stability with an annoyingly high frequency . it should be noted , though , that at least two different strategies are available for efforts to render the substitutions tolerable . the first is to create single - chain dimers of the mutant proteins [ 20 - 23 ] . although this was ineffective in the cases of the four defective mutants described here , it has in the past proven an efficient and simple means of reverting coat protein folding defects and will likely be useful for many of the other defects one might encounter . moreover , since single - chain dimers allow independent control of the amino acid sequences in the two halves of the " dimer " , it provides a means to alter by one - half the number of thiols on the virus surface , giving an added level of control over the density of modifiable sites . a second strategy for reversion of folding / stability defects is to isolate mutations at second sites that suppress those defects . a gel diffusion method that allows one to distinguish bacterial colonies that produce soluble , properly assembled coat protein from those that do the sites modified in this study were chosen because they are highly exposed on the virus surface , but a number of other sites in coat protein are also located in potentially suitable positions , and some of them are likely to be more tolerant of substitution than those tested so far . the capacity to introduce cysteines at alternative positions would allow one to alter the relative geometric arrangement of reactive sites , an additional parameter that should influence the properties of specific modified virus - like particles . the procedure outlined here serves as a guide to the identification of residues whose substitution is tolerated . wild - type ms2 coat protein has two cysteines , one at position 46 and the other at 101 . under the conditions used in this study , no evidence that these cysteines were modified by fluoroscein-5-maleimide this selectivity is a little surprising in view of the previous demonstrations that cysteine46 is somewhat susceptible to reaction with sulfhydryl - specific reagents even though , like cysteine101 , it is relatively buried within the coat protein tertiary structure . alternatively , because it is bulkier than the reagents used in the previous studies ( e.g. n - ethylmaleimide ) , the fluoroscein-5-maleimide reagent might not as easily gain access to cysteine46 . the ability to chemically modify specific sites on virus particle surfaces is a potentially powerful approach to the production of new materials for biotechnology , nanotechnology and molecular electronics . it makes possible the use of the virus - like particle as a scaffold for the attachment of a large variety of substances including metals , organics , peptides , and nucleic acids in a regular geometric array . thus , one can think of these virus - like particles as self - assembling and highly regular nanospheres , potentially susceptible to a wide range of chemical modifications at specific surface locations . they may be suitable for use in applications currently employing small spheres constructed by other , less controlled means . the ability to specifically encapsidate and protect arbitrarily chosen rnas within such particles suggests additional applications . mutations were introduced into the ms2 coat sequence using mismatched oligonucleotide primers ( from integrated dna technologies ) in a pcr - based overlap extension method . g13c ( ggc to ugc ) , g14c ( gga to ugu ) , t15c agu to ugu ) , d114c ( gau to ugu ) and g115c ( gga to ugu ) . the resulting pcr products were cloned as xbai - bamhi fragments in the t7 expression vector called pet3d thus creating a series of derivatives of the plasmid called petct . the nucleotide sequences of each of the mutant coat genes were determined at the unm center for genetics in medicine . the presence of virus - like particles in crude cell lysates was determined by electrophoresis in 1% agarose gels in 50 mm potassium phosphate , ph 7.0 as described previously . these methods include chromatography in sepharose cl-4b followed by pelleting of the virus from peak fractions by centrifugation at 25,000 rpm in the sw28 rotor overnight . electrophoresis of purified virus - like particles was conducted in 1% agarose and 40 mm tris - acetate , 2 mm edta , ph 8.0 . production of crude cell lysates , their separation into soluble and insoluble fractions , and their analysis by sds gel electrophoresis have been also detailed in previous reports . fluorescein labeling was conducted by reaction for 30 min . at room temperature in 20ul of 50 mm potassium phosphate ph7.0 , 1 mm edta , 1 mm fluorescein-5-maleimide ( from helix , inc . ) . the products were subjected to electrophoresis in agarose gels under native conditions in 40 mm tris - acetate , 2 mm edta , ph 8.0 , and in sds - polyacrylamide gels .

the ability to chemically modify the surfaces of viruses and virus - like particles makes it possible to confer properties that make them potentially useful in biotechnology , nanotechnology and molecular electronics applications . rna phages ( e.g. ms2 ) have characteristics that make them suitable scaffolds to which a variety of substances could be chemically attached in definite geometric patterns . to provide for specific chemical modification of ms2 's outer surface , cysteine residues were substituted for several amino acids present on the surface of the wild - type virus particle . some substitutions resulted in coat protein folding or stability defects , but one allowed the production of an otherwise normal virus - like particle with an accessible sulfhydryl on its surface .

Background Results Introduction of surface cysteines and their effects on coat protein structure Accessibility and reactivity of the new cysteine Discussion Conclusions Methods Acknowledgements

the ability of viruses to self - assemble into nanoscale particles of discrete size and definite geometry gives them potential utility in a variety of nano- and biotechnology applications . rna bacteriophages represent attractive systems for engineering new properties into viruses and virus - like particles . each rna phage particle is comprised of 180 copies of a single coat protein polypeptide about 130 amino acids in length , one copy of the maturase protein , and one molecule of viral genome rna . this means that virus capsids can be produced by expression of the coat gene from a plasmid in e. coli without the need for other viral components . since this hairpin can function as a packaging signal , it is straight - forward to engineer the encapsidation of an arbitrarily chosen rna by fusing it to this so - called pac site and expressing it in an e. coli strain that also produces coat protein . it is , of course , a simple matter to introduce any desired amino acid substitution by site - directed mutagenesis of the coat protein cdna clone , but systems also exist that facilitate random mutagenesis and selection of coat mutants having altered rna binding and particle assembly properties . a simple assay for correct particle assembly makes it easy to screen out those mutants that acquire undesired defects in protein folding or assembly . moreover , because coat proteins produced from a plasmid in e. coli are fully competent for particle assembly , changes in coat protein structure that are incompatible with the normal virus life cycle can be easily introduced and propagated . both virus and virus - like particles are readily produced in large quantities and high purity . here i describe the production of a bacteriophage ms2 coat protein mutant that displays a reactive thiol on the surface of the virus - like particle . it can bind a variety of metals and reacts with a large collection of organic reagents , thus making cysteines obvious targets for protein modification reactions . wild - type ms2 coat protein contains two cysteines , but they are sequestered in the interior of the protein where they should be relatively unreactive . the introduction of an accessible cysteine on the surface of the ms2 capsid therefore should create the opportunity for multivalent display of a large number of different potential ligands on its surface . based on their accessibility on the surface of the viral capsid , five different amino acids of ms2 coat protein were selected initially for cysteine substitution ( figure 1 ) . three of the five ( glycine13 , glycine14 , and threonine15 ) are located in the so - called ab - loop , a short -turn that connects the a and b -strands of coat protein . each mutant was screened by sds gel electrophoresis for the ability to produce more or less normal amounts of coat protein in the soluble fraction of cell lysates , and by agarose gel electrophoresis under native conditions for correct assembly of a virus - like particle . four of the five mutants , g13c , g14c , d114c and g115c , failed these tests ( figure 2 ) . in these cases no virus - like particles were detected and the coat proteins were found predominantly in the insoluble fraction of cell lysates . a view of the ms2 coat protein dimer with its two polypeptide chains shown as blue and red ribbons . for details of the structure of ms2 coat protein a. agarose gel electrophoresis of the soluble fractions of lysates of e. coli cells producing the wild - type ( wt ) and each of the mutant coat proteins ( lanes 16 ) . lane 1 wild - type , lane 2 g13c , lane 3 g14c ; lane 4 t15c , lane 5 g113c , lane 6 d114c . in past work it has frequently been possible to suppress the effects of mutations on ms2 coat protein folding / stability by incorporating them into so - called single - chain dimers . because of the proximity of the n - terminus of one subunit of the coat protein dimer to the c - terminus of the other subunit , it is possible to genetically fuse them into a single polypeptide chain . in an effort to revert their effects on coat protein structure , the g13c , g14c , d114c and g115c mutations were incorporated into single - chain dimer constructs . however , in none of these cases was the ability to produce active coat protein restored ( results not shown ) . in contrast to the destabilizing substitutions , the t15c mutant ( where threonine15 is replaced by cysteine ) produced significant quantities of soluble coat protein that assembled into particles with the same electrophoretic mobility as wild - type virus . assembly into a virus - sized particle was verified by the behavior of the t15c mutant upon chromatography in sepharose cl-4b . as seen in figure 3 , wild - type ms2 and the t15c mutant particles both eluted in a discrete , symmetric peak at the same position . it illustrates how the existence of the t15c mutant should make it possible to attach chemically a variety of substances in a defined geometric array on outside of the particle . introduction of cysteine at other sites would allow variations in this pattern , each of them adhering to the constraints of icosahedral geometry , but allowing different relative spacings of the functional group . elution profiles of wild - type and t15c virus - like particles from a sepharose cl-4b column . a protein roughly the size of the coat protein monomer ( lysozyme , mw about 14,000 ) elutes at position 33 . two views of a portion of the surface of the viral particle showing the exposure of threonine15 and the pattern of its display . note that the structure shown here ( downloadable as 1gav.pdb from , the protein data bank website ) is actually that of ga , a close ms2 relative with a highly similar structure t15c virus - like particles were purified from e. coli , using methods that included gel filtration chromatography on sepharose cl-4b and that were described previously for the wild - type virus - like particle . upon attempting to redissolve the pelleted t15c particles it was immediately apparent that their behavior was different from wild - type . whereas wild - type particles dissolve readily in water , the mutant capsids were insoluble . treatment with 10 mm dtt led to the immediate dissolution ( within a few minutes ) of the aggregate and to the restoration of wild - type electrophoretic behavior . when the aggregates were subjected to sds gel electrophoresis in the absence of reducing agent ( with nem included to prevent thiol - disulfide interchange during sample preparation ) about 3% of the coat protein was present in the form of a disulfide linked dimer , consistent with the idea that each capsid in the aggregate is cross - linked on average to about 5 others ( data not shown ) . agarose gel electrophoresis of ms2-t15c virus - like particles treated with dtt at the indicated concentrations . material on the right is fully reduced and possesses the electrophoretic behavior characteristic of ms2 itself ( see figures 2 and 6 ) . the accessibility of the new cysteine is further illustrated by its reaction with thiol - specific chemical reagents . for simplicity only the results obtained when capsids are reacted with fluorescein-5-maleimide are shown here , but similar results were obtained from reaction with 5,5'-dithio - bis(2-nitrobenzoic acid ) ( dtnb ) to form the 5-thio-2-nitrobenzoyl derivative , by reaction with na2so3 in the presence of dtnb to produce the thiosulfonate derivative , and when reacted with iodoacetic acid to form the carboxymethyl derivative . wild - type and t15c capsids were reacted with fluoroscein-5-maleimide under conditions described in materials and methods and the products were subjected to electrophoresis in agarose gels and photographed under uv illumination both before and after staining with ethidium bromide , which gives an orange fluorescence to all the capsids because of the rna each contains . the modification is specific for the t15c mutant wild - type ms2 remains unmodified and is abolished when the reagent is inactivated by prior addition of dtt to the reaction . when subjected to electrophoresis in sds - polyacrylamide gels a single fluorescent product staining of the gel with coomassie blue shows that attachment of fluorescein alters the mobility of coat protein , allowing an estimation of the extent of its modification . clearly , the great majority ( about 8090% ) of the t15c coat protein undergoes reaction under these conditions . failure to modify the wild - type coat protein indicates that the other cysteines ( residues 46 and 101 ) are not detectably accessible for reaction under these conditions . b. sds gel electrophoresis of the same samples shown in a. on the left is the gel stained with coomassie brilliant blue and at right it is illuminated in the uv . based on their accessibility on the surface of the viral capsid , five different amino acids of ms2 coat protein were selected initially for cysteine substitution ( figure 1 ) . three of the five ( glycine13 , glycine14 , and threonine15 ) are located in the so - called ab - loop , a short -turn that connects the a and b -strands of coat protein . each of these five amino acids was converted to cysteine by site - directed mutagenesis and the mutant genes were cloned in the plasmid called pet3d and introduced into e. coli strain bl21(de3/plyss for over - expression . each mutant was screened by sds gel electrophoresis for the ability to produce more or less normal amounts of coat protein in the soluble fraction of cell lysates , and by agarose gel electrophoresis under native conditions for correct assembly of a virus - like particle . in these cases no virus - like particles were detected and the coat proteins were found predominantly in the insoluble fraction of cell lysates . a view of the ms2 coat protein dimer with its two polypeptide chains shown as blue and red ribbons . for details of the structure of ms2 coat protein a. agarose gel electrophoresis of the soluble fractions of lysates of e. coli cells producing the wild - type ( wt ) and each of the mutant coat proteins ( lanes 16 ) . lane 1 wild - type , lane 2 g13c , lane 3 g14c ; lane 4 t15c , lane 5 g113c , lane 6 d114c . samples are labeled as in a , except for the addition of lane 0 , which is a control that produces no coat protein . in past work it has frequently been possible to suppress the effects of mutations on ms2 coat protein folding / stability by incorporating them into so - called single - chain dimers . because of the proximity of the n - terminus of one subunit of the coat protein dimer to the c - terminus of the other subunit , it is possible to genetically fuse them into a single polypeptide chain . however , in none of these cases was the ability to produce active coat protein restored ( results not shown ) . in contrast to the destabilizing substitutions , the t15c mutant ( where threonine15 is replaced by cysteine ) produced significant quantities of soluble coat protein that assembled into particles with the same electrophoretic mobility as wild - type virus . assembly into a virus - sized particle was verified by the behavior of the t15c mutant upon chromatography in sepharose cl-4b . as seen in figure 3 , wild - type ms2 and the t15c mutant particles both eluted in a discrete , symmetric peak at the same position . it illustrates how the existence of the t15c mutant should make it possible to attach chemically a variety of substances in a defined geometric array on outside of the particle . introduction of cysteine at other sites would allow variations in this pattern , each of them adhering to the constraints of icosahedral geometry , but allowing different relative spacings of the functional group . elution profiles of wild - type and t15c virus - like particles from a sepharose cl-4b column . a protein roughly the size of the coat protein monomer ( lysozyme , mw about 14,000 ) elutes at position 33 . two views of a portion of the surface of the viral particle showing the exposure of threonine15 and the pattern of its display . note that the structure shown here ( downloadable as 1gav.pdb from , the protein data bank website ) is actually that of ga , a close ms2 relative with a highly similar structure t15c virus - like particles were purified from e. coli , using methods that included gel filtration chromatography on sepharose cl-4b and that were described previously for the wild - type virus - like particle . upon attempting to redissolve the pelleted t15c particles it was immediately apparent that their behavior was different from wild - type . whereas wild - type particles dissolve readily in water , the mutant capsids were insoluble . treatment with 10 mm dtt led to the immediate dissolution ( within a few minutes ) of the aggregate and to the restoration of wild - type electrophoretic behavior . when the aggregates were subjected to sds gel electrophoresis in the absence of reducing agent ( with nem included to prevent thiol - disulfide interchange during sample preparation ) about 3% of the coat protein was present in the form of a disulfide linked dimer , consistent with the idea that each capsid in the aggregate is cross - linked on average to about 5 others ( data not shown ) . agarose gel electrophoresis of ms2-t15c virus - like particles treated with dtt at the indicated concentrations . material on the right is fully reduced and possesses the electrophoretic behavior characteristic of ms2 itself ( see figures 2 and 6 ) . the accessibility of the new cysteine is further illustrated by its reaction with thiol - specific chemical reagents . wild - type and t15c capsids were reacted with fluoroscein-5-maleimide under conditions described in materials and methods and the products were subjected to electrophoresis in agarose gels and photographed under uv illumination both before and after staining with ethidium bromide , which gives an orange fluorescence to all the capsids because of the rna each contains . the modification is specific for the t15c mutant wild - type ms2 remains unmodified and is abolished when the reagent is inactivated by prior addition of dtt to the reaction . when subjected to electrophoresis in sds - polyacrylamide gels a single fluorescent product staining of the gel with coomassie blue shows that attachment of fluorescein alters the mobility of coat protein , allowing an estimation of the extent of its modification . clearly , the great majority ( about 8090% ) of the t15c coat protein undergoes reaction under these conditions . failure to modify the wild - type coat protein indicates that the other cysteines ( residues 46 and 101 ) are not detectably accessible for reaction under these conditions . b. sds gel electrophoresis of the same samples shown in a. on the left is the gel stained with coomassie brilliant blue and at right it is illuminated in the uv . considering their locations in the coat protein structure it is not surprising that some substitutions of ab loop residues disrupted folding . genetic fusion of the subunits of the dimer was shown previously to revert the destabilizing effects of variety of mutations , including a wide range of amino acid substitutions at different locations on the -sheet , temperature - sensitive mutations occurring at numerous sites through - out the structure ( unpublished observations ) , and even insertions into the ab - loop sequence itself . it is less obvious why the substitutions at residues aspartic acid114 and glycine115 lead to folding - defects , but these residues also are involved in a turn of the polypeptide , this one connecting the two coat protein alpha - helices . as these results illustrate , amino acid substitutions can disrupt protein folding and stability with an annoyingly high frequency . the first is to create single - chain dimers of the mutant proteins [ 20 - 23 ] . although this was ineffective in the cases of the four defective mutants described here , it has in the past proven an efficient and simple means of reverting coat protein folding defects and will likely be useful for many of the other defects one might encounter . moreover , since single - chain dimers allow independent control of the amino acid sequences in the two halves of the " dimer " , it provides a means to alter by one - half the number of thiols on the virus surface , giving an added level of control over the density of modifiable sites . a gel diffusion method that allows one to distinguish bacterial colonies that produce soluble , properly assembled coat protein from those that do the sites modified in this study were chosen because they are highly exposed on the virus surface , but a number of other sites in coat protein are also located in potentially suitable positions , and some of them are likely to be more tolerant of substitution than those tested so far . the capacity to introduce cysteines at alternative positions would allow one to alter the relative geometric arrangement of reactive sites , an additional parameter that should influence the properties of specific modified virus - like particles . wild - type ms2 coat protein has two cysteines , one at position 46 and the other at 101 . under the conditions used in this study , no evidence that these cysteines were modified by fluoroscein-5-maleimide this selectivity is a little surprising in view of the previous demonstrations that cysteine46 is somewhat susceptible to reaction with sulfhydryl - specific reagents even though , like cysteine101 , it is relatively buried within the coat protein tertiary structure . alternatively , because it is bulkier than the reagents used in the previous studies ( e.g. the ability to chemically modify specific sites on virus particle surfaces is a potentially powerful approach to the production of new materials for biotechnology , nanotechnology and molecular electronics . it makes possible the use of the virus - like particle as a scaffold for the attachment of a large variety of substances including metals , organics , peptides , and nucleic acids in a regular geometric array . thus , one can think of these virus - like particles as self - assembling and highly regular nanospheres , potentially susceptible to a wide range of chemical modifications at specific surface locations . the ability to specifically encapsidate and protect arbitrarily chosen rnas within such particles suggests additional applications . the resulting pcr products were cloned as xbai - bamhi fragments in the t7 expression vector called pet3d thus creating a series of derivatives of the plasmid called petct . the presence of virus - like particles in crude cell lysates was determined by electrophoresis in 1% agarose gels in 50 mm potassium phosphate , ph 7.0 as described previously . electrophoresis of purified virus - like particles was conducted in 1% agarose and 40 mm tris - acetate , 2 mm edta , ph 8.0 .

this was a 12-month , prospective , multicenter , open - label , parallel - group , randomized , controlled clinical trial ; the full protocol was previously reported ( 16 ) . the study was approved by the ethics committee of each site and complies with the helsinki declaration . patients with type 2 diabetes not treated with insulin ( disease duration 110 years ) , aged 3575 years , and with hba1c 7.09.0% were eligible . patients were ineligible if they had insulin treatment for > 7 days , previous use of structured smbg , impending complications of diabetes , or limited life expectancy or if they were pregnant , breastfeeding , or intended to become pregnant . randomization was stratified by the diabetes treatment at enrollment ( diet only or diet plus diabetes medications ) . allocation information was sealed in sequentially numbered opaque envelopes prepared by the clinical research organization managing the trial . a commercially available educational program ( accu - chek educare ; roche diagnostics , monza , italy ) was used to provide standardized diabetes information to patients in both groups . the program is organized into subject - specific modules and includes charts and other materials to facilitate patient engagement . sessions on nutrition , physical activity , smbg , and diabetes medications were provided at baseline and additional modules were completed throughout the study . patients in the intensive structured monitoring ( ism ) group were required to perform 4-point capillary glucose measures before breakfast and lunch , 2 h after lunch , and 5 h after lunch but before dinner ( 17,18 ) 3 days / week , every week ( 2 working days [ monday friday ] and 1 weekend day [ saturday or sunday ] ) , for 12 months . ism patients were trained to interpret smbg data and were given a diary listing glycemic targets as follows : < 110 mg / dl for fasting glucose levels and glucose levels before lunch ; < 50 mg / dl as difference between postprandial and preprandial glucose levels ; and suggestions for reaching treatment goals . patients in the active control ( ac ) group were required to complete a 3-day , 4-point profile before their visits at months 6 and 12 to obtain data for comparison with the ism group . these data were not available for use by clinicians for glycemic evaluation or medication adjustments . at each follow - up visit ( months 3 , 6 , 9 , and 12 ) , investigators performed physical examinations ; recorded bmi , blood pressure , and heart rate ; and collected blood samples for hba1c measurements . hba1c for statistical analysis was measured by the central laboratory ( laboraf diagnostica e ricerca , milan , italy ) using the variant ii testing systems ( bio - rad , segrate , italy ) . at each visit , investigators prescribed diabetes medication aiming at an hba1c target < 7.0% in both groups . with ism patients , investigators reviewed and discussed the smbg and diary and reviewed and recommended changes in diet and physical activity . smbg data from ism patients were downloaded to a computer through a wireless device ( accu - chek smart - pix system ; roche diagnostics , monza , italy ) and analyzed using ad hoc software that provided easy - to - read summary statistics ( supplementary fig . investigators had the option to use a treatment algorithm ( 16 ) based on guidelines from international and national scientific societies ( 19 ) ( supplementary fig . incretin mimetics and dpp-4 inhibitors were not included in the algorithm because they were unavailable in italy when the protocol was written . once they became available , investigators were notified that they could be used for study patients . the algorithm guided changes in diabetes medications ( type or dosage ) based on mean fasting or preprandial glucose levels , differences between postprandial and preprandial glucose , and hypoglycemic events . in the ac group , smbg data were not available for viewing in patient meters and data were not downloaded until the end of the study ; therefore , adjustments of diabetes medications were based exclusively on hba1c and hypoglycemic events . two primary end points were tested in hierarchical order : change in hba1c levels from baseline to month 12 and percentage of patients reaching / maintaining the risk target ( low blood glucose index [ lbgi ] 2.5 together with high blood glucose index [ hbgi ] 5 ) from baseline to month 12 . lbgi and hbgi are summary statistics computed from smbg data shown to predict the risk of hypoglycemia and hyperglycemia , respectively ( 2023 ) . lbgi increases when the number or extent ( or both ) of low smbg measurements increases , whereas hbgi increases when the frequency or the extent ( or both ) of high smbg measurements increases ( see supplementary table 1 for computation of lbgi and hbgi ) . the hierarchical approach of primary end points avoids multiplicity issues with adjustment of type i error because the second co - primary end point is tested only if the first is statistically significant at 0.05 . secondary end points included the following : changes in hbgi and lbgi ; changes in smbg frequency ; changes in diabetes therapy ( type of medication or dosage ) ; frequency and severity of hypoglycemic episodes ; changes in blood pressure , estimated glomerular filtration rate calculated according to the creatinine - based modification of diet in renal disease equation , lipid profile , and bmi ; changes in diabetes - specific quality of life questionnaire scores ( 24 ) and diabetes - specific locus of control questionnaire scores ( 25 ) ; and study - related and diabetes - related adverse events . the diabetes - specific quality of life questionnaire used in the diabetes control and complications trial ( 24 ) , translated into italian , modified for patients with type 2 diabetes , and validated ( 26 ) , includes the following three domains : satisfaction ( score 1470 ) ; impact ( score 2892 ) ; and worry ( score 525 ) ; higher score indicates poor quality of life . the diabetes - specific locus of control questionnaire ( 25 ) , translated into italian ( 27 ) , includes the following three domains : internal ; powerful others ; and chance ( scores 636 ) ; the domain with the highest score indicates locus of control . mild hypoglycemia and moderate hypoglycemia were defined as symptoms consistent with hypoglycemia or glucose levels 60 mg / dl or 50 mg / dl , respectively , without loss of consciousness ; given some degree of overlapping between mild and moderate hypoglycemia , we present them combined as nonsevere hypoglycemia . severe hypoglycemia was defined as an event with symptoms consistent with hypoglycemia during which the person required the assistance of another person or intravenous glucose or glucagon administration . the event might be confirmed by the finding of a glucose level < 50 mg / dl . participants were informed of their risk of hypoglycemia and instructed to record any hypoglycemic event in a diary and to contact the clinic if severe or repeated nonsevere hypoglycemia occurred . in a post hoc analysis , we tested the difference in the proportion of participants in the ism group or ac group who reached clinically meaningful hba1c reductions of < 0.3 , < 0.4 , or < 0.5% . five hundred patients in each group were needed to achieve 90% power to detect a significant ( at the two - sided 5% level ) 0.3% difference between the ism and ac groups in the mean hba1c change at month 12 compared with baseline , assuming a 1.25% sd and 25% attrition ( 28 ) . primary and secondary end points were analyzed on an intent - to - treat ( itt ) basis including all randomized patients . primary and secondary end points also were analyzed in the per protocol ( pp ) population , consisting of all randomized patients who completed the study without major protocol violations and were compliant with the smbg regimen ( i.e. , 80% of the required smbg measurements in the ism group and 200 unstructured discretionary smbg measurements in the ac group , the maximum measurements recommended for these patients by the italian standards of diabetes care ) ( 29 ) . results for the pp population are reported for primary end points and selected secondary end points . statistical analyses were performed using sas ( version 9.02 , ts level 02m0 ) . the first co - primary end point was analyzed using a mixed linear model ( 30 ) with randomized group , center , visit , and randomized group - by - visit interaction as fixed effects and baseline hba1c as covariate . an unstructured variance covariance matrix was used to model the correlation between repeated measurements within each patient . restricted maximum likelihood estimates and two - sided 95% ci of the mean difference between randomized groups at month 12 were calculated using the newton - raphson algorithm . cross - sectional comparisons were performed using time - by - time contrasts programmed using the sas mixed procedure . missing data were filled in by multiple imputation assuming a missing at - random mechanism of dropout . the monte carlo markov chain technique implemented in sas proc mi was used to obtain 50 imputed datasets . rubin rules implemented in sas proc mianalyze were used to combine effect estimates and to estimate 95% cis to allow for uncertainty attributable to missing data . for the post hoc analysis , the last observation carried forward technique was used to complete missing values for patients who did not complete the study . the interaction between randomized group and center was assessed , with a two - sided p 0.10 considered statistically significant for the test of interaction . the analysis of the second co - primary end point was based on the cochran - mantel - haenszel test controlling for clinical site effects . summary statistics and two - sided 95% cis were computed for mean changes ( continuous variables ) and risk differences ( categorical variables ) . patients with type 2 diabetes not treated with insulin ( disease duration 110 years ) , aged 3575 years , and with hba1c 7.09.0% were eligible . patients were ineligible if they had insulin treatment for > 7 days , previous use of structured smbg , impending complications of diabetes , or limited life expectancy or if they were pregnant , breastfeeding , or intended to become pregnant . randomization was stratified by the diabetes treatment at enrollment ( diet only or diet plus diabetes medications ) . allocation information was sealed in sequentially numbered opaque envelopes prepared by the clinical research organization managing the trial . a commercially available educational program ( accu - chek educare ; roche diagnostics , monza , italy ) was used to provide standardized diabetes information to patients in both groups . the program is organized into subject - specific modules and includes charts and other materials to facilitate patient engagement . sessions on nutrition , physical activity , smbg , and diabetes medications were provided at baseline and additional modules were completed throughout the study . patients in the intensive structured monitoring ( ism ) group were required to perform 4-point capillary glucose measures before breakfast and lunch , 2 h after lunch , and 5 h after lunch but before dinner ( 17,18 ) 3 days / week , every week ( 2 working days [ monday friday ] and 1 weekend day [ saturday or sunday ] ) , for 12 months . ism patients were trained to interpret smbg data and were given a diary listing glycemic targets as follows : < 110 mg / dl for fasting glucose levels and glucose levels before lunch ; < 50 mg / dl as difference between postprandial and preprandial glucose levels ; and suggestions for reaching treatment goals . patients in the active control ( ac ) group were required to complete a 3-day , 4-point profile before their visits at months 6 and 12 to obtain data for comparison with the ism group . these data were not available for use by clinicians for glycemic evaluation or medication adjustments . at each follow - up visit ( months 3 , 6 , 9 , and 12 ) , investigators performed physical examinations ; recorded bmi , blood pressure , and heart rate ; and collected blood samples for hba1c measurements . hba1c for statistical analysis was measured by the central laboratory ( laboraf diagnostica e ricerca , milan , italy ) using the variant ii testing systems ( bio - rad , segrate , italy ) . at each visit , investigators prescribed diabetes medication aiming at an hba1c target < 7.0% in both groups . with ism patients , investigators reviewed and discussed the smbg and diary and reviewed and recommended changes in diet and physical activity . smbg data from ism patients were downloaded to a computer through a wireless device ( accu - chek smart - pix system ; roche diagnostics , monza , italy ) and analyzed using ad hoc software that provided easy - to - read summary statistics ( supplementary fig . investigators had the option to use a treatment algorithm ( 16 ) based on guidelines from international and national scientific societies ( 19 ) ( supplementary fig . incretin mimetics and dpp-4 inhibitors were not included in the algorithm because they were unavailable in italy when the protocol was written . once they became available , investigators were notified that they could be used for study patients . the algorithm guided changes in diabetes medications ( type or dosage ) based on mean fasting or preprandial glucose levels , differences between postprandial and preprandial glucose , and hypoglycemic events . in the ac group , smbg data were not available for viewing in patient meters and data were not downloaded until the end of the study ; therefore , adjustments of diabetes medications were based exclusively on hba1c and hypoglycemic events . two primary end points were tested in hierarchical order : change in hba1c levels from baseline to month 12 and percentage of patients reaching / maintaining the risk target ( low blood glucose index [ lbgi ] 2.5 together with high blood glucose index [ hbgi ] 5 ) from baseline to month 12 . lbgi and hbgi are summary statistics computed from smbg data shown to predict the risk of hypoglycemia and hyperglycemia , respectively ( 2023 ) . lbgi increases when the number or extent ( or both ) of low smbg measurements increases , whereas hbgi increases when the frequency or the extent ( or both ) of high smbg measurements increases ( see supplementary table 1 for computation of lbgi and hbgi ) . the hierarchical approach of primary end points avoids multiplicity issues with adjustment of type i error because the second co - primary end point is tested only if the first is statistically significant at 0.05 . secondary end points included the following : changes in hbgi and lbgi ; changes in smbg frequency ; changes in diabetes therapy ( type of medication or dosage ) ; frequency and severity of hypoglycemic episodes ; changes in blood pressure , estimated glomerular filtration rate calculated according to the creatinine - based modification of diet in renal disease equation , lipid profile , and bmi ; changes in diabetes - specific quality of life questionnaire scores ( 24 ) and diabetes - specific locus of control questionnaire scores ( 25 ) ; and study - related and diabetes - related adverse events . the diabetes - specific quality of life questionnaire used in the diabetes control and complications trial ( 24 ) , translated into italian , modified for patients with type 2 diabetes , and validated ( 26 ) , includes the following three domains : satisfaction ( score 1470 ) ; impact ( score 2892 ) ; and worry ( score 525 ) ; higher score indicates poor quality of life . the diabetes - specific locus of control questionnaire ( 25 ) , translated into italian ( 27 ) , includes the following three domains : internal ; powerful others ; and chance ( scores 636 ) ; the domain with the highest score indicates locus of control . mild hypoglycemia and moderate hypoglycemia were defined as symptoms consistent with hypoglycemia or glucose levels 60 mg / dl or 50 mg / dl , respectively , without loss of consciousness ; given some degree of overlapping between mild and moderate hypoglycemia , we present them combined as nonsevere hypoglycemia . severe hypoglycemia was defined as an event with symptoms consistent with hypoglycemia during which the person required the assistance of another person or intravenous glucose or glucagon administration . the event might be confirmed by the finding of a glucose level < 50 mg / dl . participants were informed of their risk of hypoglycemia and instructed to record any hypoglycemic event in a diary and to contact the clinic if severe or repeated nonsevere hypoglycemia occurred . in a post hoc analysis , we tested the difference in the proportion of participants in the ism group or ac group who reached clinically meaningful hba1c reductions of < 0.3 , < 0.4 , or < 0.5% . five hundred patients in each group were needed to achieve 90% power to detect a significant ( at the two - sided 5% level ) 0.3% difference between the ism and ac groups in the mean hba1c change at month 12 compared with baseline , assuming a 1.25% sd and 25% attrition ( 28 ) . primary and secondary end points were analyzed on an intent - to - treat ( itt ) basis including all randomized patients . primary and secondary end points also were analyzed in the per protocol ( pp ) population , consisting of all randomized patients who completed the study without major protocol violations and were compliant with the smbg regimen ( i.e. , 80% of the required smbg measurements in the ism group and 200 unstructured discretionary smbg measurements in the ac group , the maximum measurements recommended for these patients by the italian standards of diabetes care ) ( 29 ) . results for the pp population are reported for primary end points and selected secondary end points . statistical analyses were performed using sas ( version 9.02 , ts level 02m0 ) . the first co - primary end point was analyzed using a mixed linear model ( 30 ) with randomized group , center , visit , and randomized group - by - visit interaction as fixed effects and baseline hba1c as covariate . an unstructured variance covariance matrix was used to model the correlation between repeated measurements within each patient . restricted maximum likelihood estimates and two - sided 95% ci of the mean difference between randomized groups at month 12 cross - sectional comparisons were performed using time - by - time contrasts programmed using the sas mixed procedure . missing data were filled in by multiple imputation assuming a missing at - random mechanism of dropout . the monte carlo markov chain technique implemented in sas proc mi was used to obtain 50 imputed datasets . rubin rules implemented in sas proc mianalyze were used to combine effect estimates and to estimate 95% cis to allow for uncertainty attributable to missing data . for the post hoc analysis , the last observation carried forward technique was used to complete missing values for patients who did not complete the study . the interaction between randomized group and center was assessed , with a two - sided p 0.10 considered statistically significant for the test of interaction . the analysis of the second co - primary end point was based on the cochran - mantel - haenszel test controlling for clinical site effects . summary statistics and two - sided 95% cis were computed for mean changes ( continuous variables ) and risk differences ( categorical variables ) . patients were enrolled between may 2008 and may 2010 . of the 1,072 screened patients , 1,024 were eligible and were assigned to the ism ( n = 501 ) group or ac ( n = 523 ) group ( fig . the pp population consisted of 232 ( 46.3% ) ism patients and 321 ( 61.4% ) ac patients ; the most common reasons for exclusion were noncompliance with the smbg regimen , participant s decision to withdraw , and major violations of inclusion / exclusion criteria . the proportions of noncompleters were 14.0 and 13.6% in the ism and ac groups , respectively . the demographic , anthropometric , and metabolic characteristics of patients who withdrew from the study were similar to those of completers , except for age , which was 3 years older among completers ( p < 0.01 ) . study patients were predominantly male , obese ( bmi 30 ) , treated with oral agents , and with hba1c slightly higher than the 7.0% target ( table 1 ) . ism patients performed a median of 512 ( interquartile range , 373573 ) smbg measurements , whereas ac patients performed a median of 108 ( interquartile range , 61182 ) measurements . flow of prisma study participants . baseline characteristics of the prisma study participants by assigned intervention : itt population in the itt population , both groups showed reductions in hba1c levels ; however , over the course of the 12 months ism patients had greater reductions in hba1c than ac patients ( 0.39 vs. 0.27% , ism vs. ac , respectively ; = 0.12% ; 95% ci , 0.210 to 0.024 ; p = 0.013 ) ( fig . ism patients had an even greater hba1c reduction than ac patients ( 0.45 vs. 0.24% ; = 0.21% ; 0.331 to 0.089 ; p = 0.0007 ) ( fig . more ism patients achieved clinically meaningful reductions in hba1c ( > 0.3 , > 0.4 , or > 0.5% ) at study end than ac patients ( p < 0.025 ) ( fig . least - square mean difference in hba1c ( % ) during the study by treatment group in the itt population ( a ) and pp population ( b ) . proportion ( 95% ci ) of participants who achieved clinically meaningful hba1c reductions of > 0.3 , > 0.4 , or > 0.5% . in the itt population , a similar proportion of ism and ac patients reached / maintained the risk target at month 12 ( 74.6% [ 95% ci , 70.678.4 ] and 70.1% [ 66.074.1 ] in ism and ac patients , respectively ; p = 0.131 ) . in the pp population , a higher proportion of ism than ac patients reached / maintained the risk target ( 90.0% [ 85.493.6 ] in ism patients and 82.5% [ 77.886.5 ] in ac patients ; p = 0.038 ) . when hbgi and lbgi were analyzed as continuous variables , hbgi decreased more in ism than in ac patients at month 12 ( 0.43 ; 0.76 to 0.10 ; p = 0.011 ) , whereas no between - group difference was observed for lbgi ( 0.09 ; 0.12 to 0.20 ; p = 0.082 ) . in the itt population , the prescription of diabetes medications at visits 2 , 3 , and 4 was changed more often in ism than in ac patients ( p < 0.001 ) : 38.7% ( 95% ci , 34.343.3 ) vs. 28.0% ( 24.132.2 ) at visit 2 ; 31.8% ( 27.536.3 ) vs. 20.0% ( 16.523.9 ) at visit 3 ; 31.6% ( 27.236.2 ) vs. 20.4% ( 16.824.4 ) at visit 4 . the between - group difference did not achieve statistical significance at visit 5 ( 22.4% [ 18.626.7 ] vs. 16.7% [ 13.420.5 ] ; p = 0.143 ) ( supplementary fig . nonsevere hypoglycemia was more commonly detected in ism than in ac patients ( 1.32 vs. 0.42 events per patient year ; incidence rate ratio , 3.32 ; 95% ci , 1.964.78 ; p < 0.0001 ) . two severe hypoglycemic events occurred during the study , both in one ac patient treated with a sulfonylurea . bmi decreased both in ism and in ac patients ; however , no between - group difference over 12 months was seen in the itt population ( 0.44 [ 95% ci , 0.57 to 0.31 ] vs. 0.28 [ 95% ci : 0.40 to 0.15 ] , ism vs. ac , respectively ; = 0.16 ; 0.01 to 0.34 ; p = 0.070 ) . a between - group difference over 12 months was observed in the pp population ( 0.58 [ 0.76 to 0.40 ] vs. 0.28 [ 0.44 to 0.13 ] , ism vs. ac , respectively ; = 0.29 ; 0.060.53 ; p = 0.014 ) . all domain scores of the quality of life questionnaire improved at month 12 compared with baseline , with no differences between ism and ac patients . the locus of control questionnaire scores improved in both groups at month 12 , with a greater improvement in the chance domain in ism than in ac ( 0.92 [ sd , 0.59 ] vs. 0.10 [ sd , 0.57 ] ; p = 0.024 ) . there were no between - group differences regarding changes in systolic and diastolic blood pressures , estimated glomerular filtration rate , total cholesterol , hdl cholesterol , or ldl cholesterol from baseline to study end . however , the ac group experienced a significantly greater reduction in triglycerides than the ism group . results of additional secondary outcomes are reported ( supplementary table 2 ) . in the itt population , both groups showed reductions in hba1c levels ; however , over the course of the 12 months ism patients had greater reductions in hba1c than ac patients ( 0.39 vs. 0.27% , ism vs. ac , respectively ; = 0.12% ; 95% ci , 0.210 to 0.024 ; p = 0.013 ) ( fig . ism patients had an even greater hba1c reduction than ac patients ( 0.45 vs. 0.24% ; = 0.21% ; 0.331 to 0.089 ; p = 0.0007 ) ( fig . 2b ) . in both the itt and pp analyses , more ism patients achieved clinically meaningful reductions in hba1c ( > 0.3 , > 0.4 , or > 0.5% ) at study end than ac patients ( p < 0.025 ) ( fig . least - square mean difference in hba1c ( % ) during the study by treatment group in the itt population ( a ) and pp population ( b ) . proportion ( 95% ci ) of participants who achieved clinically meaningful hba1c reductions of > 0.3 , > 0.4 , or > 0.5% . in the itt population , a similar proportion of ism and ac patients reached / maintained the risk target at month 12 ( 74.6% [ 95% ci , 70.678.4 ] and 70.1% [ 66.074.1 ] in ism and ac patients , respectively ; p = 0.131 ) . in the pp population , a higher proportion of ism than ac patients reached / maintained the risk target ( 90.0% [ 85.493.6 ] in ism patients and 82.5% [ 77.886.5 ] in ac patients ; p = 0.038 ) . when hbgi and lbgi were analyzed as continuous variables , hbgi decreased more in ism than in ac patients at month 12 ( 0.43 ; 0.76 to 0.10 ; p = 0.011 ) , whereas no between - group difference was observed for lbgi ( 0.09 ; 0.12 to 0.20 ; p = 0.082 ) . in the itt population , the prescription of diabetes medications at visits 2 , 3 , and 4 was changed more often in ism than in ac patients ( p < 0.001 ) : 38.7% ( 95% ci , 34.343.3 ) vs. 28.0% ( 24.132.2 ) at visit 2 ; 31.8% ( 27.536.3 ) vs. 20.0% ( 16.523.9 ) at visit 3 ; 31.6% ( 27.236.2 ) vs. 20.4% ( 16.824.4 ) at visit 4 . the between - group difference did not achieve statistical significance at visit 5 ( 22.4% [ 18.626.7 ] vs. 16.7% [ 13.420.5 ] ; p = 0.143 ) ( supplementary fig . nonsevere hypoglycemia was more commonly detected in ism than in ac patients ( 1.32 vs. 0.42 events per patient year ; incidence rate ratio , 3.32 ; 95% ci , 1.964.78 ; p < 0.0001 ) . two severe hypoglycemic events occurred during the study , both in one ac patient treated with a sulfonylurea . bmi decreased both in ism and in ac patients ; however , no between - group difference over 12 months was seen in the itt population ( 0.44 [ 95% ci , 0.57 to 0.31 ] vs. 0.28 [ 95% ci : 0.40 to 0.15 ] , ism vs. ac , respectively ; = 0.16 ; 0.01 to 0.34 ; p = 0.070 ) . a between - group difference over 12 months was observed in the pp population ( 0.58 [ 0.76 to 0.40 ] vs. 0.28 [ 0.44 to 0.13 ] , ism vs. ac , respectively ; = 0.29 ; 0.060.53 ; p = 0.014 ) . all domain scores of the quality of life questionnaire improved at month 12 compared with baseline , with no differences between ism and ac patients . the locus of control questionnaire scores improved in both groups at month 12 , with a greater improvement in the chance domain in ism than in ac ( 0.92 [ sd , 0.59 ] vs. 0.10 [ sd , 0.57 ] ; p = 0.024 ) . there were no between - group differences regarding changes in systolic and diastolic blood pressures , estimated glomerular filtration rate , total cholesterol , hdl cholesterol , or ldl cholesterol from baseline to study end . however , the ac group experienced a significantly greater reduction in triglycerides than the ism group . results of additional secondary outcomes are reported ( supplementary table 2 ) . in this multicenter , randomized , clinical trial enrolling > 1,000 patients with relatively well - controlled , noninsulin - treated type 2 diabetes , we found that the use of intensive , structured smbg data by clinicians to optimize prescription of diabetes medications and by patients to modify their behaviors improved glycemic control and enabled significantly more ism patients to achieve clinically meaningful reductions of hba1c compared with discretionary , unstructured smbg data , which were available only to study patients . although the significant hba1c improvement in both groups from baseline was possibly the result of regular office visits and basic diabetes education , the additional glycemic improvement in ism participants was likely attributable to multiple factors . first , more ism than ac patients had their prescriptions of diabetes medication changed at each visit , suggesting that structured smbg data prompted clinicians to adjust therapy earlier and more intensively in contrast to the clinical inertia often seen in the management of patients with type 2 diabetes ( 3134 ) . although an analysis of the breakout of the specific changes made ( e.g. , adding a medication or increasing dose ) has not been completed , it is reasonable to assume that the treatment algorithm based on structured smbg findings ( supplementary fig . 2 ) helped clinicians select the most appropriate medication for each patient s glucose pattern . second , the greater decrease of bmi in ism patients , although significant only in the pp population , may have contributed to improving glycemic control in these patients . third , it is likely that ism patients made more effective lifestyle changes in response to smbg measurements as recommended to them throughout the study . we recognize that the between - group differences in hba1c reductions in our study may be perceived as being of modest magnitude ; however , one must consider that our patients had a baseline hba1c of 7.3% , which is close to the average hba1c of type 2 diabetic patients treated at diabetes clinics in italy ( 35 ) and lower than in most studies . it is also more challenging to lower hba1c levels in patients at these levels ( 36 ) . moreover , because international medical organizations recommend an hba1c < 7.0% as the treatment goal in type 2 diabetes ( 1 ) , based on findings from the uk prospective diabetes study , which demonstrated that any reduction in hba1c reduces the risk of complications ( 37 ) , we believe that clinicians have an obligation to utilize treatment strategies in addition to medications , including structured smbg , to help patients achieve their glycemic goals to improve clinical outcomes . when we considered the proportion of patients who achieved clinically meaningful improvements in glycemic control ( i.e. , an hba1c reduction of > 0.3 , > 0.4 , or > 0.5% ) , the proportion was significantly greater when using intensive structured smbg . we feel that this is an alternative metric for evaluating the value and utility of a behavior - based although ism patients reported a greater incidence of nonsevere hypoglycemic events , these events are likely the result of increased detection of hypoglycemia attributable to greater smbg frequency . provided the growing concerns for the increased mortality in intensively managed type 2 diabetes ( 38,39 ) , the detection of asymptomatic hypoglycemia may be an additional advantage of structured smbg . regarding hypoglycemia and weight control , for patients with hba1c close to the normal range , structured smbg is a safe treatment strategy to use when increasing diabetes medication . it is also noteworthy that in ism patients there were no changes in diabetes - specific quality of life scores , indicating no deterioration in quality of life with structured smbg , contrary to what previously has been reported ( 13 ) . furthermore , reductions in diabetes - specific locus of control chance domain scores suggest that ism patients were less likely than ac patients to attribute diabetes control to chance or fortune . as for the secondary outcomes of the study , ac patients had a greater decrease in triglycerides than ism patients , with no significant between - group differences in total cholesterol , hdl cholesterol , ldl cholesterol , and blood pressure . this finding may reflect that a greater effort either by patient or by provider in one domain of diabetes management may minimize effectiveness in other domains . the fact that ism patients may tend to decrease the amount of dietary carbohydrates and increase fat should be taken into account at the time of dietary counseling when starting intensive smbg management . because patients were treated at diabetes clinics , it may be difficult to generalize our findings to patients treated in primary practice settings who generally have less well - controlled diabetes . additionally , the large number of patients excluded from the pp analysis also makes generalization somewhat challenging , suggesting that the smbg regimen may have been too intensive or that a sizable proportion of noninsulin - treated type 2 diabetic patients may need additional support to comply with structured smbg . it is possible that less frequent use of the glycemic profiles ( e.g. , a lower number of weekly profiles ) would encourage more patients to use structured smbg but without sacrificing the beneficial effects seen in our study . it is important to note that 98 of the ac patients were excluded from the pp analysis because they tested more frequently than the protocol allowed , which suggests that many of these patients perceived a greater benefit from more frequent use of smbg . although smbg data from ac patients were not made available to clinicians for use in evaluating glycemic status or in making medication adjustments , the availability of these data to ac patients may have prompted changes in lifestyle behaviors or treatment adherence , potentially leading to improved glycemic control independent of clinician - based recommendations , especially in those patients who tested more frequently than the protocol allowed . recent studies have shown that use of structured smbg regimens positively influences patient behaviors , leading to improvements in glycemic control and other measures ( 811,18 ) . another limitation was that our study design precluded assessment of the effect of the comprehensive education provided and increased attention given to patients in both study groups , as well as the individual contributions of diet and physical exercise . furthermore , although lbgi and hbgi have been validated in patients with type 1 diabetes and insulin - treated type 2 diabetes , they have not been validated in type 2 diabetic patients treated with diet or oral diabetes medications . the modest within - group changes seen in our study suggest that these indices may not be particularly useful when studying well - controlled noninsulin - treated patients . finally , although we captured data regarding the total number of medication changes made throughout the study , we combined changes of dose and changes of prescribed medications . additional analyses of our results are being conducted to evaluate the impact of structured smbg on changes in dose , changes in type of medication prescribed , and appropriateness of these changes . in conclusion , the prisma ( prospective , randomized trial on intensive self - monitoring blood glucose management added value in noninsulin - treated type 2 diabetes mellitus patients ) study , to our knowledge the largest study of the effects of smbg in patients with type 2 diabetes , confirms the clinical usefulness and overall safety of using structured smbg to provide guidance in the prescription of diabetes medications and lifestyle changes in noninsulin - treated type 2 diabetic patients , ultimately improving glycemic control . additional studies are needed to further define and elucidate the optimal implementation of structured smbg use in these patients .

objectivewe aimed to evaluate the added value of intensive self - monitoring of blood glucose ( smbg ) , structured in timing and frequency , in noninsulin - treated patients with type 2 diabetes.research design and methodsthe 12-month , randomized , clinical trial enrolled 1,024 patients with noninsulin - treated type 2 diabetes ( median baseline hba1c , 7.3% [ iqr , 6.97.8% ] ) at 39 diabetes clinics in italy . after standardized education , 501 patients were randomized to intensive structured monitoring ( ism ) with 4-point glycemic profiles ( fasting , preprandial , 2-h postprandial , and postabsorptive measurements ) performed 3 days / week ; 523 patients were randomized to active control ( ac ) with 4-point glycemic profiles performed at baseline and at 6 and 12 months . two primary end points were tested in hierarchical order : hba1c change at 12 months and percentage of patients at risk target for low and high blood glucose index.resultsintent-to-treat analysis showed greater hba1c reductions over 12 months in ism ( 0.39% ) than in ac patients ( 0.27% ) , with a between - group difference of 0.12% ( 95% ci , 0.210 to 0.024 ; p = 0.013 ) . in the per - protocol analysis , the between - group difference was 0.21% ( 0.331 to 0.089 ; p = 0.0007 ) . more ism than ac patients achieved clinically meaningful reductions in hba1c ( > 0.3 , > 0.4 , or > 0.5% ) at study end ( p < 0.025 ) . the proportion of patients reaching / maintaining the risk target at month 12 was similar in ism ( 74.6% ) and ac ( 70.1% ) patients ( p = 0.131 ) . at visits 2 , 3 , and 4 , diabetes medications were changed more often in ism than in ac patients ( p < 0.001).conclusionsuse of structured smbg improves glycemic control and provides guidance in prescribing diabetes medications in patients with relatively well - controlled noninsulin - treated type 2 diabetes .

RESEARCH DESIGN AND METHODS Setting and participants Randomization Interventions Outcomes Statistical analysis RESULTS Changes in HbA Changes in glycemic risk Treatment intensification Incidence of hypoglycemia Changes in BMI Psychosocial measures Other secondary outcomes CONCLUSIONS

this was a 12-month , prospective , multicenter , open - label , parallel - group , randomized , controlled clinical trial ; the full protocol was previously reported ( 16 ) . patients with type 2 diabetes not treated with insulin ( disease duration 110 years ) , aged 3575 years , and with hba1c 7.09.0% were eligible . patients were ineligible if they had insulin treatment for > 7 days , previous use of structured smbg , impending complications of diabetes , or limited life expectancy or if they were pregnant , breastfeeding , or intended to become pregnant . sessions on nutrition , physical activity , smbg , and diabetes medications were provided at baseline and additional modules were completed throughout the study . patients in the intensive structured monitoring ( ism ) group were required to perform 4-point capillary glucose measures before breakfast and lunch , 2 h after lunch , and 5 h after lunch but before dinner ( 17,18 ) 3 days / week , every week ( 2 working days [ monday friday ] and 1 weekend day [ saturday or sunday ] ) , for 12 months . patients in the active control ( ac ) group were required to complete a 3-day , 4-point profile before their visits at months 6 and 12 to obtain data for comparison with the ism group . at each follow - up visit ( months 3 , 6 , 9 , and 12 ) , investigators performed physical examinations ; recorded bmi , blood pressure , and heart rate ; and collected blood samples for hba1c measurements . two primary end points were tested in hierarchical order : change in hba1c levels from baseline to month 12 and percentage of patients reaching / maintaining the risk target ( low blood glucose index [ lbgi ] 2.5 together with high blood glucose index [ hbgi ] 5 ) from baseline to month 12 . the diabetes - specific quality of life questionnaire used in the diabetes control and complications trial ( 24 ) , translated into italian , modified for patients with type 2 diabetes , and validated ( 26 ) , includes the following three domains : satisfaction ( score 1470 ) ; impact ( score 2892 ) ; and worry ( score 525 ) ; higher score indicates poor quality of life . in a post hoc analysis , we tested the difference in the proportion of participants in the ism group or ac group who reached clinically meaningful hba1c reductions of < 0.3 , < 0.4 , or < 0.5% . five hundred patients in each group were needed to achieve 90% power to detect a significant ( at the two - sided 5% level ) 0.3% difference between the ism and ac groups in the mean hba1c change at month 12 compared with baseline , assuming a 1.25% sd and 25% attrition ( 28 ) . the first co - primary end point was analyzed using a mixed linear model ( 30 ) with randomized group , center , visit , and randomized group - by - visit interaction as fixed effects and baseline hba1c as covariate . patients with type 2 diabetes not treated with insulin ( disease duration 110 years ) , aged 3575 years , and with hba1c 7.09.0% were eligible . sessions on nutrition , physical activity , smbg , and diabetes medications were provided at baseline and additional modules were completed throughout the study . patients in the intensive structured monitoring ( ism ) group were required to perform 4-point capillary glucose measures before breakfast and lunch , 2 h after lunch , and 5 h after lunch but before dinner ( 17,18 ) 3 days / week , every week ( 2 working days [ monday friday ] and 1 weekend day [ saturday or sunday ] ) , for 12 months . patients in the active control ( ac ) group were required to complete a 3-day , 4-point profile before their visits at months 6 and 12 to obtain data for comparison with the ism group . at each follow - up visit ( months 3 , 6 , 9 , and 12 ) , investigators performed physical examinations ; recorded bmi , blood pressure , and heart rate ; and collected blood samples for hba1c measurements . two primary end points were tested in hierarchical order : change in hba1c levels from baseline to month 12 and percentage of patients reaching / maintaining the risk target ( low blood glucose index [ lbgi ] 2.5 together with high blood glucose index [ hbgi ] 5 ) from baseline to month 12 . the diabetes - specific quality of life questionnaire used in the diabetes control and complications trial ( 24 ) , translated into italian , modified for patients with type 2 diabetes , and validated ( 26 ) , includes the following three domains : satisfaction ( score 1470 ) ; impact ( score 2892 ) ; and worry ( score 525 ) ; higher score indicates poor quality of life . in a post hoc analysis , we tested the difference in the proportion of participants in the ism group or ac group who reached clinically meaningful hba1c reductions of < 0.3 , < 0.4 , or < 0.5% . five hundred patients in each group were needed to achieve 90% power to detect a significant ( at the two - sided 5% level ) 0.3% difference between the ism and ac groups in the mean hba1c change at month 12 compared with baseline , assuming a 1.25% sd and 25% attrition ( 28 ) . the demographic , anthropometric , and metabolic characteristics of patients who withdrew from the study were similar to those of completers , except for age , which was 3 years older among completers ( p < 0.01 ) . baseline characteristics of the prisma study participants by assigned intervention : itt population in the itt population , both groups showed reductions in hba1c levels ; however , over the course of the 12 months ism patients had greater reductions in hba1c than ac patients ( 0.39 vs. 0.27% , ism vs. ac , respectively ; = 0.12% ; 95% ci , 0.210 to 0.024 ; p = 0.013 ) ( fig . ism patients had an even greater hba1c reduction than ac patients ( 0.45 vs. 0.24% ; = 0.21% ; 0.331 to 0.089 ; p = 0.0007 ) ( fig . more ism patients achieved clinically meaningful reductions in hba1c ( > 0.3 , > 0.4 , or > 0.5% ) at study end than ac patients ( p < 0.025 ) ( fig . least - square mean difference in hba1c ( % ) during the study by treatment group in the itt population ( a ) and pp population ( b ) . proportion ( 95% ci ) of participants who achieved clinically meaningful hba1c reductions of > 0.3 , > 0.4 , or > 0.5% . in the itt population , a similar proportion of ism and ac patients reached / maintained the risk target at month 12 ( 74.6% [ 95% ci , 70.678.4 ] and 70.1% [ 66.074.1 ] in ism and ac patients , respectively ; p = 0.131 ) . in the pp population , a higher proportion of ism than ac patients reached / maintained the risk target ( 90.0% [ 85.493.6 ] in ism patients and 82.5% [ 77.886.5 ] in ac patients ; p = 0.038 ) . when hbgi and lbgi were analyzed as continuous variables , hbgi decreased more in ism than in ac patients at month 12 ( 0.43 ; 0.76 to 0.10 ; p = 0.011 ) , whereas no between - group difference was observed for lbgi ( 0.09 ; 0.12 to 0.20 ; p = 0.082 ) . in the itt population , the prescription of diabetes medications at visits 2 , 3 , and 4 was changed more often in ism than in ac patients ( p < 0.001 ) : 38.7% ( 95% ci , 34.343.3 ) vs. 28.0% ( 24.132.2 ) at visit 2 ; 31.8% ( 27.536.3 ) vs. 20.0% ( 16.523.9 ) at visit 3 ; 31.6% ( 27.236.2 ) vs. 20.4% ( 16.824.4 ) at visit 4 . the between - group difference did not achieve statistical significance at visit 5 ( 22.4% [ 18.626.7 ] vs. 16.7% [ 13.420.5 ] ; p = 0.143 ) ( supplementary fig . nonsevere hypoglycemia was more commonly detected in ism than in ac patients ( 1.32 vs. 0.42 events per patient year ; incidence rate ratio , 3.32 ; 95% ci , 1.964.78 ; p < 0.0001 ) . bmi decreased both in ism and in ac patients ; however , no between - group difference over 12 months was seen in the itt population ( 0.44 [ 95% ci , 0.57 to 0.31 ] vs. 0.28 [ 95% ci : 0.40 to 0.15 ] , ism vs. ac , respectively ; = 0.16 ; 0.01 to 0.34 ; p = 0.070 ) . a between - group difference over 12 months was observed in the pp population ( 0.58 [ 0.76 to 0.40 ] vs. 0.28 [ 0.44 to 0.13 ] , ism vs. ac , respectively ; = 0.29 ; 0.060.53 ; p = 0.014 ) . all domain scores of the quality of life questionnaire improved at month 12 compared with baseline , with no differences between ism and ac patients . the locus of control questionnaire scores improved in both groups at month 12 , with a greater improvement in the chance domain in ism than in ac ( 0.92 [ sd , 0.59 ] vs. 0.10 [ sd , 0.57 ] ; p = 0.024 ) . there were no between - group differences regarding changes in systolic and diastolic blood pressures , estimated glomerular filtration rate , total cholesterol , hdl cholesterol , or ldl cholesterol from baseline to study end . in the itt population , both groups showed reductions in hba1c levels ; however , over the course of the 12 months ism patients had greater reductions in hba1c than ac patients ( 0.39 vs. 0.27% , ism vs. ac , respectively ; = 0.12% ; 95% ci , 0.210 to 0.024 ; p = 0.013 ) ( fig . ism patients had an even greater hba1c reduction than ac patients ( 0.45 vs. 0.24% ; = 0.21% ; 0.331 to 0.089 ; p = 0.0007 ) ( fig . in both the itt and pp analyses , more ism patients achieved clinically meaningful reductions in hba1c ( > 0.3 , > 0.4 , or > 0.5% ) at study end than ac patients ( p < 0.025 ) ( fig . least - square mean difference in hba1c ( % ) during the study by treatment group in the itt population ( a ) and pp population ( b ) . proportion ( 95% ci ) of participants who achieved clinically meaningful hba1c reductions of > 0.3 , > 0.4 , or > 0.5% . in the itt population , a similar proportion of ism and ac patients reached / maintained the risk target at month 12 ( 74.6% [ 95% ci , 70.678.4 ] and 70.1% [ 66.074.1 ] in ism and ac patients , respectively ; p = 0.131 ) . in the pp population , a higher proportion of ism than ac patients reached / maintained the risk target ( 90.0% [ 85.493.6 ] in ism patients and 82.5% [ 77.886.5 ] in ac patients ; p = 0.038 ) . when hbgi and lbgi were analyzed as continuous variables , hbgi decreased more in ism than in ac patients at month 12 ( 0.43 ; 0.76 to 0.10 ; p = 0.011 ) , whereas no between - group difference was observed for lbgi ( 0.09 ; 0.12 to 0.20 ; p = 0.082 ) . in the itt population , the prescription of diabetes medications at visits 2 , 3 , and 4 was changed more often in ism than in ac patients ( p < 0.001 ) : 38.7% ( 95% ci , 34.343.3 ) vs. 28.0% ( 24.132.2 ) at visit 2 ; 31.8% ( 27.536.3 ) vs. 20.0% ( 16.523.9 ) at visit 3 ; 31.6% ( 27.236.2 ) vs. 20.4% ( 16.824.4 ) at visit 4 . the between - group difference did not achieve statistical significance at visit 5 ( 22.4% [ 18.626.7 ] vs. 16.7% [ 13.420.5 ] ; p = 0.143 ) ( supplementary fig . nonsevere hypoglycemia was more commonly detected in ism than in ac patients ( 1.32 vs. 0.42 events per patient year ; incidence rate ratio , 3.32 ; 95% ci , 1.964.78 ; p < 0.0001 ) . bmi decreased both in ism and in ac patients ; however , no between - group difference over 12 months was seen in the itt population ( 0.44 [ 95% ci , 0.57 to 0.31 ] vs. 0.28 [ 95% ci : 0.40 to 0.15 ] , ism vs. ac , respectively ; = 0.16 ; 0.01 to 0.34 ; p = 0.070 ) . a between - group difference over 12 months was observed in the pp population ( 0.58 [ 0.76 to 0.40 ] vs. 0.28 [ 0.44 to 0.13 ] , ism vs. ac , respectively ; = 0.29 ; 0.060.53 ; p = 0.014 ) . all domain scores of the quality of life questionnaire improved at month 12 compared with baseline , with no differences between ism and ac patients . the locus of control questionnaire scores improved in both groups at month 12 , with a greater improvement in the chance domain in ism than in ac ( 0.92 [ sd , 0.59 ] vs. 0.10 [ sd , 0.57 ] ; p = 0.024 ) . there were no between - group differences regarding changes in systolic and diastolic blood pressures , estimated glomerular filtration rate , total cholesterol , hdl cholesterol , or ldl cholesterol from baseline to study end . in this multicenter , randomized , clinical trial enrolling > 1,000 patients with relatively well - controlled , noninsulin - treated type 2 diabetes , we found that the use of intensive , structured smbg data by clinicians to optimize prescription of diabetes medications and by patients to modify their behaviors improved glycemic control and enabled significantly more ism patients to achieve clinically meaningful reductions of hba1c compared with discretionary , unstructured smbg data , which were available only to study patients . first , more ism than ac patients had their prescriptions of diabetes medication changed at each visit , suggesting that structured smbg data prompted clinicians to adjust therapy earlier and more intensively in contrast to the clinical inertia often seen in the management of patients with type 2 diabetes ( 3134 ) . second , the greater decrease of bmi in ism patients , although significant only in the pp population , may have contributed to improving glycemic control in these patients . we recognize that the between - group differences in hba1c reductions in our study may be perceived as being of modest magnitude ; however , one must consider that our patients had a baseline hba1c of 7.3% , which is close to the average hba1c of type 2 diabetic patients treated at diabetes clinics in italy ( 35 ) and lower than in most studies . moreover , because international medical organizations recommend an hba1c < 7.0% as the treatment goal in type 2 diabetes ( 1 ) , based on findings from the uk prospective diabetes study , which demonstrated that any reduction in hba1c reduces the risk of complications ( 37 ) , we believe that clinicians have an obligation to utilize treatment strategies in addition to medications , including structured smbg , to help patients achieve their glycemic goals to improve clinical outcomes . when we considered the proportion of patients who achieved clinically meaningful improvements in glycemic control ( i.e. , an hba1c reduction of > 0.3 , > 0.4 , or > 0.5% ) , the proportion was significantly greater when using intensive structured smbg . provided the growing concerns for the increased mortality in intensively managed type 2 diabetes ( 38,39 ) , the detection of asymptomatic hypoglycemia may be an additional advantage of structured smbg . furthermore , reductions in diabetes - specific locus of control chance domain scores suggest that ism patients were less likely than ac patients to attribute diabetes control to chance or fortune . as for the secondary outcomes of the study , ac patients had a greater decrease in triglycerides than ism patients , with no significant between - group differences in total cholesterol , hdl cholesterol , ldl cholesterol , and blood pressure . because patients were treated at diabetes clinics , it may be difficult to generalize our findings to patients treated in primary practice settings who generally have less well - controlled diabetes . additionally , the large number of patients excluded from the pp analysis also makes generalization somewhat challenging , suggesting that the smbg regimen may have been too intensive or that a sizable proportion of noninsulin - treated type 2 diabetic patients may need additional support to comply with structured smbg . although smbg data from ac patients were not made available to clinicians for use in evaluating glycemic status or in making medication adjustments , the availability of these data to ac patients may have prompted changes in lifestyle behaviors or treatment adherence , potentially leading to improved glycemic control independent of clinician - based recommendations , especially in those patients who tested more frequently than the protocol allowed . recent studies have shown that use of structured smbg regimens positively influences patient behaviors , leading to improvements in glycemic control and other measures ( 811,18 ) . furthermore , although lbgi and hbgi have been validated in patients with type 1 diabetes and insulin - treated type 2 diabetes , they have not been validated in type 2 diabetic patients treated with diet or oral diabetes medications . the modest within - group changes seen in our study suggest that these indices may not be particularly useful when studying well - controlled noninsulin - treated patients . additional analyses of our results are being conducted to evaluate the impact of structured smbg on changes in dose , changes in type of medication prescribed , and appropriateness of these changes . in conclusion , the prisma ( prospective , randomized trial on intensive self - monitoring blood glucose management added value in noninsulin - treated type 2 diabetes mellitus patients ) study , to our knowledge the largest study of the effects of smbg in patients with type 2 diabetes , confirms the clinical usefulness and overall safety of using structured smbg to provide guidance in the prescription of diabetes medications and lifestyle changes in noninsulin - treated type 2 diabetic patients , ultimately improving glycemic control .

rf - pegs ( fluoroalkyl double - ended poly(ethylene glycol ) ) belong to a class of biocompatible and biodegradable fluorinated polymers [ 14 ] . micelles formed by rf - pegs in water consist of a hydrophobic fluorocarbon core ( rf core ) shielded by a large hydrophilic poly(ethylene glycol ) shell ( peg shell ) . certain combinations of the peg chain length and fluorocarbon size lead to sol gel two - phase coexistence and low surface erosion in water due to the high degree of cross linking of the rf - peg chains between the micelles . these properties make the rf - peg potentially useful as a drug delivery depot for controlled and sustained drug delivery [ 5 , 6 ] . previously , we have used f t1 relaxation nuclear magnetic resonance ( nmr ) and variable temperature - dependent epr to study the rf - peg s property to encapsulate hydrophobic drugs by employing a probe molecule made of free radical labeled anticancer drug , chlorambucil - tempol adduct [ 4 , 7 , 8 ] . further insight into the structure of the drug - micelle assembly was obtained through molecular dynamics simulations from these studies , we also learned that the ipdu ( isophorone diurethane ) units which were used to link the fluoroalcohol to the peg chain form the hydrophobic intermediate layer ( ipdu intermediate layer ) under aqueous conditions in addition to the already known rf core and peg shell . the ipdu intermediate layer is lipophilic in nature , and is able to hold lipophilic / hydrophobic drugs . in this study , we have used molecular diffusion nmr and f spin diffusion nmr to study the drug loading and diffusion properties of the rf - peg hydrogel for a small hydrophilic anticancer drug and its hydrophobic analog . pulsed field gradient spin echo ( pfgse ) nmr employs a spin echo pulse sequence together with pulsed field gradients to analyze the diffusion and other translational motion of molecules in a wide variety of media [ 912 ] . one of the first studies to focus on the diffusion of molecules in colloidal systems dates back to 1968 , and was conducted by tanner and stejskal . in their study , one artificial system of thin liquid layers ( mica stack ) , three different kinds of plant cells ( yeast , apple , and tobacco pith ) , and useful information regarding the model of diffusion was obtained through analyses of equations for restricted diffusion . many groups have since carried out more diffusion experiments for the studies of hydrogels [ 14 , 15 ] , protein binding polymer chain motion in gels , surfactants [ 18 , 19 ] , oil water emulsions [ 2022 ] , and separations . to circumvent the short t2 time in viscous solutions , the pulsed field gradient stimulated echo ( pfgste ) pulse sequence was developed [ 9 , 24 , 25 ] . compared with the pfgse , pfgste pulse sequence makes use of the possibly longer t1 time to observe the nmr signal in viscous media for a longer time . another pulse sequence referred to as convex ( convection compensation / excitation sculpting ) has been used to study the diffusion of solute molecules in pva hydrogels . this pulse sequence includes a water suppression step to avoid the interruption by the water signal during the diffusion measurement . many other variations of nmr diffusion techniques [ 9 , 20 , 22 , 27 ] and theoretical models have also been developed to treat hindered diffusion and restricted diffusion including diffusion in a sphere and a cylinder [ 22 , 28 , 29 ] . for instance , garasanin et al . examined the diffusion of the poly(dimethylsiloxane ) ( pdms ) fluid , its restricted diffusion inside the emulsion droplets and the brownian diffusion of the droplets . time - dependent diffusion nmr techniques were also developed to study the inhomogeneous diffusion of probe molecules in cross linked polymer gels . the theory of solute particles undergoing restricted diffusion and the corresponding mathematical approximations useful in treating such systems have well been compiled in the review by price . a theoretical model to treat diffusion coupled with chemical exchanges was reviewed by momot and kuchel . a study of nmr line shapes due to diffusive exchange and relaxation process was also reported . to compliment the information obtained from molecular diffusion nmr , we also performed f spin diffusion nmr which utilizes the dipolar interactions of abundant nuclear spins to probe spin dipolar coupled networks . thus , molecular miscibility and domain size of spin coupled networks can be probed [ 33 , 34 ] . we have used 5-fluorouracil ( fu ) , and its hydrophobic analog , 1,3-dimethyl-5-fluorouracil ( dmfu ) , as examples of small molecules to study the property of the rf - peg hydrogel as a drug delivery depot . fu belongs to a group of anticancer drugs known as antimetabolites [ 35 , 36 ] . it is used for the treatment of colon cancer , rectal cancer , breast cancer , stomach cancer , and pancreatic cancer . dmfu is a hydrophobic analog of fu with both the ring amino groups being methylated . fu and dmfu contain both proton and fluorine groups which make it convenient to use both the h and the f nmr signals to study their drug loading and diffusion characters in the rf - peg hydrogel . 6kc6 rf - peg ( c6f13-ch2ch2o - ipdu - o-(ch2ch2o-)n - ipdu - och2ch2-f13c6 ) was synthesized as described in previous methods . the term 6kc6 refers to the peg with mw 6,000 daltons ( 6 k ) and the fluoroalcohol group with six fluorinated carbon atoms ( c6 ) . either side of the peg chain is linked to an rf group through the linker ipdu . fu and dmfu with purity greater than 99% were purchased from sigma aldrich ( usa ) , and their chemical structures are given in scheme 1 . all solvents used were of analytical reagent grade purchased from fischer scientific ( usa ) . to prepare the gel samples , 100 mg of 6kc6 rf - peg and 5 mg of fu or dmfu were dissolved in 1 ml methylene chloride . then , 0.9 ml d2o was added to the mixture to make the 10% 6kc6 rf - peg hydrogel samples . the gel samples were stabilized at room temperature for more than 24 h before the nmr study . to study the drug loading properties using solid state f spin diffusion nmr , the fu and dmfu loaded hydrogel samples were freeze - dried with a labconco inc . scheme 1chemical structures of fu and dmfu chemical structures of fu and dmfu the diffusion nmr experiments were carried out with a bruker avance 600 mhz system equipped with a 5-mm diff30 water - cooled diffusion probe and a gradient amplifier capable of achieving gradient strength up to 1,200 g / cm . h pfgste nmr and pfgse nmr sequences [ 11 , 12 ] were used . the pfgse experiments were carried out with = 20 ms and = 1 ms , where denotes the gradient duration time and the distance between the two front edges of the gradient pulses . the echo intensity for each experiment was attenuated by varying the gradient strength g from 0 to 1,200 g / cm . all the signal intensities relative to the corresponding g = 0 ones were used to remove the t2 effects on the nmr signal intensities . molecular diffusions of fu and dmfu in the 6kc6 rf - peg hydrogel were observed through acquiring h nmr signal , and the diffusions of the rf - peg chains were observed through acquiring h and f nmr signals . another kind of diffusion nmr experiment using the pfgste pulse sequence was also carried out in which the echo intensity was measured by varying while keeping all the other parameters including and g constant . the advantage of this technique arises from the ability to monitor the diffusion of the probe molecule for a much longer time by using a lower gradient strength . in this manner , we could see the hindrance effect of the probe molecule due to translational barriers . for short values , however , when is increased , this effect on the nmr signal intensity can show up by approaching nonzero echo intensity while zero echo intensity is approached for free diffusion besides the variation of the diffusion curves according to the model of diffusion . in the experiment , was varied from 0 to 200 ms while the gradient strength was fixed at 75 g / cm and at 1 ms . to eliminate the t1 and t2 effects on the echoed nmr signal intensities , the signal intensity for each experiment was measured by alternating the pulse sequences with and without the gradient pulses and the ratio of the signal intensities with and without the gradients for each value samples used for this experiment include fu in d2o , fu in 6k peg d2o , fu in the 6kc6 rf - peg hydrogel and dmfu in the 6kc6 rf - peg hydrogel . in order to evaluate the locations of fu and dmfu in the rf - peg hydrogel , we carried out a f solid state spin diffusion nmr experiment for the freeze - dried fu and dmfu hydrogel samples . the drug molecules enclosed in the rf core / ipdu intermediate - layer region may show f cross peaks with the peaks of the rf group in the two - dimensional spectrum . to avoid averaging out the f f dipolar coupling a doty xc4 h / f / x triple resonance 600 mhz solid state nmr probe was used . the two - dimensional spin diffusion pulse sequence with a mixing time of 100 ms and a recycling delay of 3 s was employed . results for the pfgse experiments of fu and dmfu in the rf - peg hydrogel are shown in fig . the data are shown by plotting the natural logarithms of the normalized echo intensities e = ig / i0 ( where ig is the attenuated nmr signal intensity by the pulsed field gradient and i0 is the intensity without the attenuation ) against q(/3 ) , where q = g. only the experiments for g = 0200 g / cm were run because the nmr signal intensities decayed to lower than 10% of the original signals with gradient strengths larger than 200 g / cm . the solution for a particle s free diffusion relating the relative echo intensity e and the diffusion coefficient d is given by : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \ln \left ( { e\left ( { \delta - { { \delta } \left/ { 3 } \right.},q } \right ) } \right ) = - { q^2}\left ( { { { { \delta - \delta } } \left/ { 3 } \right . } } \right)d $ $ \end{document}where is the nuclear gyromagnetic ratio , d is the diffusion coefficient , and q = g ( , g , and have been defined in the experimental section ) [ 11 , 12 ] . all the experimental points in fig . 1 fit eq . 1 approximately . this was , of course , done without giving a clear model of diffusion but just used as a convenient way to show the data . the calculated apparent diffusion coefficients are 6.76 0.09 10 and 3.31 0.08 10 ( cm / s ) for fu and dmfu , respectively . the apparent diffusion coefficient of fu is larger than that of dmfu in the 6kc6 rf - peg hydrogel . they may show the mixed effect of the diffusion coefficients for the drug molecules in the rf core / ipdu intermediate - layer region and in the peg the diffusive exchange of the drug molecules between the two regions may also contribute to the apparent diffusion coefficients . 1semilog plots of the relative nmr signal attenuations for the diffusions of a fu in the rf - peg hydrogel and b dmfu in the rf - peg hydrogel , and their corresponding theoretical fits to eq . 1 semilog plots of the relative nmr signal attenuations for the diffusions of a fu in the rf - peg hydrogel and b dmfu in the rf - peg hydrogel , and their corresponding theoretical fits to eq . 1 to gather more experimental data in order to find the boundary effect and the exchange effect between the probe molecules in the rf core / ipdu intermediate - layer region and in the peg water phase , pfgste diffusion experiments where was varied from 0 to 200 ms while the gradient was fixed at g = 75 g / cm and = 1 ms were carried out . the results are shown in fig . 2 , where ( /3 ) is used as the unit of the horizontal axis . besides the samples of fu in 6kc6 hydrogel and dmfu in 6kc6 hydrogel , experiments for fu in 6k peg solution and fu in d2o were also carried out for the purpose of comparison . all these sets of experimental data fit the following exponential decays well : fig . 2experimental nmr signal attenuations with diffusion time ( /3 ) for dmfu in 6kc6 ( squares ) , fu in 6kc6 ( diamonds ) , fu in 6k peg ( upright triangle ) , and fu in d2o ( inverted triangles ) . the corresponding lines show the data fitting with the first - order exponential decays as shown in eq . 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e\left ( { \delta - { { \delta } \left/ { 3 } \right . } } \right ) = { y_0 } + { a_1}\exp \left [ { - { { { \left ( { \delta - { { \delta } \left/ { 3 } \right . } } \right ) } } \left/ { { { t_1 } } } \right . } } \right ] $ $ \end{document } experimental nmr signal attenuations with diffusion time ( /3 ) for dmfu in 6kc6 ( squares ) , fu in 6kc6 ( diamonds ) , fu in 6k peg ( upright triangle ) , and fu in d2o ( inverted triangles ) . the corresponding lines show the data fitting with the first - order exponential decays as shown in eq . 2 the corresponding parameters of y0 , a1 , and t1 are listed in table 1 . further discussions for the meanings of these parameters will be conducted in the discussion section . table 1parameters calculated using eq . 2 for the experimental curves in fig . 2fu in d2ofu in peg solutionfu in rf - peg hydrogeldmfu in rf - peg hydrogely00.000 0.0020.019 0.0030.078 0.0060.21 0.01a11.007 0.0040.976 0.0040.92 0.010.79 0.01t1 ( ms)40.5 0.451.4 0.638 148 2 parameters calculated using eq . 2 for the experimental curves in fig . 2 in order to evaluate the diffusion of the polymeric chains of the rf - peg itself , and thus their effects on the observed diffusions of fu and dmfu in the rf - peg hydrogels , we carried out h and f molecular diffusion nmr experiments on a sample of the rf - peg hydrogel ( 6kc6 ) without any probe molecules loaded in it . we found that even by using the maximum possible gradient ( 1,200 g / cm ) , it was hard to get any significant signal attenuations to estimate the diffusion coefficients for the rf group and the peg chains . this indicates that the hydrogel networks are quite inflexible arising from the formation of the cross linked network through the rf - peg chains . therefore , we can consider the polymeric chains as being fixed in the hydrogel while only the drug molecules did the translational motions in the hydrogel . to probe the locations of fu and dmfu in the rf - peg hydrogel , we carried out a two - dimensional ( 2d ) solid state f spin diffusion nmr experiments for the freeze - dried fu and dmfu 6kc6 rf - peg hydrogel samples , respectively . in the 2d nmr spectrum , cross - peaks appear if two nuclei are in proximity ( within some angstroms ) to each other . thus if fu or dmfu was encapsulated into the rf core / ipdu intermediate - layer region , we may see cross peaks between the peaks of the drug fluorine groups and the rf groups . otherwise , no cross peak would be observed . of course , the appearance and the intensities of the cross peaks depend on the length of the mixing time , the closeness of the atoms and the motion of the molecules . the terminal cf3 groups of the rf core appear at 81 ppm and those of the cf2 chains range from 122 to 168 ppm . the chemical shifts of the f in fu and dmfu appear at 178 and 172 ppm , respectively . the peaks in the 2d spectra are intrinsically broad due to the static condition of the samples which does not allow the dipolar interaction to be averaged out in order to detect the spin diffusions . in fig . 3a , cross peaks only appear between the cf2 and cf3f diagonal peaks , but no noticeable cross peak between the fu f peak and those of the rf groups was observed . when turned to the case of dmfu ( fig . 3b ) , in addition to the cross peaks between rf cf2 and rf cf3 , the f nucleus of dmfu also shows cross peaks with the cf2 and cf3f peaks . these spectra indicate that there was a significant portion of dmfu molecules in close contact with the rf core unit , while the amount of fu molecules in close contact with the rf core units might be much lower . 32d f spin diffusion spectra of the freeze - dried a fu in rf - peg hydrogel and b dmfu in rf - peg hydrogel 2d f spin diffusion spectra of the freeze - dried a fu in rf - peg hydrogel and b dmfu in rf - peg hydrogel figure 1 showed that the apparent diffusion coefficient of fu is larger than that of dmfu in the rf - peg hydrogel . the diffusion coefficient for a dilute suspension of spherical particles is given by the stokes einstein equation 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d = rt/(6\pi r{n_{\rm{a}}}\eta ) $ $ \end{document}where rg denotes the gas constant , t is the temperature , r is the hydrodynamic radius of a spherical particle , na is avogadro s constant , and is the viscosity of the solution . this number should not represent the ratio of the hydrodynamic radii of fu to dmfu because , as shown in scheme 1 , the molecular size difference between fu and dmfu can not be so large even though they were not spherical and the effect of hydrations to the ratio were not considered . thus , the different diffusion coefficients suggest that fu and dmfu diffused with different kinetic parameters and/or models in the rf - peg hydrogel . this hypothesis is also supported by the different curves shown in fig . 2 and their corresponding parameters in table 1 . the larger y0 of dmfu could indicate that the diffusion of dmfu was more hindered than fu . 3 have actually shown that more dmfu resided in or was in close contact with the rf core unit than fu . in order to evaluate the hindrance effect on the drug s diffusions in the rf - peg micelle , we have first reviewed the theoretical models of restricted diffusion [ 9 , 10 , 13 , 20 , 22 , 29 ] . starting to get some understanding into the possible model of diffusion , we first employed the simplest model used to treat diffusion in a reflecting sphere [ 9 , 20 , 22 ] . in this model , when the solute is in contact with a reflecting sphere or boundary , the spin is neither transported through the boundary , nor is it relaxed by the boundary . the most common approximations for this kind of diffusion include the short gradient pulse ( sgp ) and gaussian phase distribution ( gpd ) [ 9 , 13 , 39 ] . under the gpd approximation , the signal attenuation is given as an expression including the summation of the mth root of bessel equation [ 9 , 22 ] . in the condition of diffusion for a long period ( d>>r ) , this equation is reduced to the following form : 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e = \exp \left [ { { { { - { q^2}{r^2 } } } \left/ { 5 } \right . } } \right ] $ $ \end{document}where r denotes the maximum distance that a molecule can diffuse . the nmr signal attenuation is independent on but decreases significantly with the increase of r. using eq . 4 and the y0 values in table 1 , we could obtain that the maximum distances for that fu and dmfu diffused in the rf - peg hydrogel were 17.8 and 13.9 m , respectively . of course , these results are by no means right because the boundary condition between the rf core / ipdu intermediate - layer region and the peg water phase does not satisfy that defined for the restricted diffusion . from our previous study , we have learned that the diameter of the ipdu intermediate layer is about 25 in an anhydrous condition . if the diffusion of dmfu was heavily restricted within the rf core / ipdu intermediate layer , we would have seen a much severer restriction according to eq . thus , besides diffusion within the rf core / ipdu intermediate - layer region , dmfu should also have undergone longer range diffusion in the peg water phase . because dmfu is much more hydrophobic than fu , more dmfu molecules could have resided in the rf core / ipdu intermediate - layer region than the fu . ( this idea has experimentally been proven by the f spin diffusion spectra in fig . 3 . ) fu should also have behaved more or less like dmfu , but with a lesser population in the rf core / ipdu intermediate - layer region due its higher hydrophilicity . the boundary condition between the two regions could not isolate the drug molecules in each of the regions . thus , besides the diffusion in each of the regions , there should also be diffusive exchanges for the dmfu and the fu between the rf core / ipdu intermediate - layer region and the peg water phase . in synopsis , the model of diffusion slowly diffuse ( the diffusion is more hindered ) in the rf core / ipdu intermediate - layer region ( seen as a smaller compartment ) and part of them shown as a diffuse in the peg water phase ( seen as a larger compartment ) with more rapid diffusion rates . the reason for the more hindered diffusion of b is attributed to the relative immobility of the rf chains and the ipdu units . 4model of diffusion of dmfu and fu in the rf - peg hydrogel where the relative size of the peg water phase ( or region ) has been scaled down significantly relative to the rf core / ipdu intermediate - layer region . water phase and b ( gray dot ) represents fu or dmfu in the rf core / ipdu intermediate - layer region . the diffusive exchange rate constants are given as k+ and k for a to b and b to a , respectively model of diffusion of dmfu and fu in the rf - peg hydrogel where the relative size of the peg water phase ( or region ) has been scaled down significantly relative to the rf core / ipdu intermediate - layer region . a ( black dot ) represents fu or dmfu in the peg water phase and b ( gray dot ) represents fu or dmfu in the rf core / ipdu intermediate - layer region . the diffusive exchange rate constants are given as k+ and k for a to b and b to a , respectively to theoretically describe our model of diffusion , the diffusion equation should include ( 1 ) the hindered / slow diffusion of the drug molecules in the rf core / ipdu intermediate - layer region ; ( 2 ) the relatively free / fast diffusion of the drug molecules in the peg water phase ( although strictly to say , the peg water phase is not an ideally free phase ) ; and ( 3 ) the diffusive exchange between the two regions on the boundary ( or the interface ) of the two regions . bloch equations for the motions of the macroscopic nuclear magnetizations including the effect of diffusion and diffusive exchange under the time dependent pulsed field gradient can be written as [ 9 , 31 , 40 , 41 ] : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\ { \begin{gathered } \frac{{\partial { { \vec{m}}_{\rm{a}}}(\vec{r},t)}}{{\partial t } } = \gamma { { \vec{m}}_{\rm{a } } } \times \vec{b}(\vec{r},t ) - \frac{{{m_{{\rm{a}}x}}\vec{i } + { m_{{\rm{a}}y}}\vec{j}}}{{{t_2 } } } - \frac{{({m_{{\rm{a}}z } } - { m_{{\rm{a}}0}})\vec{k}}}{{{t_1 } } } + { d_{\rm{a}}}{\nabla^2}{{\vec{m}}_{\rm{a } } } - { k_{+ } } { { \vec{m}}_{\rm{a } } } + { k_{- } } { m_{\rm{b } } } \hfill \\\frac{{\partial { { \vec{m}}_{\rm{b}}}(\vec{r},t)}}{{\partial t } } = \gamma { { \vec{m}}_{\rm{b } } } \times \vec{b}(\vec{r},t ) - \frac{{{m_{{\rm{b}}x}}\vec{i } + { m_{{\rm{b}}y}}\vec{j}}}{{{t_2 } } } - \frac{{({m_{{\rm{b}}z } } - { m_{{\rm{b}}0}})\vec{k}}}{{{t_1 } } } + { d_{\rm{b}}}{\nabla^2}{{\vec{m}}_{\rm{b } } } + { k_{+ } } { { \vec{m}}_{\rm{a } } } - { k_{- } } { m_{\rm{b } } } \hfill \\\end{gathered } \right . $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \vec{m}_{\rm{a } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \vec{m}_{\rm{b } } } $ $ \end{document } denote the magnetization of the probe molecules ( dmfu or fu ) in the peg water phase and in the rf core / ipdu intermediate - layer region , respectively , k+ and k denotes the forward and reverse exchange rate constants for a to b , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vec{b}(\vec{r},t ) $ $ \end{document } denotes the magnetic field including the static magnetic field and the time dependent pulsed field gradient field . the nmr line shapes resulting from the bloch equations where no time - dependent pulsed field gradient is included have been treated by belton and hills . their theoretical treatment considers how the line shape is changed due to the relaxation effect on the boundary and the diffusive exchange between the two compartments of their model system . their treatment relies on the distinguished chemical shifts of the probe molecules in the two compartments . the distinguished chemical shifts , however , rarely exist for systems without selectively adding a paramagnetic relaxation reagent . here , we use the pulsed field gradient technique to attenuate the nmr signal intensities in order to probe the properties of diffusion for our system . the chemical shift distinction of the nmr signals of the two components is not necessary using this method . the f molecular diffusion experiment has shown that the rf core was quite immobile in space . thus , after the drug molecules entered the rf core / ipdu intermediate - layer region , their translational freedom can be treated solely as the diffusion of the drug molecules in this region . in addition , the drug s diffusion in this region was hindered due to the relative immobility of the rf chains and the ipdu units . in contrast to the much smaller size of the rf core / ipdu intermediate - layer region , as a suitable approximation , the probe molecules in the peg water phase can diffuse in a much longer range and the diffusion is much less hindered . it would be complicated to find the theoretical solution ( more accurately to say approximation ) to eq . thus , we will discuss a few approximations specifically pertaining to the rf - peg hydrogel system . furthermore , all the relaxation effects in these approximations will not be included as they have been removed in the signal intensities as shown in figs . 1 and 2 . assuming that the diffusions of both a and b were free in each of the regions without any diffusive exchange , then eq . 5 will be reduced to two independent bloch equations for a and b , respectively . the solution of each of them is an exponential decay function . because the nmr signals of a and b are indistinguishable , the solution for eq . 5 would be described by the sum of their two double exponential decays : 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e\left ( { \delta - { { \delta } \left/ { 3 } \right.},q } \right ) = { e_a}(0)\exp \left [ { - { q^2}\left ( { \delta - { { \delta } \left/ { 3 } \right . } } \right){d_a } } \right ] + { e_b}(0)\exp \left [ { - { q^2}\left ( { \delta - { { \delta } \left/ { 3 } \right . } } \right){d_b } } \right ] $ $ \end{document } this , of course , does not represent the empirical result as shown in eq . 2 , where only one exponential decay appears . water phase , eq . 6 will be reduced to a single exponential decay ( the first term ) . if all of the drug molecules were restricted in the rf core / ipdu intermediate - layer region , at a long diffusion duration ( d>>r ) , the gpd approximation would result in the solution as shown in eq . the equation to describe the diffusions of the combination of the two cases without any diffusive exchange between the two regions would be : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e\left ( { \delta - { { \delta } \left/ { { 3,q } } \right . } } \right ) = { e_a}(0)\exp \left [ { - { q^2}\left ( { \delta - { { \delta } \left/ { 3 } \right . } } \right){d_a } } \right ] + { e_b}(0)\exp \left [ { \left . { { { { - { q^2}{r^2 } } } \left/ { 5 } \right . } } \right ) } \right ] $ $ \end{document}where ea(0 ) and eb(0 ) represent the normalized signal intensities ( i.e. , ea(0 ) + eb(0 ) = 1 ) corresponding to the drug s populations in the two regions . since r is very small for the rf core / ipdu intermediate - layer region , the second term is approximately equal to be eb(0 ) . thus , eq . 7 becomes 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e\left ( { \delta - { { \delta } \left/ { 3 } \right.},q } \right ) = { e_a}(0)\exp \left [ { - { q^2}\left ( { \delta - { { \delta } \left/ { 3 } \right . } } \right){d_a } } \right ] + { e_b}(0 ) $ $ \end{document } this could also represent the approximation , if the diffusion in the rf core / ipdu intermediate - layer region is very slow . this equation represents the empirical solution of eq . 2 . bear in mind that until this point , we have assumed that the diffusive exchange would not have existed . however , for diffusion limited exchange , if the diffusion coefficient db is much smaller than da and thus the exchange is very slow , this solution is a fair approximation for eq . it has been known that for the case of free diffusions of a and b in one phase with the existence of molecular exchange between them , the solution for eq . 5 is : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } e(\delta - \delta /3,q ) = { e_{\rm{a}}}(\delta - \delta /3,q ) + { e_{\rm{b}}}(\delta - \delta /3,q ) \\= - { e_{\rm{a}}}(0)\frac{{{{({k^{+ } } ) } ^2 } + ( { k^{- } } ) { d^{- } } { q^2 } - { k^{+ } } \omega } } { { 2{k_{- } } \omega } } \exp \left [ { - ( \delta - \delta /3)\frac{1}{2}\left ( { { k^{+ } } + { d^{+ } } { q^2 } + \omega } \right ) } \right ] \\+ { e_{\rm{b}}}(0)\frac{{{{({k^{+ } } ) } ^2 } + ( { k^{- } } ) { d^{- } } { q^2 } + { k^{+ } } \omega } } { { 2{k_{- } } \omega } } \exp \left [ { - ( \delta - \delta /3)\frac{1}{2}\left ( { { k^{+ } } + { d^{+ } } { q^2 } - \omega } \right ) } \right ] \\\end{gathered } $ $ \end{document}where k = k+ + k , k = k+k- , q = g , d = da + db , d = dadb and = [ ( k + qd ) + 4k+k ] . plot of e( /3,q ) versus ( /3 ) would show the mixed double exponential decays at a short diffusion time . with a longer diffusion time , the curve would tend to show the single exponential decay for the component with slow diffusion . this case can be related to our system by making the following conditions : ( 1 ) b is carried by the rf core / ipdu intermediate - layer region in the peg water phase ; ( 2 ) the diffusion of the rf core / ipdu intermediate - layer region is zero ( which has been shown by the f diffusion experiment ) ; ( 3 ) the diffusion of b in the rf core / ipdu intermediate - layer region is slow due to the relative immobility of the rf chains and the ipdu units ; ( 4 ) the diffusive exchange between a and b exists . ( 9 ) becomes 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } e(\delta - \delta /3,q ) = - { e_{\rm{a}}}(0)\frac{{{{({k^{+ } } ) } ^2 } + ( { k^{- } } ) { d_{\rm{a}}}{q^2 } - { k^{+ } } \omega } } { { 2{k_{- } } \omega } } \exp \left [ { - ( \delta - \delta /3)\frac{1}{2}({k^{+ } } + { d_{\rm{a}}}{q^2 } + \omega ) } \right ] \\+ { e_{\rm{b}}}(0)\frac{{{{({k^{+ } } ) } ^2 } + ( { k^{- } } ) { d_{\rm{a}}}{q^2 } + { k^{+ } } \omega } } { { 2{k_{- } } \omega } } \exp \left [ { - ( \delta - \delta /3)\frac{1}{2}({k^{+ } } + { d_{\rm{a}}}{q^2 } - \omega ) } \right ] \\\end{gathered } $ $ \end{document}where = [ ( k + qda ) + 4k+k- ] . if 4k+k in is small and thus can be ignored , then eq . 10 becomes 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } e(\delta - \delta /3,q ) = - { e_{\rm{a}}}(0)\frac{{{{({k^{+ } } ) } ^2 } + ( { k^{- } } ) { d_{\rm{a}}}{q^2 } - { k^{+ } } \omega } } { { 2{k_{- } } \omega } } \exp \left [ { - ( \delta - \delta /3)({k_{+ } } + { d_{\rm{a}}}{q^2 } ) } \right ] \\+ { e_{\rm{b}}}(0)\frac{{{{({k^{+ } } ) } ^2 } + ( { k^{- } } ) { d_{\rm{a}}}{q^2 } + { k^{+ } } \omega } } { { 2{k_{- } } \omega } } \exp [ - ( \delta - \delta /3){k_{- } } ] \\\end{gathered } $ $ \end{document } the second term decays much slower than the first term due to the vanished db . when k ( and also k+ due to the exchangeable equilibrium ) approaches zero , eq . ( 11 ) becomes 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e(\delta - \delta /3,q ) = { e_{\rm{a}}}(0)\exp \left [ { - ( \delta - \delta /3){d_{\rm{a}}}{q^2 } } \right ] + { e_{\rm{b}}}(0 ) $ $ \end{document } it is reasonable that the diffusion of the drug molecules in the rf core / ipdu intermediate - layer region is slow due to the relative immobility of the rf chains and the ipdu units . thus , if the diffusive exchange is diffusion limited for the drug molecules in the rf core / ipdu intermediate - layer region , the exchange rate constants are small . based on these arguments , eq . the exact case of the molecular diffusion in the rf - peg hydrogel , the diffusion coefficient of b may not be zero , although it should be small and also the rate constants of the diffusive exchange is not zero although they should be small as well . if the diffusive exchange rates were very rapid , the curve in fig . 2 for the dmfu s diffusion should approach to zero but not to a nonzero value . we would expect that the attenuation of the nmr signal intensity for drug s diffusion in the rf - peg hydrogel can be more accurately represented by the hybridization of eqs . , we are not going to pursue the more accurate solution further . comparing eq . 8 or eq . 12 with eq . 2 , we obtain eb(0 ) y0 = 0.21 for dmfu ( table 1 ) . this indicates that about 21% of the dmfu molecules were populated in the rf core / ipdu intermediate - layer region , while 79% of the dmfu molecules were populated in the peg similarly , 7.8% of the fu molecules were populated in the rf core / ipdu intermediate - layer region , while 82% of the molecules were populated in the peg fu is more hydrophilic than dmfu , and thus it is reasonable that more dmfu could have resided in the rf core / ipdu intermediate - layer region than fu . 3b did not show the cross peaks of fu with the rf groups , it does not necessarily mean that there were not fu in the rf core / ipdu intermediate - layer region . most of the 7.8% fu could reside in the ipdu intermediate layer . although the rf core / ipdu intermediate - layer region and the peg chains could also hinder the diffusions of the drug molecules in the peg water phase , the effect could be much smaller than the hindrance to the diffusion of the probe molecules in the rf core / ipdu intermediate - layer region . the y0 values of fu s diffusion in the peg solution ( y0 = 0.019 ) and in water ( y0 = 0.000 ) show that the hindrance effects of peg and water are quite small . for the current approximation , eq . 12 could be used to estimate the diffusion coefficients of dmfu and fu in the peg they were found to be 5.18 10 and 6.54 10 cm / s , respectively . 1 are 3.31 10 and 6.76 10 cm / s , respectively . because the value was kept small ( 20 ms ) for the experiments done for fig . 1 , which may not allow too much time for considerable diffusive exchange , the apparent diffusion coefficients calculated from fig . 1 should be closer to the true values than those calculated from fig . 2 using eq . 8 or eq . in summary , based on the results of molecular diffusion and solid state spin diffusion experiments , we have proposed the model of diffusion of small molecules in the rf - peg hydrogel . we have seen that a greater percentage of dmfu molecules resided in the rf core / ipdu intermediate - layer region than that of fu . water phase through the diffusive exchange process between the rf core / ipdu intermediate - layer region and the peg water phase . this provides a piece of useful information for drug delivery applications regarding the drug loading and diffusion properties of the rf - peg hydrogel . we would expect that small hydrophobic drugs can be held by the rf core / ipdu intermediate - layer region to a much greater extent than hydrophilic drugs and hydrophobic drugs may tend to degrade more from the gel surface to the body together with the rf - peg micelles . in contrast to hydrophobic drugs , small hydrophilic drugs tend to diffuse from the peg our previous studies on a larger hydrophobic drug , chlorambucil - tempol adduct , shows that the drug was encapsulated in the ipdu intermediate layer [ 4 , 7 ] . besides the drug loading capacity of the ipdu intermediate layer , the rf core may also play a role to host the dmfu molecules as can be seen from the spin diffusion spectrum in fig . the smaller molecular size , the hydrophobicity , and the presence of the fluorine atom in the molecule may have helped to do so . we have learned that the drug loading property in the rf - peg hydrogel is dependent on the compatibility of the hydrophobic core - layer region of the rf - peg hydrogel with the size , chemical composition , and hydrophobicity of the drugs . this opens the door to design particular polymer units to form the intermediate layer between the rf core and the peg shell for customized drug delivery . this study also demonstrates that the nmr techniques used in this research are useful to probe the loading and diffusion properties of drugs in polymeric micelles .

rf - peg ( fluoroalkyl double - ended poly(ethylene glycol ) ) hydrogel is potentially useful as a drug delivery depot due to its advanced properties of sol gel two - phase coexistence and low surface erosion . in this study , 1h molecular diffusion nuclear magnetic resonance ( nmr ) and 19f spin diffusion nmr were used to probe the drug loading and diffusion properties of the rf - peg hydrogel for small anticancer drugs , 5-fluorouracil ( fu ) and its hydrophobic analog , 1,3-dimethyl-5-fluorouracil ( dmfu ) . it was found that fu has a larger apparent diffusion coefficient than that of dmfu , and the diffusion of the latter was more hindered . the result of 19f spin diffusion nmr for the corresponding freeze - dried samples indicates that a larger portion of dmfu resided in the rf core / ipdu intermediate - layer region ( where ipdu refers to isophorone diurethane , as a linker to interconnect the rf group and the peg chain ) than that of fu while the opposite is true in the peg water phase . to understand the experimental data , a diffusion model was proposed to include : ( 1 ) hindered diffusion of the drug molecules in the rf core / ipdu - intermediate - layer region ; ( 2 ) relatively free diffusion of the drug molecules in the peg - water phase ( or region ) ; and ( 3 ) diffusive exchange of the probe molecules between the above two regions . this study also shows that molecular diffusion nmr combined with spin diffusion nmr is useful in studying the drug loading and diffusion properties in hydrogels for the purpose of drug delivery applications .

Introduction Experimental Results Discussion Conclusion

rf - pegs ( fluoroalkyl double - ended poly(ethylene glycol ) ) belong to a class of biocompatible and biodegradable fluorinated polymers [ 14 ] . certain combinations of the peg chain length and fluorocarbon size lead to sol gel two - phase coexistence and low surface erosion in water due to the high degree of cross linking of the rf - peg chains between the micelles . these properties make the rf - peg potentially useful as a drug delivery depot for controlled and sustained drug delivery [ 5 , 6 ] . previously , we have used f t1 relaxation nuclear magnetic resonance ( nmr ) and variable temperature - dependent epr to study the rf - peg s property to encapsulate hydrophobic drugs by employing a probe molecule made of free radical labeled anticancer drug , chlorambucil - tempol adduct [ 4 , 7 , 8 ] . further insight into the structure of the drug - micelle assembly was obtained through molecular dynamics simulations from these studies , we also learned that the ipdu ( isophorone diurethane ) units which were used to link the fluoroalcohol to the peg chain form the hydrophobic intermediate layer ( ipdu intermediate layer ) under aqueous conditions in addition to the already known rf core and peg shell . in this study , we have used molecular diffusion nmr and f spin diffusion nmr to study the drug loading and diffusion properties of the rf - peg hydrogel for a small hydrophilic anticancer drug and its hydrophobic analog . we have used 5-fluorouracil ( fu ) , and its hydrophobic analog , 1,3-dimethyl-5-fluorouracil ( dmfu ) , as examples of small molecules to study the property of the rf - peg hydrogel as a drug delivery depot . fu and dmfu contain both proton and fluorine groups which make it convenient to use both the h and the f nmr signals to study their drug loading and diffusion characters in the rf - peg hydrogel . to study the drug loading properties using solid state f spin diffusion nmr , the fu and dmfu loaded hydrogel samples were freeze - dried with a labconco inc . molecular diffusions of fu and dmfu in the 6kc6 rf - peg hydrogel were observed through acquiring h nmr signal , and the diffusions of the rf - peg chains were observed through acquiring h and f nmr signals . to eliminate the t1 and t2 effects on the echoed nmr signal intensities , the signal intensity for each experiment was measured by alternating the pulse sequences with and without the gradient pulses and the ratio of the signal intensities with and without the gradients for each value samples used for this experiment include fu in d2o , fu in 6k peg d2o , fu in the 6kc6 rf - peg hydrogel and dmfu in the 6kc6 rf - peg hydrogel . in order to evaluate the locations of fu and dmfu in the rf - peg hydrogel , we carried out a f solid state spin diffusion nmr experiment for the freeze - dried fu and dmfu hydrogel samples . the drug molecules enclosed in the rf core / ipdu intermediate - layer region may show f cross peaks with the peaks of the rf group in the two - dimensional spectrum . results for the pfgse experiments of fu and dmfu in the rf - peg hydrogel are shown in fig . the apparent diffusion coefficient of fu is larger than that of dmfu in the 6kc6 rf - peg hydrogel . they may show the mixed effect of the diffusion coefficients for the drug molecules in the rf core / ipdu intermediate - layer region and in the peg the diffusive exchange of the drug molecules between the two regions may also contribute to the apparent diffusion coefficients . 1semilog plots of the relative nmr signal attenuations for the diffusions of a fu in the rf - peg hydrogel and b dmfu in the rf - peg hydrogel , and their corresponding theoretical fits to eq . 1 semilog plots of the relative nmr signal attenuations for the diffusions of a fu in the rf - peg hydrogel and b dmfu in the rf - peg hydrogel , and their corresponding theoretical fits to eq . 1 to gather more experimental data in order to find the boundary effect and the exchange effect between the probe molecules in the rf core / ipdu intermediate - layer region and in the peg water phase , pfgste diffusion experiments where was varied from 0 to 200 ms while the gradient was fixed at g = 75 g / cm and = 1 ms were carried out . 2 in order to evaluate the diffusion of the polymeric chains of the rf - peg itself , and thus their effects on the observed diffusions of fu and dmfu in the rf - peg hydrogels , we carried out h and f molecular diffusion nmr experiments on a sample of the rf - peg hydrogel ( 6kc6 ) without any probe molecules loaded in it . we found that even by using the maximum possible gradient ( 1,200 g / cm ) , it was hard to get any significant signal attenuations to estimate the diffusion coefficients for the rf group and the peg chains . to probe the locations of fu and dmfu in the rf - peg hydrogel , we carried out a two - dimensional ( 2d ) solid state f spin diffusion nmr experiments for the freeze - dried fu and dmfu 6kc6 rf - peg hydrogel samples , respectively . thus if fu or dmfu was encapsulated into the rf core / ipdu intermediate - layer region , we may see cross peaks between the peaks of the drug fluorine groups and the rf groups . these spectra indicate that there was a significant portion of dmfu molecules in close contact with the rf core unit , while the amount of fu molecules in close contact with the rf core units might be much lower . 32d f spin diffusion spectra of the freeze - dried a fu in rf - peg hydrogel and b dmfu in rf - peg hydrogel 2d f spin diffusion spectra of the freeze - dried a fu in rf - peg hydrogel and b dmfu in rf - peg hydrogel figure 1 showed that the apparent diffusion coefficient of fu is larger than that of dmfu in the rf - peg hydrogel . thus , the different diffusion coefficients suggest that fu and dmfu diffused with different kinetic parameters and/or models in the rf - peg hydrogel . 4 and the y0 values in table 1 , we could obtain that the maximum distances for that fu and dmfu diffused in the rf - peg hydrogel were 17.8 and 13.9 m , respectively . of course , these results are by no means right because the boundary condition between the rf core / ipdu intermediate - layer region and the peg water phase does not satisfy that defined for the restricted diffusion . if the diffusion of dmfu was heavily restricted within the rf core / ipdu intermediate layer , we would have seen a much severer restriction according to eq . thus , besides diffusion within the rf core / ipdu intermediate - layer region , dmfu should also have undergone longer range diffusion in the peg water phase . because dmfu is much more hydrophobic than fu , more dmfu molecules could have resided in the rf core / ipdu intermediate - layer region than the fu . fu should also have behaved more or less like dmfu , but with a lesser population in the rf core / ipdu intermediate - layer region due its higher hydrophilicity . the boundary condition between the two regions could not isolate the drug molecules in each of the regions . thus , besides the diffusion in each of the regions , there should also be diffusive exchanges for the dmfu and the fu between the rf core / ipdu intermediate - layer region and the peg water phase . in synopsis , the model of diffusion slowly diffuse ( the diffusion is more hindered ) in the rf core / ipdu intermediate - layer region ( seen as a smaller compartment ) and part of them shown as a diffuse in the peg water phase ( seen as a larger compartment ) with more rapid diffusion rates . the reason for the more hindered diffusion of b is attributed to the relative immobility of the rf chains and the ipdu units . 4model of diffusion of dmfu and fu in the rf - peg hydrogel where the relative size of the peg water phase ( or region ) has been scaled down significantly relative to the rf core / ipdu intermediate - layer region . water phase and b ( gray dot ) represents fu or dmfu in the rf core / ipdu intermediate - layer region . the diffusive exchange rate constants are given as k+ and k for a to b and b to a , respectively model of diffusion of dmfu and fu in the rf - peg hydrogel where the relative size of the peg water phase ( or region ) has been scaled down significantly relative to the rf core / ipdu intermediate - layer region . a ( black dot ) represents fu or dmfu in the peg water phase and b ( gray dot ) represents fu or dmfu in the rf core / ipdu intermediate - layer region . the diffusive exchange rate constants are given as k+ and k for a to b and b to a , respectively to theoretically describe our model of diffusion , the diffusion equation should include ( 1 ) the hindered / slow diffusion of the drug molecules in the rf core / ipdu intermediate - layer region ; ( 2 ) the relatively free / fast diffusion of the drug molecules in the peg water phase ( although strictly to say , the peg water phase is not an ideally free phase ) ; and ( 3 ) the diffusive exchange between the two regions on the boundary ( or the interface ) of the two regions . $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \vec{m}_{\rm{a } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \vec{m}_{\rm{b } } } $ $ \end{document } denote the magnetization of the probe molecules ( dmfu or fu ) in the peg water phase and in the rf core / ipdu intermediate - layer region , respectively , k+ and k denotes the forward and reverse exchange rate constants for a to b , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vec{b}(\vec{r},t ) $ $ \end{document } denotes the magnetic field including the static magnetic field and the time dependent pulsed field gradient field . thus , after the drug molecules entered the rf core / ipdu intermediate - layer region , their translational freedom can be treated solely as the diffusion of the drug molecules in this region . in addition , the drug s diffusion in this region was hindered due to the relative immobility of the rf chains and the ipdu units . in contrast to the much smaller size of the rf core / ipdu intermediate - layer region , as a suitable approximation , the probe molecules in the peg water phase can diffuse in a much longer range and the diffusion is much less hindered . if all of the drug molecules were restricted in the rf core / ipdu intermediate - layer region , at a long diffusion duration ( d>>r ) , the gpd approximation would result in the solution as shown in eq . since r is very small for the rf core / ipdu intermediate - layer region , the second term is approximately equal to be eb(0 ) . } \right){d_a } } \right ] + { e_b}(0 ) $ $ \end{document } this could also represent the approximation , if the diffusion in the rf core / ipdu intermediate - layer region is very slow . this case can be related to our system by making the following conditions : ( 1 ) b is carried by the rf core / ipdu intermediate - layer region in the peg water phase ; ( 2 ) the diffusion of the rf core / ipdu intermediate - layer region is zero ( which has been shown by the f diffusion experiment ) ; ( 3 ) the diffusion of b in the rf core / ipdu intermediate - layer region is slow due to the relative immobility of the rf chains and the ipdu units ; ( 4 ) the diffusive exchange between a and b exists . ( 11 ) becomes 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e(\delta - \delta /3,q ) = { e_{\rm{a}}}(0)\exp \left [ { - ( \delta - \delta /3){d_{\rm{a}}}{q^2 } } \right ] + { e_{\rm{b}}}(0 ) $ $ \end{document } it is reasonable that the diffusion of the drug molecules in the rf core / ipdu intermediate - layer region is slow due to the relative immobility of the rf chains and the ipdu units . thus , if the diffusive exchange is diffusion limited for the drug molecules in the rf core / ipdu intermediate - layer region , the exchange rate constants are small . the exact case of the molecular diffusion in the rf - peg hydrogel , the diffusion coefficient of b may not be zero , although it should be small and also the rate constants of the diffusive exchange is not zero although they should be small as well . we would expect that the attenuation of the nmr signal intensity for drug s diffusion in the rf - peg hydrogel can be more accurately represented by the hybridization of eqs . this indicates that about 21% of the dmfu molecules were populated in the rf core / ipdu intermediate - layer region , while 79% of the dmfu molecules were populated in the peg similarly , 7.8% of the fu molecules were populated in the rf core / ipdu intermediate - layer region , while 82% of the molecules were populated in the peg fu is more hydrophilic than dmfu , and thus it is reasonable that more dmfu could have resided in the rf core / ipdu intermediate - layer region than fu . 3b did not show the cross peaks of fu with the rf groups , it does not necessarily mean that there were not fu in the rf core / ipdu intermediate - layer region . although the rf core / ipdu intermediate - layer region and the peg chains could also hinder the diffusions of the drug molecules in the peg water phase , the effect could be much smaller than the hindrance to the diffusion of the probe molecules in the rf core / ipdu intermediate - layer region . in summary , based on the results of molecular diffusion and solid state spin diffusion experiments , we have proposed the model of diffusion of small molecules in the rf - peg hydrogel . we have seen that a greater percentage of dmfu molecules resided in the rf core / ipdu intermediate - layer region than that of fu . water phase through the diffusive exchange process between the rf core / ipdu intermediate - layer region and the peg water phase . this provides a piece of useful information for drug delivery applications regarding the drug loading and diffusion properties of the rf - peg hydrogel . we would expect that small hydrophobic drugs can be held by the rf core / ipdu intermediate - layer region to a much greater extent than hydrophilic drugs and hydrophobic drugs may tend to degrade more from the gel surface to the body together with the rf - peg micelles . in contrast to hydrophobic drugs , small hydrophilic drugs tend to diffuse from the peg our previous studies on a larger hydrophobic drug , chlorambucil - tempol adduct , shows that the drug was encapsulated in the ipdu intermediate layer [ 4 , 7 ] . besides the drug loading capacity of the ipdu intermediate layer , the rf core may also play a role to host the dmfu molecules as can be seen from the spin diffusion spectrum in fig . we have learned that the drug loading property in the rf - peg hydrogel is dependent on the compatibility of the hydrophobic core - layer region of the rf - peg hydrogel with the size , chemical composition , and hydrophobicity of the drugs . this opens the door to design particular polymer units to form the intermediate layer between the rf core and the peg shell for customized drug delivery . this study also demonstrates that the nmr techniques used in this research are useful to probe the loading and diffusion properties of drugs in polymeric micelles .

trioxacarcins were first isolated in 1981 from the marine - derived micro - organism streptomyces bottropensis do-45 ( 1,2 ) . they are cytotoxic against various cancer cell lines , active against gram - positive and gram - negative bacteria and exhibit anti - malarial activity ( 3 ) . trioxacarcin a ( figure 1 ) contains a complex ring system that is attached to sugars at both ends , at the 4- and 13-positions , and causes it to exhibit intensive green fluorescence in solution ; in powder form it is yellow . nucleophilic attack of n7 of guanine opens the epoxide ( 1) to form a covalent bond to the guanine ( 2) in a dna molecule ( figure 2 ) . this alkylation is favored when a thymine is located on the 3-side of guanine and does not take place when the guanine is terminal ( 4 ) . cleavage of this trioxacarcindna ( 3) complex at 373 k results in the natural product gutingimycin ( 4) ( 3,5 ) , named after the ancient name for the city of gttingen , leaving an abasic dna . presumably , this cleavage takes place under milder conditions in vivo . figure 1.schematic diagram of trioxacarcin a. figure 2.proposed mechanism for the reaction of trioxacarcin a with dna . only the epoxide group of trioxacarcin a ( 1 ) is shown , the rest of the molecule is described by r1 and r2 . schematic diagram of trioxacarcin a. proposed mechanism for the reaction of trioxacarcin a with dna . only the epoxide group of trioxacarcin a ( 1 ) is shown , the rest of the molecule is described by r1 and r2 . anthracyclines resemble trioxacarcin a in that they also contain a planar aromatic ring system with one or more sugars attached to it , although they can only intercalate but not bind covalently to dna . some of them are used for the treatment of various cancer types , e.g. daunomycin (= daunorubicin ) against leukemia . in dna daunomycin complexes , the positively charged amino sugar of daunomycin is positioned in the minor groove of dna ( 6 ) . the anthracycline nogalamycin has bulky sugar residues at both ends of the molecule that interact with both grooves of dna ( 7 ) . pluramycin antibiotics ( 8) such as hedamycin ( 9 ) and altromycin b ( 10 ) , and also psorospermin ( 11 ) , are even more similar to trioxacarcin a because they contain in addition one or more epoxides and so can both intercalate and alkylate dna ; like trioxacarcin they bind covalently to the guanine n7 . however , no crystal structures have been reported of such covalent antibiotic dna complexes . to throw more light onto the stereochemical requirements for the covalent bond formation and subsequent elimination of a nucleobase , we have determined the structure of trioxacarin a covalently bound to an oligonucleotide to 1.78 resolution by x - ray diffraction . trioxacarcin a was obtained from the marine streptomyces sp . isolate b8652 by fermentation ( 12 ) . oligonucleotides were purchased already purified by hplc from biomers.net gmbh and used without further purification . crystals were grown at 40c in hanging drops by vapor diffusion ; we found by experiment that the higher than usual temperature produced better quality single crystals . the solution in the reservoir contained 1.55 m tri - ammonium citrate ( ph 7.0 ) and 30% v / v dmso . drug solution contained 2.5 mm dna ( single - strand concentration ) , 2.8 mm trioxacarcin a and 25% v / v methanol . drug solution was prepared by mixing trioxacarcin a stock solution ( containing 5.0 mm trioxacarcin a and 50% v / v methanol ) with a 5.6 mm dna solution in a 1:1 ratio at room temperature and incubation for 3 days at 4c . the hanging drops prepared from 1 l dna - drug solution and 2 l reservoir solution were equilibrated against 500 l reservoir solution . yellow tetragonal crystals grew within 24 h to a size of 0.2 0.1 0.05 mm . for data collection at 100 k in a nitrogen gas stream , the crystals were transferred to a cryosolution containing 1.55 m tri - ammonium citrate ( ph 7.0 ) , 30% v / v dmso and 15% v / v glycerol . three different oligonucleotides were used to obtain crystals as described above : native d(aaccggtt ) and brominated d(aaccggut ) and d(aaccggtu ) . in the brominated oligonucleotides thymidine the dna drug complex crystallized in space group p4122 with unit - cell a = b = 37.60 and c = 91.62 . data were collected at beamline 14.2 at bessy , berlin with a mar-165 ccd detector . the crystals containing brominated oligonucleotides were used for two br - mad experiments . from both crystals , peak and inflection datasets were collected , 180 frames for each dataset with a -rotation of 1 per frame ( see table 1 for crystallographic details ) . in order to avoid radiation damage , an 0.53 mm aluminium filter was used to decrease the intensity of the direct beam . in addition , a native dataset was collected to 1.78 resolution from a crystal containing the native oligonucleotide . table 1.data collection , phasing and refinement statisticsnatived(aaccggut)d(aaccggtu)crystal data space groupp4122p4122p4122 a , 37.6037.6037.56 c , 91.6291.2190.97peakinflectionpeakinflectiondiffraction data wavelength , 0.920390.920320.920470.920330.92042 resolution limit , 1.672.182.182.392.39 total reflections103 55245 88546 39236 33329 272 unique reflections82253789379029172756completeness , % overall99.089.798.798.893.4 outermost resolution shell94.789.398.590.759.4i/(i ) overall27.8113.214.613.814.8 outermost resolution shell2.924.54.22.71.5phasing resolution , 2.6 pseudo - free cc after dm0.61refinement reflections used6488 resolution , 1.78 rwork22.0 rfree26.5rms deviation bond length , 0.016 bond angles , 2.5average b factor ( all atoms ) , 53.16 data collection , phasing and refinement statistics the datasets were integrated with hkl2000 ( 13 ) and the space group determined by xprep ( bruker axs , madison wi , usa ) . shelxd ( 14 ) was used for substructure solution by searching for two bromine atoms with a resolution cutoff at 2.6 . shelxe ( 15 ) was employed for phasing and density modification . in the experimental map , the position of thymine could be deduced from the bromine positions from the substructure solution and the anomalous maps calculated using shelxe . it was also possible to recognize the drug in the experimental map ( figure 3a ) . the structure was refined isotropically with tls constraints against the native dataset with refmac ( 17 ) to rwork = 22.0% and rfree = 26.5% ( figure 3b ) . figures 3 and 57 were generated using pymol ( delano scientific llc , south san francisco ca , usa ) . figure 3.experimental map ( a ) and map after final refinement ( b ) , both contoured at a 1 level . the carbon atoms of the trioxacarcin are shown in light orange . experimental map ( a ) and map after final refinement ( b ) , both contoured at a 1 level . as predicted in the study of the sequence specificity ( 4 ) , trioxacarcin a binds covalently to d(aaccggtt ) by alkylating the n7 position of the guanine that is followed by a thymine . unexpectedly , this thymine is flipped out of the duplex dna and the adenine originally paired with it now forms a base pair with the following thymine in the sequence . the aromatic rings a and b of the drug are involved in stacking interactions with the dna . as found for nogalamycin ( 7 ) , trioxacarcin a interacts with both grooves of the dna ( figure 5 ) ; the 4-sugar is positioned in the minor groove , the 13-sugar in the major groove . figure 4.simplified view of the dna trioxacarcin duplex with residue names trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . on the bottom is the minor groove enclosing the 4-sugar , on the bottom the major groove containing the 13-sugar ( left side ) and the two 16-methoxy groups ( right - side ) . on the extreme upper left - side , the terminal a(101 ) trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . on the bottom is the minor groove enclosing the 4-sugar , on the bottom the major groove containing the 13-sugar ( left side ) and the two 16-methoxy groups ( right - side ) . on the extreme upper left - side , the terminal a(101 ) can be seen . since the duplex does not lie on a 2-fold axis , the two self - complementary strands are crystallographically independent , with some small structural differences caused by interactions of the residues a(1 ) , a(101 ) , t(7 ) and t(107 ) , which are no longer base - paired within the duplex , with different symmetry related residues in the crystal . residue a(101 ) does not appear to make specific contacts to neighboring molecules and is highly disordered , whereas n1 of a(1 ) accepts an hydrogen bond ( 2.7 ) from a symmetry equivalent of 4-oh of trioxacarcin(109 ) . the general conformations of both strands are fairly similar except for residues a(1 ) and a(101 ) that are positioned at the 5-terminus of the oligonucleotide and do not base pair within the duplex . the dna trioxacarcin duplex shows a distorted b - dna geometry with watson crick base pairing . in table 2 , the sugar - phosphate and glycosyl torsion angles are compared with the usual ranges and mean values for b - dna ( 19 ) . the flipping - out of t(7 ) is most evident in its torsion angle of 82 [ 74 for t(107 ) ] that differs by about 180 from the standard value . the other torsion angles all lie in ranges typically observed in b - dna structures . table 2.backbone and glycosyl torsion anglesangle , degreesresiduea2 ( next to intercalation)28217660155254280209a10230818050155251270224c3 ( next to intercalation)28817046150206192276c10328715752147218188274c430814953133168265240c10429813956134183238242g530719147151180262262g10531118148149181268259g6 ( next to intercalation)29318351144238264273g10630318546141233267270t7 ( flipped - out)3021596114725182252t1072811846916125474258t8 ( next to intercalation)27617251143232t10823618481130221b - dna ( range)270330130200208070180160270150300200300b - dna ( mean)298176 ( i)48128 ( i)184 ( i)265 ( i)258 ( a / g)146 ( ii)144 ( ii)246 ( ii)174 ( ii)241 ( t / c)271 ( ii)torsion angles are defined as : o3 * -p - o5 * -c5 * , : p - o5 * -c5 * -c4 * , : o5 * -c5 * -c4 * -c3 * , : c5 * -c4 * -c3 * -o3 * , : c4 * -c3 * -o3 * -p , : c3 * -o3 * -p - o5 * , (purines ) : o4 * -c1 * -n9-c4 , (pyrimidines ) : o4 * -c1 * -n1-c2 . for the mean values bi and bii conformations are distinguished , in the case of the glycosyl torsion angle also purines and pyrimidines for the bi conformation . backbone and glycosyl torsion angles torsion angles are defined as : o3 * -p - o5 * -c5 * , : p - o5 * -c5 * -c4 * , : o5 * -c5 * -c4 * -c3 * , : c5 * -c4 * -c3 * -o3 * , : c4 * -c3 * -o3 * -p , : c3 * -o3 * -p - o5 * , (purines ) : o4 * -c1 * -n9-c4 , (pyrimidines ) : o4 * -c1 * -n1-c2 . for the mean values bi and bii conformations are distinguished , in the case of the glycosyl torsion angle also purines and pyrimidines for the bi conformation . in addition to the covalent bond , trioxacarcin a forms direct and water mediated hydrogen bonds with the dna ( figure 6 ) . there is an hydrogen bond between the 2-oh of trioxacarcin and o4 of the deoxyribose attached to the cytosine opposite to the alkylated guanine [ 2.8 for trioxacarcin(9 ) and 2.7 for trioxacarcin(109 ) ] . in the minor groove , n2 of the alkylated guanine donates an hydrogen bond to 3-oh of the 4-sugar of trioxacarcin ( 3.1 for both trioxacarcins ) . there is also a water - mediated hydrogen bond between n3 of the alkylated guanine and 3-oh . the 4-sugar interacts only with the alkylated guanine . in the major groove , the 1-oxygen of the 13-sugar of trioxacarcin(109 ) accepts an hydrogen bond ( 3.2 ) from n6 of residue a(2 ) . in the case of trioxacarcin(9 ) and a(102 ) , the corresponding hydrogen bond is mediated by a water molecule , giving rise to a small difference between the two strands . the 13-sugar is also involved in an internal hydrogen bond [ 3.4 for trioxacarcin(9 ) and 3.1 for trioxacarcin(109 ) ] between 3-oh and 14-oh ( figure 6 ) , stabilizing the oh - group formed by the nucleophilic attack on the epoxide ring . the 13-sugar of the trioxacarcin is held parallel to the trioxacarcin chromophore by this internal hydrogen bond , whereas the 4-sugar is orientated perpendicular to it . base pair g(105)-c(4 ) is at the top , followed by base pair g(106)-c(3 ) . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . base pair g(105)-c(4 ) is at the top , followed by base pair g(106)-c(3 ) . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . hydrogen bonds are indicated with dashed lines . for the formation of gutingimycin , the guanine sugar bond between n9 and c1 ( figure 7a ) has to break and the antibiotic has to leave the dna duplex taking the guanine with it . sugar bond breaks whilst the double strand is still intact , gutingimycin would then only be attached to the abasic dna by two hydrogen bonds because one hydrogen bond involved a symmetry equivalent that would not be relevant in solution and the other two hydrogen bonds that held it in place are between the 4-sugar of the trioxacarcin and the guanine . after leaving , the guanine rotates by about 180 to give the conformation observed in the crystal structure of gutingimycin ( 5 ) that is stabilized by stacking interactions between the guanine and the chromophore ; this would inhibit re - intercalation of the gutingimycin into the dna . figure 7b shows the crystal structure of gutingimycin ( 5 ) in a similar orientation to the bound trioxacarcin in the dna drug duplex ( figure 7a ) , illustrating the 180 rotation of the guanine base . n2 of this guanine lies above ring c in both structures . in the dna drug complex , n3 is positioned above ring b and n9 above ring a , whereas in gutingimycin n1 is above ring b and o6 above ring a. the orientation of the sugars is almost identical in both structures since they are held in place by the same internal hydrogen bonds . the carbon atoms of the upper base pair g(106)-c(3 ) are drawn in green , the ones of the lower base pair t(108)-a(2 ) in turquoise . the trioxacarcin is positioned between the two base pairs , with its carbon atoms colored in light orange . the carbon atoms of the flipped - out base t(107 ) are drawn in gray . ( b ) the small - molecule crystal structure of gutingimycin crystallized without dna ( 5 ) . the carbon atoms of the upper base pair g(106)-c(3 ) are drawn in green , the ones of the lower base pair t(108)-a(2 ) in turquoise . the trioxacarcin is positioned between the two base pairs , with its carbon atoms colored in light orange . the carbon atoms of the flipped - out base t(107 ) are drawn in gray . ( b ) the small - molecule crystal structure of gutingimycin crystallized without dna ( 5 ) . one trioxacarcin is involved in stacking interactions with g(106 ) and a(2 ) ( figure 7a ) , the other with g(6 ) and a(102 ) . residues t(108 ) and t(8 ) interact slightly with the antibiotics but c(3 ) and c(103 ) do not . in the case of daunomycin or nogalamycin the long axis of the aglycone is nearly perpendicular to that of the base pairs ( 6,7 ) , but this is not the case for trioxacarcin , which is constrained by the alkylation site . the aromatic rings a and b of the trioxacarcins lie below the atoms n9 , n3 and c1 of residues g(106 ) and g(6 ) . the long axis of the trioxacarcin aglycone is nearly parallel to that of the alkylated guanine ( figure 7a ) and runs close to the sugar - phosphate backbone of this guanine . this orientation brings the 10-methoxy groups of the trioxacarcins approximately into the positions where the deoxyriboses of residues t(107 ) and t(7 ) would lie if they were not flipped out . the flipped - out thymines are positioned near the 6-methyl groups of the trioxacarcins , the distance between c2 of t(7 ) and 6-methyl of trioxacarcin(9 ) is 3.3 [ 3.6 for the corresponding atoms of t(107 ) and trioxacarcin(109 ) ] . the following analysis is based on standard nomenclature of nucleic acid structure parameters ( 20 ) . they are not planar ; t(108)-a(2 ) is buckled by 9 and g(106)-c(3 ) by 10 [ t(8)-a(102 ) by 6 and g(6)-c(103 ) by 13 ] . this effect is well known for intercalators and explained by the need to maximize van der waals contacts . the helical twist angles from base pair t(108)-a(2 ) to base pair a(102)-t(8 ) are 48 , 35 , 40 , 37 and 45 ; for b - dna 37 is typical ( 20 ) . noncovalent intercalators such as daunomycin or nogalamycin usually unwind dna ( reducing these angles ) , whereas the intercalation of trioxacarcin combined with the flipping - out of a base in one of the two strands leads to an increased helical twist and an opening of the base pair ( rotation of the bases relative to each other in the plane of the base - pair ) for t(108)-a(2 ) of 10 [ 8 for t(8)-a(102 ) ] towards the major groove . the angle between base pairs t(108)-a(2 ) and g(106)-c(3 ) ( tilt ) is 12 and opens towards the strand with the flipped - out thymine t(107 ) [ corresponding values for t(8)-a(102)/g(6)-c(103 ) ] , also visible in figure 6 . another distortion induced by the trioxacarcin is a displacement along the long axis of base pairs t(108)-a(2 ) and g(106)-c(3 ) ( slide ) by 2.0 [ 2.2 for t(8)-a(102)/g(6)-c(103 ) ] , also visible in figure 7a . all these distortions are within the ranges observed for both complexed and uncomplexed double - stranded dna structures . structures of anthracyclines intercalated in dna without the formation of covalent bonds invariably show the drug intercalated at the 5-side of a guanine , usually between c and g or t and g. nmr studies of hedamycin dna ( 9,21,22 ) , altromycin dna ( 10 ) and psorospermin dna ( 11 ) complexes , in which a covalent bond is formed by nucleophilic attack of the guanine n7 on an epoxide as in trioxacarcin , showed a similar intercalation site to that observed for the anthracyclines , namely on the 5-side of the alkylated guanine , without a flipped - out nucleobase . the crystal structure of trioxacarcin bound to d(aaccggtt ) reported here reveals a quite different intercalation on the 3-side of guanine combined with a flipped - out thymine on the same side . this result is consistent with sequence selectivity studies ( 4 ) that report a preferred adduct formation with 5-aatttgtaatt or 5-aattagtaatt compared to 5-aatttgaaatt or 5-aattagaaatt , showing that only a variation on the 3-side of the alkylated guanine influences drug binding . in view of the evidence from the crystal structure that a(102 ) and a(2 ) pair with the thymine after the flipped - out thymine , we predict that trioxacarcin a should react preferentially with the dna sequence 5-gtt . in contrast , in the pluramycin and related complexes it is always the base on the 5-side of the alkylated guanine that influences the sequence specificity , consistent with the observed intercalation site . since trioxacarcin a does not react with a guanine positioned at the 3-terminus of a dna - oligonucleotide ( 4 ) , it appears that docking of the antibiotic , guided by the hydrogen bonds discussed above and the location of the bulky sugars in the minor and major grooves , with preliminary noncovalent binding to the dna - helix is a prerequisite for the reaction of the trioxacarcin a with a guanine base . it has to be taken into account that trioxacarcin a would have to thread into the dna backbone , either by transient melting or by unwinding of the helix ( 7,23 ) . the flipping - out of bases plays an important role in certain dna protein interactions ; for example in the dna repair enzyme uracil - dna glycosylase ( udg ) the discrimination between uracil and thymine is initiated by thermally induced opening of ta and ua base pairs ( 24 ) . base - pair dynamics may also be connected with the sequence selectivity of trioxacarcin a. an unresolved question is how streptomycetes protect their own dna from the drug . this study has revealed a unique structure for a dna drug complex that combines intercalation , alkylation and base flip - out . it provides insight into the mechanism of the formation of gutingimycin with the abstraction of a guanine base from dna and adds to our understanding of dna manipulation by antibiotics . as predicted in the study of the sequence specificity ( 4 ) , trioxacarcin a binds covalently to d(aaccggtt ) by alkylating the n7 position of the guanine that is followed by a thymine . unexpectedly , this thymine is flipped out of the duplex dna and the adenine originally paired with it now forms a base pair with the following thymine in the sequence . the aromatic rings a and b of the drug are involved in stacking interactions with the dna . as found for nogalamycin ( 7 ) , trioxacarcin a interacts with both grooves of the dna ( figure 5 ) ; the 4-sugar is positioned in the minor groove , the 13-sugar in the major groove . figure 4.simplified view of the dna trioxacarcin duplex with residue names trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . on the bottom is the minor groove enclosing the 4-sugar , on the bottom the major groove containing the 13-sugar ( left side ) and the two 16-methoxy groups ( right - side ) . on the extreme upper left - side , the terminal a(101 ) trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . on the bottom is the minor groove enclosing the 4-sugar , on the bottom the major groove containing the 13-sugar ( left side ) and the two 16-methoxy groups ( right - side ) . on the extreme upper left - side , the terminal a(101 ) can be seen . since the duplex does not lie on a 2-fold axis , the two self - complementary strands are crystallographically independent , with some small structural differences caused by interactions of the residues a(1 ) , a(101 ) , t(7 ) and t(107 ) , which are no longer base - paired within the duplex , with different symmetry related residues in the crystal . residue a(101 ) does not appear to make specific contacts to neighboring molecules and is highly disordered , whereas n1 of a(1 ) accepts an hydrogen bond ( 2.7 ) from a symmetry equivalent of 4-oh of trioxacarcin(109 ) . the general conformations of both strands are fairly similar except for residues a(1 ) and a(101 ) that are positioned at the 5-terminus of the oligonucleotide and do not base pair within the duplex . the dna trioxacarcin duplex shows a distorted b - dna geometry with watson crick base pairing . in table 2 , the sugar - phosphate and glycosyl torsion angles are compared with the usual ranges and mean values for b - dna ( 19 ) . the flipping - out of t(7 ) is most evident in its torsion angle of 82 [ 74 for t(107 ) ] that differs by about 180 from the standard value . the other torsion angles all lie in ranges typically observed in b - dna structures . table 2.backbone and glycosyl torsion anglesangle , degreesresiduea2 ( next to intercalation)28217660155254280209a10230818050155251270224c3 ( next to intercalation)28817046150206192276c10328715752147218188274c430814953133168265240c10429813956134183238242g530719147151180262262g10531118148149181268259g6 ( next to intercalation)29318351144238264273g10630318546141233267270t7 ( flipped - out)3021596114725182252t1072811846916125474258t8 ( next to intercalation)27617251143232t10823618481130221b - dna ( range)270330130200208070180160270150300200300b - dna ( mean)298176 ( i)48128 ( i)184 ( i)265 ( i)258 ( a / g)146 ( ii)144 ( ii)246 ( ii)174 ( ii)241 ( t / c)271 ( ii)torsion angles are defined as : o3 * -p - o5 * -c5 * , : p - o5 * -c5 * -c4 * , : o5 * -c5 * -c4 * -c3 * , : c5 * -c4 * -c3 * -o3 * , : c4 * -c3 * -o3 * -p , : c3 * -o3 * -p - o5 * , (purines ) : o4 * -c1 * -n9-c4 , (pyrimidines ) : o4 * -c1 * -n1-c2 . for the mean values bi and bii conformations are distinguished , in the case of the glycosyl torsion angle also purines and pyrimidines for the bi conformation . backbone and glycosyl torsion angles torsion angles are defined as : o3 * -p - o5 * -c5 * , : p - o5 * -c5 * -c4 * , : o5 * -c5 * -c4 * -c3 * , : c5 * -c4 * -c3 * -o3 * , : c4 * -c3 * -o3 * -p , : c3 * -o3 * -p - o5 * , (purines ) : o4 * -c1 * -n9-c4 , (pyrimidines ) : o4 * -c1 * -n1-c2 . for the mean values bi and bii conformations are distinguished , in the case of the glycosyl torsion angle also purines and pyrimidines for the bi conformation . in addition to the covalent bond , trioxacarcin a forms direct and water mediated hydrogen bonds with the dna ( figure 6 ) . there is an hydrogen bond between the 2-oh of trioxacarcin and o4 of the deoxyribose attached to the cytosine opposite to the alkylated guanine [ 2.8 for trioxacarcin(9 ) and 2.7 for trioxacarcin(109 ) ] . in the minor groove , n2 of the alkylated guanine donates an hydrogen bond to 3-oh of the 4-sugar of trioxacarcin ( 3.1 for both trioxacarcins ) . there is also a water - mediated hydrogen bond between n3 of the alkylated guanine and 3-oh . the 4-sugar interacts only with the alkylated guanine . in the major groove , the 1-oxygen of the 13-sugar of trioxacarcin(109 ) accepts an hydrogen bond ( 3.2 ) from n6 of residue a(2 ) . in the case of trioxacarcin(9 ) and a(102 ) , the corresponding hydrogen bond is mediated by a water molecule , giving rise to a small difference between the two strands . the 13-sugar is also involved in an internal hydrogen bond [ 3.4 for trioxacarcin(9 ) and 3.1 for trioxacarcin(109 ) ] between 3-oh and 14-oh ( figure 6 ) , stabilizing the oh - group formed by the nucleophilic attack on the epoxide ring . the 13-sugar of the trioxacarcin is held parallel to the trioxacarcin chromophore by this internal hydrogen bond , whereas the 4-sugar is orientated perpendicular to it . base pair g(105)-c(4 ) is at the top , followed by base pair g(106)-c(3 ) . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . base pair g(105)-c(4 ) is at the top , followed by base pair g(106)-c(3 ) . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . hydrogen bonds are indicated with dashed lines . for the formation of gutingimycin , the guanine sugar bond between n9 and c1 ( figure 7a ) has to break and the antibiotic has to leave the dna duplex taking the guanine with it . assuming that the guanine sugar bond breaks whilst the double strand is still intact , gutingimycin would then only be attached to the abasic dna by two hydrogen bonds because one hydrogen bond involved a symmetry equivalent that would not be relevant in solution and the other two hydrogen bonds that held it in place are between the 4-sugar of the trioxacarcin and the guanine . after leaving , the guanine rotates by about 180 to give the conformation observed in the crystal structure of gutingimycin ( 5 ) that is stabilized by stacking interactions between the guanine and the chromophore ; this would inhibit re - intercalation of the gutingimycin into the dna . figure 7b shows the crystal structure of gutingimycin ( 5 ) in a similar orientation to the bound trioxacarcin in the dna drug duplex ( figure 7a ) , illustrating the 180 rotation of the guanine base . n2 of this guanine lies above ring c in both structures . in the dna drug complex , n3 is positioned above ring b and n9 above ring a , whereas in gutingimycin n1 is above ring b and o6 above ring a. the orientation of the sugars is almost identical in both structures since they are held in place by the same internal hydrogen bonds . the carbon atoms of the upper base pair g(106)-c(3 ) are drawn in green , the ones of the lower base pair t(108)-a(2 ) in turquoise . the trioxacarcin is positioned between the two base pairs , with its carbon atoms colored in light orange . the carbon atoms of the flipped - out base t(107 ) are drawn in gray . ( b ) the small - molecule crystal structure of gutingimycin crystallized without dna ( 5 ) . the carbon atoms of the upper base pair g(106)-c(3 ) are drawn in green , the ones of the lower base pair t(108)-a(2 ) in turquoise . the trioxacarcin is positioned between the two base pairs , with its carbon atoms colored in light orange . the carbon atoms of the flipped - out base t(107 ) are drawn in gray . ( b ) the small - molecule crystal structure of gutingimycin crystallized without dna ( 5 ) . one trioxacarcin is involved in stacking interactions with g(106 ) and a(2 ) ( figure 7a ) , the other with g(6 ) and a(102 ) . residues t(108 ) and t(8 ) interact slightly with the antibiotics but c(3 ) and c(103 ) do not . in the case of daunomycin or nogalamycin the long axis of the aglycone is nearly perpendicular to that of the base pairs ( 6,7 ) , but this is not the case for trioxacarcin , which is constrained by the alkylation site . the aromatic rings a and b of the trioxacarcins lie below the atoms n9 , n3 and c1 of residues g(106 ) and g(6 ) . the long axis of the trioxacarcin aglycone is nearly parallel to that of the alkylated guanine ( figure 7a ) and runs close to the sugar - phosphate backbone of this guanine . this orientation brings the 10-methoxy groups of the trioxacarcins approximately into the positions where the deoxyriboses of residues t(107 ) and t(7 ) would lie if they were not flipped out . the flipped - out thymines are positioned near the 6-methyl groups of the trioxacarcins , the distance between c2 of t(7 ) and 6-methyl of trioxacarcin(9 ) is 3.3 [ 3.6 for the corresponding atoms of t(107 ) and trioxacarcin(109 ) ] . the following analysis is based on standard nomenclature of nucleic acid structure parameters ( 20 ) . the base pairs of the dna trioxacarcin duplex are distorted in several different ways . at the intercalation site they are not planar ; t(108)-a(2 ) is buckled by 9 and g(106)-c(3 ) by 10 [ t(8)-a(102 ) by 6 and g(6)-c(103 ) by 13 ] . this effect is well known for intercalators and explained by the need to maximize van der waals contacts . the helical twist angles from base pair t(108)-a(2 ) to base pair a(102)-t(8 ) are 48 , 35 , 40 , 37 and 45 ; for b - dna 37 is typical ( 20 ) . noncovalent intercalators such as daunomycin or nogalamycin usually unwind dna ( reducing these angles ) , whereas the intercalation of trioxacarcin combined with the flipping - out of a base in one of the two strands leads to an increased helical twist and an opening of the base pair ( rotation of the bases relative to each other in the plane of the base - pair ) for t(108)-a(2 ) of 10 [ 8 for t(8)-a(102 ) ] towards the major groove . the angle between base pairs t(108)-a(2 ) and g(106)-c(3 ) ( tilt ) is 12 and opens towards the strand with the flipped - out thymine t(107 ) [ corresponding values for t(8)-a(102)/g(6)-c(103 ) ] , also visible in figure 6 . another distortion induced by the trioxacarcin is a displacement along the long axis of base pairs t(108)-a(2 ) and g(106)-c(3 ) ( slide ) by 2.0 [ 2.2 for t(8)-a(102)/g(6)-c(103 ) ] , also visible in figure 7a . all these distortions are within the ranges observed for both complexed and uncomplexed double - stranded dna structures . structures of anthracyclines intercalated in dna without the formation of covalent bonds invariably show the drug intercalated at the 5-side of a guanine , usually between c and g or t and g. nmr studies of hedamycin dna ( 9,21,22 ) , altromycin dna ( 10 ) and psorospermin dna ( 11 ) complexes , in which a covalent bond is formed by nucleophilic attack of the guanine n7 on an epoxide as in trioxacarcin , showed a similar intercalation site to that observed for the anthracyclines , namely on the 5-side of the alkylated guanine , without a flipped - out nucleobase . the crystal structure of trioxacarcin bound to d(aaccggtt ) reported here reveals a quite different intercalation on the 3-side of guanine combined with a flipped - out thymine on the same side . this result is consistent with sequence selectivity studies ( 4 ) that report a preferred adduct formation with 5-aatttgtaatt or 5-aattagtaatt compared to 5-aatttgaaatt or 5-aattagaaatt , showing that only a variation on the 3-side of the alkylated guanine influences drug binding . in view of the evidence from the crystal structure that a(102 ) and a(2 ) pair with the thymine after the flipped - out thymine , we predict that trioxacarcin a should react preferentially with the dna sequence 5-gtt . in contrast , in the pluramycin and related complexes it is always the base on the 5-side of the alkylated guanine that influences the sequence specificity , consistent with the observed intercalation site . since trioxacarcin a does not react with a guanine positioned at the 3-terminus of a dna - oligonucleotide ( 4 ) , it appears that docking of the antibiotic , guided by the hydrogen bonds discussed above and the location of the bulky sugars in the minor and major grooves , with preliminary noncovalent binding to the dna - helix is a prerequisite for the reaction of the trioxacarcin a with a guanine base . it has to be taken into account that trioxacarcin a would have to thread into the dna backbone , either by transient melting or by unwinding of the helix ( 7,23 ) . the flipping - out of bases plays an important role in certain dna protein interactions ; for example in the dna repair enzyme uracil - dna glycosylase ( udg ) the discrimination between uracil and thymine is initiated by thermally induced opening of ta and ua base pairs ( 24 ) . base - pair dynamics may also be connected with the sequence selectivity of trioxacarcin a. an unresolved question is how streptomycetes protect their own dna from the drug . this study has revealed a unique structure for a dna drug complex that combines intercalation , alkylation and base flip - out . it provides insight into the mechanism of the formation of gutingimycin with the abstraction of a guanine base from dna and adds to our understanding of dna manipulation by antibiotics .

we report a crystal structure that shows an antibiotic that extracts a nucleobase from a dna molecule caught in the act after forming a covalent bond but before departing with the base . the structure of trioxacarcin a covalently bound to double - stranded d(aaccggtt ) was determined to 1.78 resolution by mad phasing employing brominated oligonucleotides . the dna drug complex has a unique structure that combines alkylation ( at the n7 position of a guanine ) , intercalation ( on the 3-side of the alkylated guanine ) , and base flip - out . an antibiotic - induced flipping - out of a single , nonterminal nucleobase from a dna duplex was observed for the first time in a crystal structure .

INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION Overall structure AntibioticDNA interactions Distortion of the DNA Comparison with related DNAantibiotic complexes

trioxacarcin a ( figure 1 ) contains a complex ring system that is attached to sugars at both ends , at the 4- and 13-positions , and causes it to exhibit intensive green fluorescence in solution ; in powder form it is yellow . nucleophilic attack of n7 of guanine opens the epoxide ( 1) to form a covalent bond to the guanine ( 2) in a dna molecule ( figure 2 ) . cleavage of this trioxacarcindna ( 3) complex at 373 k results in the natural product gutingimycin ( 4) ( 3,5 ) , named after the ancient name for the city of gttingen , leaving an abasic dna . figure 1.schematic diagram of trioxacarcin a. figure 2.proposed mechanism for the reaction of trioxacarcin a with dna . only the epoxide group of trioxacarcin a ( 1 ) is shown , the rest of the molecule is described by r1 and r2 . schematic diagram of trioxacarcin a. proposed mechanism for the reaction of trioxacarcin a with dna . only the epoxide group of trioxacarcin a ( 1 ) is shown , the rest of the molecule is described by r1 and r2 . to throw more light onto the stereochemical requirements for the covalent bond formation and subsequent elimination of a nucleobase , we have determined the structure of trioxacarin a covalently bound to an oligonucleotide to 1.78 resolution by x - ray diffraction . in the brominated oligonucleotides thymidine the dna drug complex crystallized in space group p4122 with unit - cell a = b = 37.60 and c = 91.62 . in addition , a native dataset was collected to 1.78 resolution from a crystal containing the native oligonucleotide . as predicted in the study of the sequence specificity ( 4 ) , trioxacarcin a binds covalently to d(aaccggtt ) by alkylating the n7 position of the guanine that is followed by a thymine . unexpectedly , this thymine is flipped out of the duplex dna and the adenine originally paired with it now forms a base pair with the following thymine in the sequence . the aromatic rings a and b of the drug are involved in stacking interactions with the dna . as found for nogalamycin ( 7 ) , trioxacarcin a interacts with both grooves of the dna ( figure 5 ) ; the 4-sugar is positioned in the minor groove , the 13-sugar in the major groove . figure 4.simplified view of the dna trioxacarcin duplex with residue names trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . on the extreme upper left - side , the terminal a(101 ) trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . since the duplex does not lie on a 2-fold axis , the two self - complementary strands are crystallographically independent , with some small structural differences caused by interactions of the residues a(1 ) , a(101 ) , t(7 ) and t(107 ) , which are no longer base - paired within the duplex , with different symmetry related residues in the crystal . the flipping - out of t(7 ) is most evident in its torsion angle of 82 [ 74 for t(107 ) ] that differs by about 180 from the standard value . in addition to the covalent bond , trioxacarcin a forms direct and water mediated hydrogen bonds with the dna ( figure 6 ) . there is an hydrogen bond between the 2-oh of trioxacarcin and o4 of the deoxyribose attached to the cytosine opposite to the alkylated guanine [ 2.8 for trioxacarcin(9 ) and 2.7 for trioxacarcin(109 ) ] . in the minor groove , n2 of the alkylated guanine donates an hydrogen bond to 3-oh of the 4-sugar of trioxacarcin ( 3.1 for both trioxacarcins ) . there is also a water - mediated hydrogen bond between n3 of the alkylated guanine and 3-oh . the 4-sugar interacts only with the alkylated guanine . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . for the formation of gutingimycin , the guanine sugar bond between n9 and c1 ( figure 7a ) has to break and the antibiotic has to leave the dna duplex taking the guanine with it . after leaving , the guanine rotates by about 180 to give the conformation observed in the crystal structure of gutingimycin ( 5 ) that is stabilized by stacking interactions between the guanine and the chromophore ; this would inhibit re - intercalation of the gutingimycin into the dna . figure 7b shows the crystal structure of gutingimycin ( 5 ) in a similar orientation to the bound trioxacarcin in the dna drug duplex ( figure 7a ) , illustrating the 180 rotation of the guanine base . in the dna drug complex , n3 is positioned above ring b and n9 above ring a , whereas in gutingimycin n1 is above ring b and o6 above ring a. the orientation of the sugars is almost identical in both structures since they are held in place by the same internal hydrogen bonds . in the case of daunomycin or nogalamycin the long axis of the aglycone is nearly perpendicular to that of the base pairs ( 6,7 ) , but this is not the case for trioxacarcin , which is constrained by the alkylation site . noncovalent intercalators such as daunomycin or nogalamycin usually unwind dna ( reducing these angles ) , whereas the intercalation of trioxacarcin combined with the flipping - out of a base in one of the two strands leads to an increased helical twist and an opening of the base pair ( rotation of the bases relative to each other in the plane of the base - pair ) for t(108)-a(2 ) of 10 [ 8 for t(8)-a(102 ) ] towards the major groove . structures of anthracyclines intercalated in dna without the formation of covalent bonds invariably show the drug intercalated at the 5-side of a guanine , usually between c and g or t and g. nmr studies of hedamycin dna ( 9,21,22 ) , altromycin dna ( 10 ) and psorospermin dna ( 11 ) complexes , in which a covalent bond is formed by nucleophilic attack of the guanine n7 on an epoxide as in trioxacarcin , showed a similar intercalation site to that observed for the anthracyclines , namely on the 5-side of the alkylated guanine , without a flipped - out nucleobase . the crystal structure of trioxacarcin bound to d(aaccggtt ) reported here reveals a quite different intercalation on the 3-side of guanine combined with a flipped - out thymine on the same side . this result is consistent with sequence selectivity studies ( 4 ) that report a preferred adduct formation with 5-aatttgtaatt or 5-aattagtaatt compared to 5-aatttgaaatt or 5-aattagaaatt , showing that only a variation on the 3-side of the alkylated guanine influences drug binding . in view of the evidence from the crystal structure that a(102 ) and a(2 ) pair with the thymine after the flipped - out thymine , we predict that trioxacarcin a should react preferentially with the dna sequence 5-gtt . in contrast , in the pluramycin and related complexes it is always the base on the 5-side of the alkylated guanine that influences the sequence specificity , consistent with the observed intercalation site . since trioxacarcin a does not react with a guanine positioned at the 3-terminus of a dna - oligonucleotide ( 4 ) , it appears that docking of the antibiotic , guided by the hydrogen bonds discussed above and the location of the bulky sugars in the minor and major grooves , with preliminary noncovalent binding to the dna - helix is a prerequisite for the reaction of the trioxacarcin a with a guanine base . it has to be taken into account that trioxacarcin a would have to thread into the dna backbone , either by transient melting or by unwinding of the helix ( 7,23 ) . the flipping - out of bases plays an important role in certain dna protein interactions ; for example in the dna repair enzyme uracil - dna glycosylase ( udg ) the discrimination between uracil and thymine is initiated by thermally induced opening of ta and ua base pairs ( 24 ) . this study has revealed a unique structure for a dna drug complex that combines intercalation , alkylation and base flip - out . it provides insight into the mechanism of the formation of gutingimycin with the abstraction of a guanine base from dna and adds to our understanding of dna manipulation by antibiotics . as predicted in the study of the sequence specificity ( 4 ) , trioxacarcin a binds covalently to d(aaccggtt ) by alkylating the n7 position of the guanine that is followed by a thymine . unexpectedly , this thymine is flipped out of the duplex dna and the adenine originally paired with it now forms a base pair with the following thymine in the sequence . as found for nogalamycin ( 7 ) , trioxacarcin a interacts with both grooves of the dna ( figure 5 ) ; the 4-sugar is positioned in the minor groove , the 13-sugar in the major groove . figure 4.simplified view of the dna trioxacarcin duplex with residue names trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . on the extreme upper left - side , the terminal a(101 ) trioxacarcin is shown in red ( oxygen atoms ) and light orange ( carbon atoms ) ; the dna is shown in light gray except for the phosphorus atoms that are shown in black and the two residues containing the flipped - out thymines in green . the flipping - out of t(7 ) is most evident in its torsion angle of 82 [ 74 for t(107 ) ] that differs by about 180 from the standard value . for the mean values bi and bii conformations are distinguished , in the case of the glycosyl torsion angle also purines and pyrimidines for the bi conformation . in addition to the covalent bond , trioxacarcin a forms direct and water mediated hydrogen bonds with the dna ( figure 6 ) . there is an hydrogen bond between the 2-oh of trioxacarcin and o4 of the deoxyribose attached to the cytosine opposite to the alkylated guanine [ 2.8 for trioxacarcin(9 ) and 2.7 for trioxacarcin(109 ) ] . in the minor groove , n2 of the alkylated guanine donates an hydrogen bond to 3-oh of the 4-sugar of trioxacarcin ( 3.1 for both trioxacarcins ) . there is also a water - mediated hydrogen bond between n3 of the alkylated guanine and 3-oh . the 4-sugar interacts only with the alkylated guanine . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . trioxacarcin a is bound to g(106 ) and intercalates on its 3-side between base pair g(106)-c(3 ) and base pair t(108)-a(2 ) , which is at the bottom . for the formation of gutingimycin , the guanine sugar bond between n9 and c1 ( figure 7a ) has to break and the antibiotic has to leave the dna duplex taking the guanine with it . after leaving , the guanine rotates by about 180 to give the conformation observed in the crystal structure of gutingimycin ( 5 ) that is stabilized by stacking interactions between the guanine and the chromophore ; this would inhibit re - intercalation of the gutingimycin into the dna . figure 7b shows the crystal structure of gutingimycin ( 5 ) in a similar orientation to the bound trioxacarcin in the dna drug duplex ( figure 7a ) , illustrating the 180 rotation of the guanine base . in the dna drug complex , n3 is positioned above ring b and n9 above ring a , whereas in gutingimycin n1 is above ring b and o6 above ring a. the orientation of the sugars is almost identical in both structures since they are held in place by the same internal hydrogen bonds . in the case of daunomycin or nogalamycin the long axis of the aglycone is nearly perpendicular to that of the base pairs ( 6,7 ) , but this is not the case for trioxacarcin , which is constrained by the alkylation site . the long axis of the trioxacarcin aglycone is nearly parallel to that of the alkylated guanine ( figure 7a ) and runs close to the sugar - phosphate backbone of this guanine . the flipped - out thymines are positioned near the 6-methyl groups of the trioxacarcins , the distance between c2 of t(7 ) and 6-methyl of trioxacarcin(9 ) is 3.3 [ 3.6 for the corresponding atoms of t(107 ) and trioxacarcin(109 ) ] . noncovalent intercalators such as daunomycin or nogalamycin usually unwind dna ( reducing these angles ) , whereas the intercalation of trioxacarcin combined with the flipping - out of a base in one of the two strands leads to an increased helical twist and an opening of the base pair ( rotation of the bases relative to each other in the plane of the base - pair ) for t(108)-a(2 ) of 10 [ 8 for t(8)-a(102 ) ] towards the major groove . all these distortions are within the ranges observed for both complexed and uncomplexed double - stranded dna structures . structures of anthracyclines intercalated in dna without the formation of covalent bonds invariably show the drug intercalated at the 5-side of a guanine , usually between c and g or t and g. nmr studies of hedamycin dna ( 9,21,22 ) , altromycin dna ( 10 ) and psorospermin dna ( 11 ) complexes , in which a covalent bond is formed by nucleophilic attack of the guanine n7 on an epoxide as in trioxacarcin , showed a similar intercalation site to that observed for the anthracyclines , namely on the 5-side of the alkylated guanine , without a flipped - out nucleobase . the crystal structure of trioxacarcin bound to d(aaccggtt ) reported here reveals a quite different intercalation on the 3-side of guanine combined with a flipped - out thymine on the same side . this result is consistent with sequence selectivity studies ( 4 ) that report a preferred adduct formation with 5-aatttgtaatt or 5-aattagtaatt compared to 5-aatttgaaatt or 5-aattagaaatt , showing that only a variation on the 3-side of the alkylated guanine influences drug binding . in view of the evidence from the crystal structure that a(102 ) and a(2 ) pair with the thymine after the flipped - out thymine , we predict that trioxacarcin a should react preferentially with the dna sequence 5-gtt . in contrast , in the pluramycin and related complexes it is always the base on the 5-side of the alkylated guanine that influences the sequence specificity , consistent with the observed intercalation site . since trioxacarcin a does not react with a guanine positioned at the 3-terminus of a dna - oligonucleotide ( 4 ) , it appears that docking of the antibiotic , guided by the hydrogen bonds discussed above and the location of the bulky sugars in the minor and major grooves , with preliminary noncovalent binding to the dna - helix is a prerequisite for the reaction of the trioxacarcin a with a guanine base . the flipping - out of bases plays an important role in certain dna protein interactions ; for example in the dna repair enzyme uracil - dna glycosylase ( udg ) the discrimination between uracil and thymine is initiated by thermally induced opening of ta and ua base pairs ( 24 ) . base - pair dynamics may also be connected with the sequence selectivity of trioxacarcin a. an unresolved question is how streptomycetes protect their own dna from the drug . this study has revealed a unique structure for a dna drug complex that combines intercalation , alkylation and base flip - out . it provides insight into the mechanism of the formation of gutingimycin with the abstraction of a guanine base from dna and adds to our understanding of dna manipulation by antibiotics .

protein deacetylases counter the action of protein acetyl transferases ( verdin and ott , 2015 ) by removing acetyl groups added to lysine residues by the latter . this large family of enzymes is often referred to as histone deacetylases ( hdacs ) , but histones are certainly not their only substrates . in mammals , hdacs are broadly divided into four classes ( i iv ) ( yang and seto , 2008 ) . class i hdacs ( hdacs 1 , 2 , 3 and 8) are homologous with the yeast reduced potassium deficiency 3 ( rpd3 ) , are ubiquitously expressed , and are primarily localized to the nucleus . class ii hdacs ( namely hdacs 4 , 5 , 6 , 7 , 9 and 10 ) , which are homologues of yeast histone deacetylase 1 ( hda1 ) , shuttle between the nucleus and the cytoplasm , and are more tissue specific in terms of expression . hdac11 has only fairly low homology to both rpd3 and hda1 , and has been proposed to constitute a separate class iv ( gregoretti et al . , 2004 ) sirtuins are a family of proteins with homologies to the silence information regulator 2 ( sir2 ) gene of the yeast s. cerevisiae ( brachmann et al . , 1995 ) , and are not structurally homologous to the other hdacs . lysine deacetylation by sirtuins is coupled to the cleavage of a key intermediate of cellular energy metabolism , nicotinamide adenine dinucleotide ( nad ) , into nicotinamide and 1-o - acetyl - adp - ribose ( tanner et al . , 2000 ) or 2- and 3-o - acetyl - adp - ribose ( jackson and denu , 2002 ) . the activities of sirtuins are thus obligatorily dependent on cellular nad , inhibited by the reaction product nicotinamide , and effectively influenced by cellular metabolic and redox states . homologues of yeast sir2 exist in lower model organisms , such as the nematode c. elegans sir-2.1 and the fruit fly d. melanogaster sir2 . the mammalian genome has seven sirtuin paralogues ( sirt1 - 7 ) , with sirt1 having the highest homology to yeast sir2 ( haigis and guarente , 2006 ; haigis and sinclair , 2010 ) . in yeast , sir2p regulates chromatin silencing , and reduces the accumulation of rdna circles during yeast replicative aging ( sinclair and guarente , 1997 ) . sir2 and its metazoan orthologues have been prominently associated with lifespan extension ( howitz et al . , 2003 ; tissenbaum and guarente , 2001 ) , in particular that induced by caloric or dietary restriction ( cr / dr ) ( rogina and helfand , 2004 ; wood et al . , 2004 ) . a large number of studies have connected sirt1 expression and/or activation by compounds such as resveratrol , with increase health span and lifespan ( guarente , 2011 ) . however , the notion that sirt1 is a mediator of cr effects and a longevity factor in mammals and humans has been controversial in two major ways . there were doubts about the specificity and effectiveness of natural and synthetic sirt1 activators such as resveratrol and srt1720 ( kaeberlein et al . sirt1 , at least in certain contexts ( longo , 2009 ; tang , 2006 ) , may also promote aging . sirt1 s role in longevity has been more extensively investigated in yeast and lower invertebrate models , and although the notion of sirt1 as a longevity gene has a great deal of support , there were also controversies and evidence to the contrary ( burnett et al . , 2011 ) . sirt1 s knockout phenotype was difficult to interpret , as the mice often died early due largely to developmental defects ( cheng et al . , 2003 ; mcburney et al . , 2003 earlier work on transgenic over - expression of sirt1 in mice did not result in a significant increase in lifespan ( herranz et al . , 2010 ) . more recent findings have , however , provided some reconciliation of the contrasting results and opinions . the mode of action of sirtuin - activating compounds ( stacs ) , possibly via an allosteric mechanism , has been better clarified ( hubbard et al . , 2013 ) . additionally , both the transgenic expression of sirt1 ( satoh et al . , 2013 ) and the specific activator srt1720 ( mitchell et al . , 2014 ) have eventually been shown to increase lifespan in mice . indeed , dissection of sirt1 s role in cr and aging is difficult because sirt1 has multiple physiological functions as well as wide - ranging roles in pathological settings . this is due to its astonishingly large repertoire of targets , many of which are key transcription factors associated with important aspects of cell survival and metabolism . prominent amongst these are the tumor suppressor tp53 ( vaziri et al . , 2001 ) , the forkhead box class o ( foxo ) family members ( brunet et al . , 2004 ) , and nuclear factor b ( nf-b ) ( yeung et al . , 2004 ) , all key regulators of cell death and survival . sirt1 represses a key metabolic regulator , peroxisome proliferator - activated receptor gamma ( ppar ) ( picard et al . , 2004 ; sugden et al . , 2010 ) , via deacetylation of ppar coactivator-1 ( pgc-1 ) ( gerhart - hines et al . , 2007 , 2005 ; rodgers et al . , 2005 ) , which regulates energy metabolism in skeletal muscle , adipose tissues and the liver . sirt1 has also been implicated in cellular metabolism through its activation of amp - dependent kinase ( ampk ) via liver kinase b1 ( lkb1 ) ( hou et al . , 2008 ; lan et al . , 2008 ; zu et al . , 2010 ) , as well as suppression of the mammalian target of rapamycin ( mtor ) pathway ( ghosh et al . , 2010 ; hong et al . of the seven mammalian sirtuins , three ( sirt3 , sirt4 and sirt5 ) have a primary mitochondrial localization ( michishita et al . , 2005 ) . sirt1 , on the other hand , is primarily nuclear , but its activities have large bearings on mitochondrial biogenesis and turnover . in the ensuing paragraphs we first look at some evidence for the predominantly nuclear sirt1 being physically localized at the mitochondria . sirt1 is primarily found in the nucleus of most cell types ( michishita et al . , 2005 ) , and it is not just a bona fide histone deacetylase ( vaquero et al . , 2006 ) but also deacetylates histone modifying enzymes such as the histone methyl - transferase suv39h1 ( vaquero et al . , 2007 ) . both sirt1 and suv39h1 are required for the epigenetic control of the rdna locus for rrna synthesis ( murayama et al . , 2008 ) , and sirt1 befitting its nuclear localization , a majority of sirt1 s known function is associated with deacetylation of transcription factors , as mentioned in the section above . as per all nuclear proteins sirt1 , however , appears to possess both nuclear localization signals and nuclear export signals , and could shuttle between the cytoplasm and the nucleus ( tanno et al . , 2007 ) . sirt1 s cytoplasmic appearance has been associated with enhanced apoptotic cell death ( jin et al . , 2007 ) , and also shown to be due to an increased protein stability via oncogenic signaling through the insulin - like growth factor-1-phosphoinositide 3-kinase ( igf-1/pi3 kinase ) axis in cancer cells ( byles et al . , 2010 ) . on the other hand , sirt1 does have cytosolic substrates whose deacetylation by sirt1 results in distinct physiological or functional consequences . for instance , the cytoplasmic acetyl - coa synthetase ( hallows et al . , 2006 ) and the actin binding protein cortactin ( zhang et al . , 2009 ) are both deacetylated by sirt1 , and these influence cytosolic fatty acid synthesis and cytoskeletal changes during cell migration , respectively . sirt1 and its substrate , pgc-1 , regulate aspects of energy metabolism through the mitochondria , but this was initially thought to occur through their influence on nuclear localized transcription . a rather interesting aspect of sirt1 s activity and function that is less well defined is its extranuclear localization , particularly at the mitochondria ( aquilano et al . , 2012 ) . this was first shown by aquilano and colleagues using both confocal imaging and subcellular fractionation analysis ( aquilano et al . , 2010 ) . both sirt1 and the nuclear transcription factor pgc-1 could be found in the mitochondria of human cell lines and platelets , as well as in various mouse organs . within the mitochondria , both deacetylase and its substrate are associated with the mitochondrial dna ( mtdna ) nucleoids ( bogenhagen , 2012 ) , as well as with the mitochondrial transcription factor a ( tfam ) , a key mitochondrial gene transcription factor and regulator of mtdna copy number ( campbell et al . , 2012 ) . these findings are fascinating , as they suggest that sirt1 and pgc-1 may also directly affect mitochondrial transcription . however , the degree of mitochondrial sirt1 and pgc-1 s functionality in this regard is still uncertain . in the sections that follow , we shall outline findings that point to how sirt1 mediates aspects of mitochondrial biogenesis and turnover , as well as the controversies associated with these findings . mitochondria are dynamic organelles , and inheritance occurs by partitioning existing mitochondria between daughter cells in dividing cells ( mishra and chan , 2014 ) . however , within all cells , either dividing or terminally differentiated , the mitochondria population is constantly renewed , and the steady state number of this organelle represents an equilibrium between mitochondrial biogenesis and eventual degradation through the process of mitophagy ( stotland and gottlieb , 2015 ; vega et al . , 2015 ) . mitochondrial biogenesis is orchestrated by the peroxisome proliferator - activated receptor gamma coactivator-1 ( pgc-1 ) family of transcriptional coactivators ( austin and st - pierre , 2012 ) . the pgc-1 family consists of three members , namely , pgc-1 , pgc-1 and the pgc related coactivator ( prc ) . pgc-1 , in particular , is often cited as a master regulator of this process . pgc-1 co - activates the transcription of nuclear respiratory factor ( nrf ) 1 and 2 , which , in turn , regulate the transcription of tfam . tfam translocates to mitochondrial matrix where it stimulates mitochondrial dna replication and mitochondrial gene expression . pgc-1 undergoes several modes of post - translational modification , which includes acetylation and phosphorylation . acetylation of pgc-1 occurs at several of its lysine residues and is catalyzed by the ubiquitous histone acetyl transferase general control of amino acid synthesis 5 ( gcn5 ) ( dominy et al . conversely , deacetylation of pgc-1 was shown by several studies to be dependent on sirt1 activity ( amat et al . , 2009 ; dominy et al . , 2010 ; gerhart - hines et al . , 2007 ; gurd , 2011 ; , 2006 ; nemoto et al . , 2005 ; olmos et al . , 2013 ; rodgers et al . , 2005 ) , which increases pgc-1 s transcriptional activity . pgc-1 is phosphorylated by both mitogen - activated protein kinase p38 ( barger et al . , 2001 ) , and the amp - dependent kinase ( ampk ) in skeletal muscle ( leick et al . , 2010 ) , which also stabilizes the protein and increases its activity . on the other hand , phosphorylation of pgc-1 by akt / protein kinase b ( pkb ) , downstream of insulin signaling in the liver , is known to decrease its stability and transcriptional activity ( li et al . , 2007 ) . given the importance of its acetylation status to pgc-1 activity , the connection between sirt1 and pgc-1 has been a focus of investigations into metabolic regulation and mitochondrial biogenesis . finkel s group has provided clear biochemical evidence that sirt1 physically and functionally interacts with pgc-1 ( nemoto et al . , 2005 ) , and this interaction could be distinguished by a single amino acid mutation in the putative adp - ribosyltransferase domain of sirt1 . this mutation abolishes sirt1 s interaction with pgc-1 , but not with two other major substrates , p53 and foxo3a . in what contexts a rather simplified view in this regard is that sirt1 acts a metabolic and redox sensor of changes in nutrient and energy status , such as those occurring during cr ( cant and auwerx , 2009 ) . cr has been shown to significantly increase mitochondrial biogenesis in multiple tissues in mice ( nisoli et al . , 2005 ) and early work from puigserver s group showed that sirt1 is induced during fasting , and induced sirt1 interacts with pgc-1 to increase the expression of hepatic gluconeogenic genes ( rodgers et al . , 2005 ) . the group also showed that fasting induced pgc-1 deacetylation by sirt1 in skeletal muscle , which is required for the activation of mitochondrial fatty acid oxidation genes ( gerhart - hines et al . , 2007 ) . auwerx s group , on the other hand , showed that resveratrol treatment of mice increased running time and oxygen consumption by the skeletal muscles , which is accompanied by the induction of genes for oxidative phosphorylation and mitochondrial biogenesis ( lagouge et al . , 2006 ) . the authors attributed these changes to activation of sirt1 , and a consequential decrease in pgc-1 acetylation and activity . the authors also showed that resveratrol treatment protected mice against diet - induced - obesity and insulin resistance . a more specific sirt1 activator , srt1720 , likewise enhanced muscle endurance and protected mice from diet - induced obesity and insulin resistance by enhancing oxidative metabolism in skeletal muscle , liver , and brown adipose tissue ( feige et al . , 2008 ) . auwerx and colleagues also found that deletion of the poly(adp - ribose ) polymerase-1(parp-1 ) , a major nad - consuming enzyme , resulted in increased sirt1 activity . parp-1 knockout mice have a higher mitochondrial content , an increase in energy expenditure , and were protected against metabolic disease ( bai et al . , 2011 ) . the work of anderson and colleagues ( anderson et al . , 2008 ) provided dynamical insights as to how sirt1 may regulate pgc-1 action . the latter s nuclear transcription activity is enhanced by sirt1-dependent nuclear accumulation and is counter regulated by glycogen synthase kinase beta ( gsk3 ) , which targets pgc-1 for intranuclear proteasomal degradation . despite the apparently convincing functional connection between sirt1 and pgc-1 , whether sirt1 is critically or directly involved in mitochondrial biogenesis has been controversial . gurd and colleagues ( gurd et al . , 2009 ) examined the relationship between sirt1 levels and activity , pgc-1 and markers of mitochondrial density ( such as cytochrome c oxidase subunit iv , coxiv ) in skeletal and heart muscles , and surprisingly found a largely negative correlation between sirt1 levels and the rest of the parameters . in fact , overexpression of sirt1 in muscle downregulated pgc-1 and mttfa levels , and while muscle stimulation and the ampk activator aicar upregulated pgc-1 and cox iv , sirt1 level was downregulated . the group further showed that while nuclear sirt1 activity correlated with indices of mitochondrial density , nuclear sirt1 protein levels were negatively correlated in skeletal and heart muscles ( gurd et al . , 2011 ) . while training did not alter muscle or nuclear sirt1 protein content in human muscle , it did increase muscle and nuclear pgc-1 and sirt1 activity . thus , it is an increased nuclear sirt1 activity that may actually contribute to exercise - stimulated mitochondrial biogenesis in muscle . from a more fundamental perspective , hancock and colleagues ( hancock et al . , 2011 ) have reexamined the claim that cr resulted in dramatic increases in mitochondrial biogenesis in multiple mouse tissues ( nisoli et al . , 2005 ) . in contrast to previous work , the authors found no elevation in pgc-1 levels after cr , and no evidence of any significant elevation in mitochondrial biogenesis markers in heart , brain , skeletal muscle , liver and adipose tissues . although the authors did not report any measurement of sirt1 level / activity status or pgc-1 s acetylation status , the study does challenge the reproducibility of the cr - induced mitochondrial phenomenon . another study directly questioned the role of sirt1 in exercise - induced mitochondrial biogenesis through pgc-1 decetylation ( philp et al . , 2011 ) . floxed and crossed with cre driven by muscle creatine kinase promoter ) , and found that skeletal muscle endurance , mitochondrial oxidative metabolism , and mitochondrial biogenesis were all not impaired . moreover , mice deficient in muscle sirt1 are similar to wild - type mice with regard to pgc-1 expression , nuclear translocation , activity , and deacetylation status . the authors further suggest that it is really the changes in gcn5 acetyltransferase activity that are important in regulating pgc-1 activity after exercise , and sirt1 may not be relevant . another recent study added to the controversy by showing that resveratrol feeding had no effect on mitochondrial proteins in muscle ( higashida et al . , 2013 ) , while high levels of resveratrol activated ampk in c2c12 myotubes in culture , which did increase mitochondrial protein levels . the group further showed that sirt1 silencing or a dominant negative mutant did increase pgc-1 acetylation , and over - expression of sirt1 reduced it . however , the latter actually suppressing , instead of enhanced pgc-1 s co - activator activity , with a consequential reduction in mitochondrial proteins . the discussion above highlights a particularly controversial point pertaining to the effect of resveratrol on pgc-1 and mitochondrial biogenesis , as well as the mode of involvement of the key cellular nutrient and energy status sensor ampk ( hardie , 2011 ) . using an inducible knockout model , sinclair s group has shown that intact sirt1 in mice is required for resveratrol s activity on mitochondrial biogenesis and function ( price et al . , 2012 ) . the authors also showed that with moderate levels of resveratrol , ampk activation in the system was sirt1-dependent . although higher levels of resveratrol could activate ampk in a sirt1-independent manner , improvement in mitochondrial function in both cases of moderate and high dosages of resveratrol was not observed in the absence of sirt1 . that sirt1 may act upstream of ampk has support in a previous report , which showed that sirt1 could potentially deacetylate and activate the major ampk activating kinase , serine / threonine kinase 11 ( stk11 ) ( or liver kinase b1 , lkb1 ) ( lan et al . , 2008 ) . resveratrol s pharmacological effect is multifaceted , and amongst its known effects is the direct activation of ampk ( dasgupta and milbrandt , 2007 ) and inhibition of camp - degrading phosphodiesterases ( park et al . , 2012 ) . the work of dasgupta and milbrandt indicates that resveratrol could directly activate ampk , at least in certain cell types , in a manner that is completely independent of sirt1 activity ( dasgupta and milbrandt , 2007 ) . another report has also shown that ampk deficient mice are fairly resistant to the metabolic improvement effect of resveratrol ( um et al . , 2010 ) . a role for ampk in mitochondrial biogenesis in skeletal muscle and other tissues kukidome et al . , 2006 ; reznick and shulman , 2006 ; reznick et al . , 2007 ; zong et al . , 2002 ) . in skeletal muscle , ampk has been shown to directly phosphorylate pgc-1 ( jger et al . , 2007 ) , and the phosphorylation of thr-177 and ser-538 is required for the pgc-1-dependent induction of its own promoter . it was also demonstrated that a single administration of the ampk activator aicar increased the mrna expression of all pgc-1 isoforms ( tadaishi et al . it is therefore clear that any compound or physiological regimes that elevate ampk levels may also influence pgc-1 levels / activity and consequent mitochondrial biogenesis , in a manner that may be largely independent of sirt1 . while some results , such as those of price et al . ( 2012 ) are in support of sirt1 acting upstream of ampk , others have demonstrated just the opposite . auwerx s group has shown that ampk could at least exert an influence on sirt1 activity by increasing cellular nad levels ( cant et al . , 2009 ; 2010 ) . the work of park et al . ( 2012 ) indicated that resveratrol inhibits camp phosphodiesterases , thus leading to elevated camp levels and the activation of ampk and camp - inducible factors such as the rap exchange factor directly activated by camp 1 ( epac1 ) . the authors showed that epac1 is required for resveratrol - mediated increases in nad levels , which were abolished by silencing of epac1 . what could be discerned from these remarkably different reports is that the connection between sirt1 , ampk and pgc-1 is nonlinear , is definitely cell / tissue - type dependent and is influenced by energy and nutrient status . given sirt1 s role in mitochondrial biogenesis discussed above , it might appear paradoxical that sirt1 has also been recently implicated in the opposite process , namely , the destruction of damaged or aged mitochondria via mitophagy ( yoshii and mizushima , 2015 ) . a detailed examination of mitophagy and its mechanism is beyond the scope of this review , and the reader is referred to several excellent recent review articles on this topic ( durcan and fon , 2015 ; eiyama and okamoto , 2015 ; hamacher - brady and brady , 2015 ; yoshii and mizushima , 2015 ) . mitophagy is critically dependent on two factors - the pten - induced putative kinase 1 ( pink1 ) and the e3 ubiquitin ligase parkin . these proteins act in sensing the functional and health statuses of mitochondria , and mark damaged mitochondria for autophagic disposal ( eiyama and okamoto , 2015 ) . sirt1 s regulation of general macroautophagy is well known ( ng and tang , 2013 ) and is broadly viewed as a cellular protective mechanism against stress and death insults ( ou et al . , 2014 ; suzuki and bartlett , 2014 ) . a connection between sirt1 and mitophagy was first shown by hwang s group , when the authors found that nicotinamide treatment of primary human fibroblasts extended their replicative lifespan apparently by accelerating autophagic degradation of mitochondria ( kang and hwang , 2009 ) . nicotinamide decreased mitochondrial mass and increased mitochondrial membrane potential , with elevations of the autophagosome marker light chain 3 ( lc3)-ii and levels of proteins that regulate mitochondrial fusion and fission . a subsequent paper from the hwang lab indeed suggested that the mitophagic effect of nicotinamide is mediated through an increase of the nad / nadh ratio and sirt1 activation ( jang et al . , 2012 ) , and mitochondrial dysfunction in xeroderma pigmentosum group a ( xpa ) , a nucleotide excision dna repair disorder , is characterized by defective mitophagy ( fang et al . , 2014 ) . this is apparently due to hyperactivation of the dna damage sensor parp-1 , a major nad consuming enzyme , and the consequent lowering of sirt1 activity . another recent report indicated that the sirt1 activator resveratrol acts through a signaling axis that involves sirt1-sirt3 and pink1/parkin - mediated mitophagy , resulting in cardio - protection ( das et al . , 2014 ) . furthermore , loss of sirt1 in the luminal epithelium in human prostate cancer delayed parkin translocation to the mitochondria and reduced mitophagy ( di sante et al . , 2015 ) . a particularly interesting aspect of the findings discussed above is sirt1 s known role in protection against neurodegenerative disorders ( ng et al . , 2015 ; tang , 2009 ; zhang et al . , 2011 ) . over the years , sirt1 activity has been extensively associated with neuroprotection , and autophagy ( or more specifically , mitophagy ) induction could be part of its protective repertoire . sirt1 activity is known to attenuate -synuclein aggregation and toxicity , a hallmark of parkinson s disease ( pd ) ( albani et al . , 2009 ; donmez et al . , 2012 ; sampaio - marques et al . , 2012 ) . the mitophagy regulators pink1 and parkin are both encoded by pd susceptibility genes that are mutated in patients with juvenile or early - onset parkinsonism ( mullin and schapira , 2015 ) . interestingly , a genetic screen in drosophila found that ectopic expression of the fly sir2 rescued mitochondrial defects in pink1-null mutants ( but not parkin mutants ) ( koh et al . , 2012 ) . a better known connection between sirt1 and neurodegenerative diseases is not a mitophagy factor , but rather pgc-1 ( rna - vrs and weydt , 2010 ; tsunemi and la spada , 2012 ) . pgc-1 suppression promotes -synuclein accumulation in cellular models of pd ( ebrahim et al . , 2010 ) . the zn - finger protein parkin - interacting substrate ( paris , or znf746 ) accumulates in cellular models of parkin inactivation and in the human pd brain ( shin et al . , 2011 ) . paris represses the expression of pgc-1 and nrf-1 , and transgenic overexpression of paris leads to the selective loss of dopaminergic neurons in the substantia nigra , the disease - susceptible neuron group in pd . another report indicated that loss of parkin in cells from patients with early - onset pd increased pgc-1 , but the elevated pgc-1 appeared transcriptionally non - functional ( pacelli et al . , 2011 ) . meta - analysis of gene expression profiles and molecular pathways clearly implicated pgc-1-responsive genes amongst those downregulated in pd co - transduction of adenovirus carrying human pgc-1 in rat e17 mid - brain neuron cultures attenuated dopaminergic neuron loss induced by either mutant -synuclein , or the mitochondrial respiratory chain complex i - inhibitor rotenone . transgenic mice that over - express pgc-1 in substantia nigra s dopaminergic neurons are resistant to degeneration induced by the neurotoxin mptp ( mud et al . , 2012 ) . loss of pgc-1 is also a well - known feature in huntington s disease ( hd ) , a hereditary disorder where aggregation of mutant huntingtin protein with an extended polyglutamine tract causes striatal neuron loss ( ross and tabrizi , 2011 ) . nuclear accumulation of mutant huntingtin repressed pgc-1 gene transcription by associating with its promoter ( cui et al . , 2006 ) , and over - expression of pgc-1 could partially reverse the toxicity of mutant huntingtin in cultured striatal neurons . interestingly , nicotinamide could upregulate pgc-1 and the neuroprotective brain - derived neurotrophic factor ( bdnf ) in a transgenic mouse model of hd , and improve motor deficits ( hathorn et al . , 2011 ) . another study with inducible pgc-1 expression in hd mice not only rescued the hd phenotype and spared neurons , but could result in significant elimination of mutant huntingtin aggregates ( la spada , 2012 ) . given pgc-1 s beneficial effect on a multitude of neurodegenerative pathologies , it is likely that the neuroprotective effect of sirt1 may , at least in part , be exerted through activation of pgc-1. while all of the above results appear to attest to the promise of pgc-1 as a drug target in pd and hd , one must avoid generalizing pgc-1 s potential beneficial effects on other neurodegenerative diseases . in particular , a recent report has shown that crossing of an alzheimer s disease ( ad ) mouse model ( tg19959 mice ) with transgenic strains over - expressing human pgc-1 exacerbated amyloid and tau accumulation ( dumont et al . , sirt1 s protective activity against the detriments of mitochondrial dysfunction involves other sirt1 substrates , particularly transcription factors that influence mitochondrial activity . one such group of factors are members of the forkhead box o ( foxo ) transcription factor family ( webb and brunet , 2014 ) . sirt1 is known to deacetylate members of the foxo family , particularly in response to stress ( brunet et al . , 2004 ) . drosophila sir2 expression prevented the demise of fly dopaminergic neurons in pink1-null mutants in a dfoxo - dependent manner ( koh et al . , 2012 ) . activation of foxo3a , in particular , has been shown to inhibit mitochondrial gene expression , causing a reduction in mitochondrial dna copy number and respiratory activity ( ferber et al . , 2012 ) . in fact , a declined in nuclear nad , and a consequentially diminished sirt1 activity , was also found to underlie a specific loss of mitochondrial - encoded subunits of the oxidative phosphorylation system ( gomes et al . , 2013 ) . interestingly , foxo3a could translocate into the mitochondria where it could potentially be a substrate of mitochondrial sirt3 ( jacobs et al . , foxo3a mitochondrial translocation was also shown to occur during gtp - induced erythroid differentiation ( meshkini and yazdanparast , 2012 ) . recent work in c. elegans has indicated that worm sir2.1 mediates longevity via the mitochondrial unfolded protein response ( upr ) and the activation of the worm foxo orthologue daf-16 ( mouchiroud et al . , 2013 ) . it was further shown that inhibition of the mitochondrial chaperone trap1 generates a foxo - dependent retrograde cell protective signal from the mitochondria to the nucleus ( kim et al . , 2015 ) . sirt1 s modulation of foxo activity could therefore influence mitochondria function via gene expression in the nucleus , as well as affecting retrograde signaling from the mitochondria to the nucleus . a few other sirt1-modified transcription factors could also have a role , either directly or indirectly , in mitochondrial activity and function . sirt1 deacetylates and represses the activity of the hypoxia inducible factor 1-alpha ( hif-1 ) ( lim et al . , 2010 ) . hif-1 is known to repress mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 ( pdk1 ) ( papandreou et al . , 2006 ) , and sirt1 activity may thus influence mitochondrial respiratory function via the former . one prominent transcription factor substrate of sirt1 is the tumor suppressor tp53 ( vaziri et al . , 2001 ) , and sirt1 deacetylation of p53 suppresses its transactivation capacity and promotes cell survival ( luo et al . , 2001 ) interestingly , it has been shown that mouse embryonic stem ( mes ) cells produces endogenous reactive oxygen species ( ros ) , which causes p53 translocation into the mitochondria in the presence of sirt1 , but induces p53 nuclear translocation in sirt1-null mes cells ( han et al . , 2008 ) . sirt1 activity may therefore influence mitochondria - mediated apoptosis by influencing mitochondrial translocation of factors such as p53 . another transcription factor that is deacetylated by sirt1 which has known roles in mitochondrial physiology is the nuclear factor erythroid 2-related factor 2 ( nrf2 ) ( kawai et al . , 2011 ) . nrf2 and its inhibitor , the kelch - like ech associated protein 1 ( keap1 ) , are master regulators of the cellular anti - oxidative response , and have key roles in cellular bioenergetics ( dinkova - kostova et al . , 2015 ) . it is clear from these examples that sirt1 activity exerts a complex and multi - effector mediated influence on mitochondrial functions . in this review , i discussed evidence for sirt1 s regulation of mitochondrial biogenesis and turnover , particularly in relation to pgc-1 ( see fig . the emerging view in the field is that sirt1 activity could exert important influence on mitochondrial function , but the degree of influence is likely cell type- and physiological context - dependent . sirt1 activation could promote mitochondrial biogenesis in conditions of energy deficiency associated with disease and injury . on the other hand , it could also have an important role in triggering the demise or turnover of damaged mitochondria . should one pathway occur preferentially over the other , which pathway will predominate would likely depend on the nature of disease / injury , the relevant sirt1 substrates available , and the cell s energetic and metabolic status , such as the availability of nad . for example , factors that result in direct mitochondrial damage may induce a higher rate of mitophagy . in terminally differentiated cells with acute energy requirements such as neurons , sustaining a steady supply of mitochondria and salvaging whatever that remains functional could be important to ensure survival . in dividing cells , a quick mitophagic clearance of damaged mitochondria may be more urgent so that these are not passed down to daughter cells during division . on the whole , sirt1 s regulation of mitochondrial biogenesis and mitophagy could thus act in concert for mitochondria quality maintenance . the large regulatory repertoire of sirt1 could potentially be exploited for therapeutics , but a better understanding of the molecular factors it influences and their dynamic interactions would be necessary .

sirt1 is the most prominent and extensively studied member of sirtuins , the family of mammalian class iii histone deacetylases heavily implicated in health span and longevity . although primarily a nuclear protein , sirt1 s deacetylation of peroxisome proliferator - activated receptor gamma coactivator-1 ( pgc-1 ) has been extensively implicated in metabolic control and mitochondrial biogenesis , which was proposed to partially underlie sirt1 s role in caloric restriction and impacts on longevity . the notion of sirt1 s regulation of pgc-1 activity and its role in mitochondrial biogenesis has , however , been controversial . interestingly , sirt1 also appears to be important for the turnover of defective mitochondria by mitophagy . i discuss here evidences for sirt1 s regulation of mitochondrial biogenesis and turnover , in relation to pgc-1 deacetylation and various aspects of cellular physiology and disease .

INTRODUCTION SIRT1S MITOCHONDRIAL LOCALIZATION SIRT1S DEACETYLATION OF PGC - 1 - METABOLIC REGULATION AND MITOCHONDRIAL BIOGENESIS THE CONNECTION BETWEEN SIRT1, AMPK AND PGC-1 IN REGULATING MITOCHONDRIAL FUNCTION SIRT1 AND MITOPHAGY SIRT1, PGC-1 AND NEURODEGENERATIVE DISEASES OTHER SIRT1-REGULATED FACTORS/SUBSTRATES IN MITOCHONDRIAL FUNCTION CONCLUSION

this large family of enzymes is often referred to as histone deacetylases ( hdacs ) , but histones are certainly not their only substrates . class ii hdacs ( namely hdacs 4 , 5 , 6 , 7 , 9 and 10 ) , which are homologues of yeast histone deacetylase 1 ( hda1 ) , shuttle between the nucleus and the cytoplasm , and are more tissue specific in terms of expression . hdac11 has only fairly low homology to both rpd3 and hda1 , and has been proposed to constitute a separate class iv ( gregoretti et al . lysine deacetylation by sirtuins is coupled to the cleavage of a key intermediate of cellular energy metabolism , nicotinamide adenine dinucleotide ( nad ) , into nicotinamide and 1-o - acetyl - adp - ribose ( tanner et al . the activities of sirtuins are thus obligatorily dependent on cellular nad , inhibited by the reaction product nicotinamide , and effectively influenced by cellular metabolic and redox states . sir2 and its metazoan orthologues have been prominently associated with lifespan extension ( howitz et al . , 2003 ; tissenbaum and guarente , 2001 ) , in particular that induced by caloric or dietary restriction ( cr / dr ) ( rogina and helfand , 2004 ; wood et al . a large number of studies have connected sirt1 expression and/or activation by compounds such as resveratrol , with increase health span and lifespan ( guarente , 2011 ) . however , the notion that sirt1 is a mediator of cr effects and a longevity factor in mammals and humans has been controversial in two major ways . sirt1 s role in longevity has been more extensively investigated in yeast and lower invertebrate models , and although the notion of sirt1 as a longevity gene has a great deal of support , there were also controversies and evidence to the contrary ( burnett et al . sirt1 s knockout phenotype was difficult to interpret , as the mice often died early due largely to developmental defects ( cheng et al . , 2003 earlier work on transgenic over - expression of sirt1 in mice did not result in a significant increase in lifespan ( herranz et al . more recent findings have , however , provided some reconciliation of the contrasting results and opinions . the mode of action of sirtuin - activating compounds ( stacs ) , possibly via an allosteric mechanism , has been better clarified ( hubbard et al . indeed , dissection of sirt1 s role in cr and aging is difficult because sirt1 has multiple physiological functions as well as wide - ranging roles in pathological settings . sirt1 represses a key metabolic regulator , peroxisome proliferator - activated receptor gamma ( ppar ) ( picard et al . , 2010 ) , via deacetylation of ppar coactivator-1 ( pgc-1 ) ( gerhart - hines et al . , 2005 ) , which regulates energy metabolism in skeletal muscle , adipose tissues and the liver . sirt1 has also been implicated in cellular metabolism through its activation of amp - dependent kinase ( ampk ) via liver kinase b1 ( lkb1 ) ( hou et al . of the seven mammalian sirtuins , three ( sirt3 , sirt4 and sirt5 ) have a primary mitochondrial localization ( michishita et al . sirt1 , on the other hand , is primarily nuclear , but its activities have large bearings on mitochondrial biogenesis and turnover . in the ensuing paragraphs we first look at some evidence for the predominantly nuclear sirt1 being physically localized at the mitochondria . sirt1 is primarily found in the nucleus of most cell types ( michishita et al . both sirt1 and suv39h1 are required for the epigenetic control of the rdna locus for rrna synthesis ( murayama et al . , 2008 ) , and sirt1 befitting its nuclear localization , a majority of sirt1 s known function is associated with deacetylation of transcription factors , as mentioned in the section above . as per all nuclear proteins sirt1 , however , appears to possess both nuclear localization signals and nuclear export signals , and could shuttle between the cytoplasm and the nucleus ( tanno et al . sirt1 s cytoplasmic appearance has been associated with enhanced apoptotic cell death ( jin et al . , 2007 ) , and also shown to be due to an increased protein stability via oncogenic signaling through the insulin - like growth factor-1-phosphoinositide 3-kinase ( igf-1/pi3 kinase ) axis in cancer cells ( byles et al . on the other hand , sirt1 does have cytosolic substrates whose deacetylation by sirt1 results in distinct physiological or functional consequences . for instance , the cytoplasmic acetyl - coa synthetase ( hallows et al . sirt1 and its substrate , pgc-1 , regulate aspects of energy metabolism through the mitochondria , but this was initially thought to occur through their influence on nuclear localized transcription . a rather interesting aspect of sirt1 s activity and function that is less well defined is its extranuclear localization , particularly at the mitochondria ( aquilano et al . within the mitochondria , both deacetylase and its substrate are associated with the mitochondrial dna ( mtdna ) nucleoids ( bogenhagen , 2012 ) , as well as with the mitochondrial transcription factor a ( tfam ) , a key mitochondrial gene transcription factor and regulator of mtdna copy number ( campbell et al . however , the degree of mitochondrial sirt1 and pgc-1 s functionality in this regard is still uncertain . in the sections that follow , we shall outline findings that point to how sirt1 mediates aspects of mitochondrial biogenesis and turnover , as well as the controversies associated with these findings . however , within all cells , either dividing or terminally differentiated , the mitochondria population is constantly renewed , and the steady state number of this organelle represents an equilibrium between mitochondrial biogenesis and eventual degradation through the process of mitophagy ( stotland and gottlieb , 2015 ; vega et al . mitochondrial biogenesis is orchestrated by the peroxisome proliferator - activated receptor gamma coactivator-1 ( pgc-1 ) family of transcriptional coactivators ( austin and st - pierre , 2012 ) . pgc-1 , in particular , is often cited as a master regulator of this process . pgc-1 co - activates the transcription of nuclear respiratory factor ( nrf ) 1 and 2 , which , in turn , regulate the transcription of tfam . tfam translocates to mitochondrial matrix where it stimulates mitochondrial dna replication and mitochondrial gene expression . pgc-1 undergoes several modes of post - translational modification , which includes acetylation and phosphorylation . acetylation of pgc-1 occurs at several of its lysine residues and is catalyzed by the ubiquitous histone acetyl transferase general control of amino acid synthesis 5 ( gcn5 ) ( dominy et al . conversely , deacetylation of pgc-1 was shown by several studies to be dependent on sirt1 activity ( amat et al . , 2005 ) , which increases pgc-1 s transcriptional activity . pgc-1 is phosphorylated by both mitogen - activated protein kinase p38 ( barger et al . on the other hand , phosphorylation of pgc-1 by akt / protein kinase b ( pkb ) , downstream of insulin signaling in the liver , is known to decrease its stability and transcriptional activity ( li et al . given the importance of its acetylation status to pgc-1 activity , the connection between sirt1 and pgc-1 has been a focus of investigations into metabolic regulation and mitochondrial biogenesis . , 2005 ) , and this interaction could be distinguished by a single amino acid mutation in the putative adp - ribosyltransferase domain of sirt1 . this mutation abolishes sirt1 s interaction with pgc-1 , but not with two other major substrates , p53 and foxo3a . cr has been shown to significantly increase mitochondrial biogenesis in multiple tissues in mice ( nisoli et al . , 2005 ) and early work from puigserver s group showed that sirt1 is induced during fasting , and induced sirt1 interacts with pgc-1 to increase the expression of hepatic gluconeogenic genes ( rodgers et al . the group also showed that fasting induced pgc-1 deacetylation by sirt1 in skeletal muscle , which is required for the activation of mitochondrial fatty acid oxidation genes ( gerhart - hines et al . auwerx s group , on the other hand , showed that resveratrol treatment of mice increased running time and oxygen consumption by the skeletal muscles , which is accompanied by the induction of genes for oxidative phosphorylation and mitochondrial biogenesis ( lagouge et al . the authors attributed these changes to activation of sirt1 , and a consequential decrease in pgc-1 acetylation and activity . the latter s nuclear transcription activity is enhanced by sirt1-dependent nuclear accumulation and is counter regulated by glycogen synthase kinase beta ( gsk3 ) , which targets pgc-1 for intranuclear proteasomal degradation . despite the apparently convincing functional connection between sirt1 and pgc-1 , whether sirt1 is critically or directly involved in mitochondrial biogenesis has been controversial . , 2009 ) examined the relationship between sirt1 levels and activity , pgc-1 and markers of mitochondrial density ( such as cytochrome c oxidase subunit iv , coxiv ) in skeletal and heart muscles , and surprisingly found a largely negative correlation between sirt1 levels and the rest of the parameters . in fact , overexpression of sirt1 in muscle downregulated pgc-1 and mttfa levels , and while muscle stimulation and the ampk activator aicar upregulated pgc-1 and cox iv , sirt1 level was downregulated . the group further showed that while nuclear sirt1 activity correlated with indices of mitochondrial density , nuclear sirt1 protein levels were negatively correlated in skeletal and heart muscles ( gurd et al . thus , it is an increased nuclear sirt1 activity that may actually contribute to exercise - stimulated mitochondrial biogenesis in muscle . , 2011 ) have reexamined the claim that cr resulted in dramatic increases in mitochondrial biogenesis in multiple mouse tissues ( nisoli et al . in contrast to previous work , the authors found no elevation in pgc-1 levels after cr , and no evidence of any significant elevation in mitochondrial biogenesis markers in heart , brain , skeletal muscle , liver and adipose tissues . although the authors did not report any measurement of sirt1 level / activity status or pgc-1 s acetylation status , the study does challenge the reproducibility of the cr - induced mitochondrial phenomenon . another study directly questioned the role of sirt1 in exercise - induced mitochondrial biogenesis through pgc-1 decetylation ( philp et al . floxed and crossed with cre driven by muscle creatine kinase promoter ) , and found that skeletal muscle endurance , mitochondrial oxidative metabolism , and mitochondrial biogenesis were all not impaired . the authors further suggest that it is really the changes in gcn5 acetyltransferase activity that are important in regulating pgc-1 activity after exercise , and sirt1 may not be relevant . the group further showed that sirt1 silencing or a dominant negative mutant did increase pgc-1 acetylation , and over - expression of sirt1 reduced it . however , the latter actually suppressing , instead of enhanced pgc-1 s co - activator activity , with a consequential reduction in mitochondrial proteins . the discussion above highlights a particularly controversial point pertaining to the effect of resveratrol on pgc-1 and mitochondrial biogenesis , as well as the mode of involvement of the key cellular nutrient and energy status sensor ampk ( hardie , 2011 ) . using an inducible knockout model , sinclair s group has shown that intact sirt1 in mice is required for resveratrol s activity on mitochondrial biogenesis and function ( price et al . although higher levels of resveratrol could activate ampk in a sirt1-independent manner , improvement in mitochondrial function in both cases of moderate and high dosages of resveratrol was not observed in the absence of sirt1 . that sirt1 may act upstream of ampk has support in a previous report , which showed that sirt1 could potentially deacetylate and activate the major ampk activating kinase , serine / threonine kinase 11 ( stk11 ) ( or liver kinase b1 , lkb1 ) ( lan et al . the work of dasgupta and milbrandt indicates that resveratrol could directly activate ampk , at least in certain cell types , in a manner that is completely independent of sirt1 activity ( dasgupta and milbrandt , 2007 ) . a role for ampk in mitochondrial biogenesis in skeletal muscle and other tissues kukidome et al . it is therefore clear that any compound or physiological regimes that elevate ampk levels may also influence pgc-1 levels / activity and consequent mitochondrial biogenesis , in a manner that may be largely independent of sirt1 . ( 2012 ) are in support of sirt1 acting upstream of ampk , others have demonstrated just the opposite . the authors showed that epac1 is required for resveratrol - mediated increases in nad levels , which were abolished by silencing of epac1 . given sirt1 s role in mitochondrial biogenesis discussed above , it might appear paradoxical that sirt1 has also been recently implicated in the opposite process , namely , the destruction of damaged or aged mitochondria via mitophagy ( yoshii and mizushima , 2015 ) . sirt1 s regulation of general macroautophagy is well known ( ng and tang , 2013 ) and is broadly viewed as a cellular protective mechanism against stress and death insults ( ou et al . , 2012 ) , and mitochondrial dysfunction in xeroderma pigmentosum group a ( xpa ) , a nucleotide excision dna repair disorder , is characterized by defective mitophagy ( fang et al . a particularly interesting aspect of the findings discussed above is sirt1 s known role in protection against neurodegenerative disorders ( ng et al . over the years , sirt1 activity has been extensively associated with neuroprotection , and autophagy ( or more specifically , mitophagy ) induction could be part of its protective repertoire . interestingly , a genetic screen in drosophila found that ectopic expression of the fly sir2 rescued mitochondrial defects in pink1-null mutants ( but not parkin mutants ) ( koh et al . paris represses the expression of pgc-1 and nrf-1 , and transgenic overexpression of paris leads to the selective loss of dopaminergic neurons in the substantia nigra , the disease - susceptible neuron group in pd . loss of pgc-1 is also a well - known feature in huntington s disease ( hd ) , a hereditary disorder where aggregation of mutant huntingtin protein with an extended polyglutamine tract causes striatal neuron loss ( ross and tabrizi , 2011 ) . , 2006 ) , and over - expression of pgc-1 could partially reverse the toxicity of mutant huntingtin in cultured striatal neurons . interestingly , nicotinamide could upregulate pgc-1 and the neuroprotective brain - derived neurotrophic factor ( bdnf ) in a transgenic mouse model of hd , and improve motor deficits ( hathorn et al . given pgc-1 s beneficial effect on a multitude of neurodegenerative pathologies , it is likely that the neuroprotective effect of sirt1 may , at least in part , be exerted through activation of pgc-1. while all of the above results appear to attest to the promise of pgc-1 as a drug target in pd and hd , one must avoid generalizing pgc-1 s potential beneficial effects on other neurodegenerative diseases . , sirt1 s protective activity against the detriments of mitochondrial dysfunction involves other sirt1 substrates , particularly transcription factors that influence mitochondrial activity . sirt1 is known to deacetylate members of the foxo family , particularly in response to stress ( brunet et al . activation of foxo3a , in particular , has been shown to inhibit mitochondrial gene expression , causing a reduction in mitochondrial dna copy number and respiratory activity ( ferber et al . in fact , a declined in nuclear nad , and a consequentially diminished sirt1 activity , was also found to underlie a specific loss of mitochondrial - encoded subunits of the oxidative phosphorylation system ( gomes et al . interestingly , foxo3a could translocate into the mitochondria where it could potentially be a substrate of mitochondrial sirt3 ( jacobs et al . a few other sirt1-modified transcription factors could also have a role , either directly or indirectly , in mitochondrial activity and function . one prominent transcription factor substrate of sirt1 is the tumor suppressor tp53 ( vaziri et al . , 2001 ) , and sirt1 deacetylation of p53 suppresses its transactivation capacity and promotes cell survival ( luo et al . , 2001 ) interestingly , it has been shown that mouse embryonic stem ( mes ) cells produces endogenous reactive oxygen species ( ros ) , which causes p53 translocation into the mitochondria in the presence of sirt1 , but induces p53 nuclear translocation in sirt1-null mes cells ( han et al . another transcription factor that is deacetylated by sirt1 which has known roles in mitochondrial physiology is the nuclear factor erythroid 2-related factor 2 ( nrf2 ) ( kawai et al . nrf2 and its inhibitor , the kelch - like ech associated protein 1 ( keap1 ) , are master regulators of the cellular anti - oxidative response , and have key roles in cellular bioenergetics ( dinkova - kostova et al . in this review , i discussed evidence for sirt1 s regulation of mitochondrial biogenesis and turnover , particularly in relation to pgc-1 ( see fig . sirt1 activation could promote mitochondrial biogenesis in conditions of energy deficiency associated with disease and injury . on the other hand , it could also have an important role in triggering the demise or turnover of damaged mitochondria . should one pathway occur preferentially over the other , which pathway will predominate would likely depend on the nature of disease / injury , the relevant sirt1 substrates available , and the cell s energetic and metabolic status , such as the availability of nad . in terminally differentiated cells with acute energy requirements such as neurons , sustaining a steady supply of mitochondria and salvaging whatever that remains functional could be important to ensure survival . on the whole , sirt1 s regulation of mitochondrial biogenesis and mitophagy could thus act in concert for mitochondria quality maintenance . the large regulatory repertoire of sirt1 could potentially be exploited for therapeutics , but a better understanding of the molecular factors it influences and their dynamic interactions would be necessary .

acute otitis media is one of the most common childhood infections and the leading cause for children to visit a doctor . most children experience at least one episode of otitis media ; in 5085% of the children acute otitis media is diagnosed at least once in the first 3 years of life [ 2 , 3 ] . in addition to host factors like birth weight , gestational age and craniofacial abnormalities , environmental factors like passive smoking , day care attendance , socioeconomic status , pacifier use and breast - feeding have been studied . frequency of colonization with bacterial airway pathogens was shown to be associated with otitis media in the first years of life [ 48 ] . streptococcus pneumoniae , haemophilus influenzae and moraxella catarrhalis are the most frequently cultured pathogens in acute otitis media . early age of colonization seems to increases the risk of otitis media [ 4 , 8 , 9 ] . syrjnen et al . have suggested that carriage of newly acquired bacteria is associated with otitis media . our aim was to study in a population - based prospective cohort the risk factors for otitis media in children with special emphasis on the role of colonization with s. pneumoniae , h. influenzae and m. catarrhalis in the first year of life . this study was embedded in the generation r study , a population - based prospective cohort study from fetal life until young adulthood . the generation r study has been described in detail previously [ 11 , 12 ] . detailed assessments of fetal and postnatal growth and development were conducted in 1,232 dutch pregnant women and their children in the generation r focus study . the medical ethics committee of the erasmus medical centre , rotterdam , has approved the study . written informed consent we obtained data on birth weight , gestational age and gender from midwives and hospital registries . information about maternal smoking , siblings , educational level of the mother , day care attendance , breast - feeding , pacifier use and otitis media in the first year of life was obtained by postal questionnaires at the infants age of 6 , 12 and 24 months . mothers were asked whether their children suffered from fever in the prior period ( no , yes ) , and subsequently whether this period of fever was accompanied by earache and whether they had visited a doctor . the diagnosis otitis media in the period from 0 to 6 months of age and the period from 6 to 12 months of age was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the 6 months before reaching the age of respectively 6 and 12 months . likewise , the diagnosis of otitis media in the second year of life was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the year before reaching the age of 24 months . furthermore , otitis - proneness was defined as having four or more episodes by 1 year of age , six or more by 2 years of age , or placement of myringotomy tubes , similar to the definition used by faden et al . . risk factors for otitis media in the second year of life were studied in the whole cohort . in the focus cohort , samples were taken at the infants age of 1.5 , 6 , and 14 months to detect nasopharyngeal bacterial carriage . nasopharyngeal samples were taken with rayon tipped dacron pernasal swabs ( copan italia , brescia , italy ) and transported in amies transport medium and plated within 6 h of sampling on a blood agar plate with 5% sheep blood , a chocolate agar plate and a heamophilus selective agarplate . the plates are kept at 35c in a co2 rich environment for 2 days . all bacteria were determined by use of standard methods . in the whole cohort associations of birth weight , gestational age , gender , maternal smoking , siblings , educational level of mother , day care attendance , duration of breast - feeding , pacifier use and otitis media in the first year of life with otitis media in the second year of life were assessed by logistic regression models resulting in odds ratios ( or ) with their 95% confidence interval ( ci ) . additionally , a multivariate logistic regression model was performed to assess the association of each risk factor separately with otitis media in the second year of life was adjusted for all the other risk factors resulting in adjusted odds ratios ( aor ) . in the focus cohort association of bacterial carriage in the first year of life with otitis media in the second year of life was assessed for the airway pathogens s. pneumoniae , m. catarrhalis and h. influenzae . the analyses were performed using frequency of specific bacterial carriage in the first year of life , and at ages of first bacterial carriage . furthermore , we did the same analysis with the three bacteria grouped as any airway pathogen . additionally , regression models were adjusted including all determinants presented in table 1 . furthermore , possible interaction between otitis media in the first year of life and bacterial carriage and the risk of otitis media in the second year of life was tested . according to the national dutch vaccination policy all children were vaccinated against h. influenzae b , but non of the children were vaccinated against pneumocci.table 1population descriptiveotitis media 1224 months of agen = 5,323non = 2,808 ( 52.8%)yesn = 2,515 ( 47.2%)oraorbirthweight3,438 ( 574)3,422 ( 577)3,456 ( 570)1.11 * ( 1.011.22)1.07 ( 0.841.37)gestational age40.0 ( 25.343.4)40.0 ( 25.343.3)40.1 ( 27.143.4)1.02 ( 0.991.05)1.04 ( 0.971.12)gender male ( ref)2,643 ( 49.7)48.4%51.0% female2,680 ( 50.3)51.6%49.0%0.90 ( 0.811.00)0.89 ( 0.721.11)smoking no ( ref)3,110 ( 84.9)85.1%84.6% yes554 ( 1 . 15)14.9%15.4%1.04 ( 0.871.25)1.16 ( 0.861.55)siblings no ( ref)2,159 ( 58.9)62.9%54.1% yes1,508 ( 41.1)37.1%45.9%1.44 * * * ( 1.261.65)1.42 * ( 1.131.79)educational level mother low292 ( 5.8)4.7%7.0% middle1,911 ( 37.8)37.3%38.4%0.69 * * ( 0.540.88)0.84 ( 0.361.95 ) high2,851 ( 56.4)58.1%54.5%0.62 * * * ( 0.490.80)1.03 ( 0.442.40)day care no ( ref)730 ( 22.3)21.3%23.5% yes2,543 ( 77.7)78.7%76.5%0.88 ( 0.751.04)0.86 ( 0.661.13)breastfeeding never ( ref)450 ( 19.0)17.7%20.4% less than 3 months1,031 ( 43.5)46.3%40.3%0.76 * ( 0.610.95)0.86 ( 0.631.16 ) more than 3 months890 ( 37.5)36.0%39.3%0.95 ( 0.761.19)1.14 ( 0.831.55)pacifier use no ( ref)1,242 ( 34.4)34.5%34.4% yes2,364 ( 65.6)65.5%65.6%1.01 ( 0.881.16)0.95 ( 0.751.20)otitis from 0 to 6 months no ( ref)3,309 ( 91.9)94.7%88.5% yes292 ( 8.1)5.3%11.5%2.34 * * * ( 1.823.00)1.83 * * ( 1.242.71)otitis from 6 to 12 months no ( ref)2,808 ( 52.8)67.4%45.8% yes2,515 ( 47.2)32.6%54.2%2.44 * * * ( 2.162.76)2.72 * * * ( 2.183.38)values of birth weight is mean ( standard deviation ) , gestational age is median ( range ) . other values are absolute numbers ( percentages ) . data were missing on birth weight ( n = 8) , gestational age ( n = 1 ) , maternal smoking ( n = 1,659 ) , siblings ( n = 1,656 ) , educational level of mother ( n = 269 ) , day care attendance ( n = 2,050 ) , breast feeding ( n = 2,952 ) , pacifier use ( n = 1,717 ) , otitis from 0 to 6 months ( n = 1,722 ) and otitis from 6 to 12 months ( n = 815 ) . birthweight entered in analyses in kg . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 population descriptive values of birth weight is mean ( standard deviation ) , gestational age is median ( range ) . data were missing on birth weight ( n = 8) , gestational age ( n = 1 ) , maternal smoking ( n = 1,659 ) , siblings ( n = 1,656 ) , educational level of mother ( n = 269 ) , day care attendance ( n = 2,050 ) , breast feeding ( n = 2,952 ) , pacifier use ( n = 1,717 ) , otitis from 0 to 6 months ( n = 1,722 ) and otitis from 6 to 12 months ( n = 815 ) . birthweight entered in analyses in kg . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 differences of infant characteristics between infants with and without data on otitis media in the second year of life were assessed by the independent sample t - test for continuous normal distributed variables , non - parametric mann whitney test for continuous non - normal distributed variables and the chi - square test for categorical variables . differences of frequency of otitis media in the second year of life between groups with missing risk factors and complete data were assessed in the same way . the statistical analyses were performed using the statistical package of social sciences version 11.0 for windows ( spss inc , chicago , il , usa ) . this study was embedded in the generation r study , a population - based prospective cohort study from fetal life until young adulthood . the generation r study has been described in detail previously [ 11 , 12 ] . detailed assessments of fetal and postnatal growth and development were conducted in 1,232 dutch pregnant women and their children in the generation r focus study . the medical ethics committee of the erasmus medical centre , rotterdam , has approved the study . written informed consent we obtained data on birth weight , gestational age and gender from midwives and hospital registries . information about maternal smoking , siblings , educational level of the mother , day care attendance , breast - feeding , pacifier use and otitis media in the first year of life was obtained by postal questionnaires at the infants age of 6 , 12 and 24 months . mothers were asked whether their children suffered from fever in the prior period ( no , yes ) , and subsequently whether this period of fever was accompanied by earache and whether they had visited a doctor . the diagnosis otitis media in the period from 0 to 6 months of age and the period from 6 to 12 months of age was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the 6 months before reaching the age of respectively 6 and 12 months . likewise , the diagnosis of otitis media in the second year of life was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the year before reaching the age of 24 months . furthermore , otitis - proneness was defined as having four or more episodes by 1 year of age , six or more by 2 years of age , or placement of myringotomy tubes , similar to the definition used by faden et al . . risk factors for otitis media in the second year of life were studied in the whole cohort . in the focus cohort , samples were taken at the infants age of 1.5 , 6 , and 14 months to detect nasopharyngeal bacterial carriage . nasopharyngeal samples were taken with rayon tipped dacron pernasal swabs ( copan italia , brescia , italy ) and transported in amies transport medium and plated within 6 h of sampling on a blood agar plate with 5% sheep blood , a chocolate agar plate and a heamophilus selective agarplate . the plates are kept at 35c in a co2 rich environment for 2 days . in the whole cohort associations of birth weight , gestational age , gender , maternal smoking , siblings , educational level of mother , day care attendance , duration of breast - feeding , pacifier use and otitis media in the first year of life with otitis media in the second year of life were assessed by logistic regression models resulting in odds ratios ( or ) with their 95% confidence interval ( ci ) . additionally , a multivariate logistic regression model was performed to assess the association of each risk factor separately with otitis media in the second year of life was adjusted for all the other risk factors resulting in adjusted odds ratios ( aor ) . in the focus cohort association of bacterial carriage in the first year of life with otitis media in the second year of life was assessed for the airway pathogens s. pneumoniae , m. catarrhalis and h. influenzae . the analyses were performed using frequency of specific bacterial carriage in the first year of life , and at ages of first bacterial carriage . furthermore , we did the same analysis with the three bacteria grouped as any airway pathogen . additionally , regression models were adjusted including all determinants presented in table 1 . furthermore , possible interaction between otitis media in the first year of life and bacterial carriage and the risk of otitis media in the second year of life was tested . according to the national dutch vaccination policy all children were vaccinated against h. influenzae b , but non of the children were vaccinated against pneumocci.table 1population descriptiveotitis media 1224 months of agen = 5,323non = 2,808 ( 52.8%)yesn = 2,515 ( 47.2%)oraorbirthweight3,438 ( 574)3,422 ( 577)3,456 ( 570)1.11 * ( 1.011.22)1.07 ( 0.841.37)gestational age40.0 ( 25.343.4)40.0 ( 25.343.3)40.1 ( 27.143.4)1.02 ( 0.991.05)1.04 ( 0.971.12)gender male ( ref)2,643 ( 49.7)48.4%51.0% female2,680 ( 50.3)51.6%49.0%0.90 ( 0.811.00)0.89 ( 0.721.11)smoking no ( ref)3,110 ( 84.9)85.1%84.6% yes554 ( 1 . 15)14.9%15.4%1.04 ( 0.871.25)1.16 ( 0.861.55)siblings no ( ref)2,159 ( 58.9)62.9%54.1% yes1,508 ( 41.1)37.1%45.9%1.44 * * * ( 1.261.65)1.42 * ( 1.131.79)educational level mother low292 ( 5.8)4.7%7.0% middle1,911 ( 37.8)37.3%38.4%0.69 * * ( 0.540.88)0.84 ( 0.361.95 ) high2,851 ( 56.4)58.1%54.5%0.62 * * * ( 0.490.80)1.03 ( 0.442.40)day care no ( ref)730 ( 22.3)21.3%23.5% yes2,543 ( 77.7)78.7%76.5%0.88 ( 0.751.04)0.86 ( 0.661.13)breastfeeding never ( ref)450 ( 19.0)17.7%20.4% less than 3 months1,031 ( 43.5)46.3%40.3%0.76 * ( 0.610.95)0.86 ( 0.631.16 ) more than 3 months890 ( 37.5)36.0%39.3%0.95 ( 0.761.19)1.14 ( 0.831.55)pacifier use no ( ref)1,242 ( 34.4)34.5%34.4% yes2,364 ( 65.6)65.5%65.6%1.01 ( 0.881.16)0.95 ( 0.751.20)otitis from 0 to 6 months no ( ref)3,309 ( 91.9)94.7%88.5% yes292 ( 8.1)5.3%11.5%2.34 * * * ( 1.823.00)1.83 * * ( 1.242.71)otitis from 6 to 12 months no ( ref)2,808 ( 52.8)67.4%45.8% yes2,515 ( 47.2)32.6%54.2%2.44 * * * ( 2.162.76)2.72 * * * ( 2.183.38)values of birth weight is mean ( standard deviation ) , gestational age is median ( range ) . data were missing on birth weight ( n = 8) , gestational age ( n = 1 ) , maternal smoking ( n = 1,659 ) , siblings ( n = 1,656 ) , educational level of mother ( n = 269 ) , day care attendance ( n = 2,050 ) , breast feeding ( n = 2,952 ) , pacifier use ( n = 1,717 ) , otitis from 0 to 6 months ( n = 1,722 ) and otitis from 6 to 12 months ( n = 815 ) . birthweight entered in analyses in kg . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 population descriptive values of birth weight is mean ( standard deviation ) , gestational age is median ( range ) . data were missing on birth weight ( n = 8) , gestational age ( n = 1 ) , maternal smoking ( n = 1,659 ) , siblings ( n = 1,656 ) , educational level of mother ( n = 269 ) , day care attendance ( n = 2,050 ) , breast feeding ( n = 2,952 ) , pacifier use ( n = 1,717 ) , otitis from 0 to 6 months ( n = 1,722 ) and otitis from 6 to 12 months ( n = 815 ) . birthweight entered in analyses in kg . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 differences of infant characteristics between infants with and without data on otitis media in the second year of life were assessed by the independent sample t - test for continuous normal distributed variables , non - parametric mann whitney test for continuous non - normal distributed variables and the chi - square test for categorical variables . differences of frequency of otitis media in the second year of life between groups with missing risk factors and complete data were assessed in the same way . the statistical analyses were performed using the statistical package of social sciences version 11.0 for windows ( spss inc , chicago , il , usa ) . in the generation r cohort , 7,295 children , with a delivery date from april 2002 until january 2006 , participated in the postnatal phase of the study . data on otitis media in the second year of life was available in 5,323 ( response rate 73% ) children . characteristics of the children of the whole cohort and their association with otitis media in the second year of life are presented in table 1 . having siblings is associated with an increased risk of otitis media in the second year of life ( aor 1.42 , ci 1.131.79 ) . furthermore , the occurrence of otitis media during the follow - up period between 0 and 6 months and the occurrence of otitis media during the follow - up period between 6 and 12 months of age was associated with the occurrence of otitis media in the second year of life ( respectively , aor 1.83 , ci 1.242.71 and aor 2.72 , ci 2.183.38 ) . bacterial carriage was studied in the focus cohort , in which 1,079 children , with a delivery date from february 2003 until august 2005 , participated . the children were planned to visit the generation r focus study research centre at the age of 1.5 month ( response rate 81.8% ) , 6 months ( response rate 81.6% ) and 14 months ( response rate 80.0% ) . of the infants 379 ( 43.3% ) had at least one episode of otitis media in the second year of life . in table 2 the associations between the frequency of bacterial carriage in the first year of life and otitis media in the second year of life are presented . we analysed the association between bacterial carriage in the first year of life and otitis media in the second year of life in two different ways . first , we defined carriage by adding the three sampling time points and assessed whether the frequency of bacterial carriage was associated with otitis media . second , we assessed whether early age of bacterial carriage was associated with otitis media ( data not shown ) . in addition to this , we assessed whether bacterial carriage in the first year of life was associated with otitis - proneness . again , no associations were observed ( data not shown).table 2association between bacterial carriage in the first year of life and otitis media in the second year of lifeotitis media 1224 months of agen ( % ) no ( n = 384 ) ( % ) yes ( n = 287 ) ( % ) oraorstreptococcus pneumoniae never ( reference)316 ( 47.1)47.147.0referencereference once241 ( 35.9)35.436.61.04 ( 0.741.45)0.64 ( 0.351.17 ) 2 or more114 ( 17.0)17.416.40.94 ( 0.611.45)0.72 ( 0.331.57)moraxella catarrhalis never ( reference)354 ( 52.8)54.750.2referencereference once240 ( 35.8)33.139.41.30 ( 0.931.81)1.57 ( 0.882.80 ) 2 or more77 ( 11.5)12.210.50.93 ( 0.561.54)0.91 ( 0.382.15)haemophilus influenzae never ( reference)381 ( 56.8)58.354.7referencereference 1224 ( 33.4)31.535.91.22 ( 0.871.69)1.08 ( 0.601.96 ) once66 ( 9.8)10.29.40.99 ( 0.581.68)1.16 ( 0.443.08)airway pathogen never ( reference)136 ( 20.3)20.619.9referencereference once163 ( 24.3)25.323.00.94 ( 0.591.50)0.58 ( 0.251.35 ) 2 or more372 ( 55.4)54.257.11.09 ( 0.731.63)0.86 ( 0.401.84)or univariate model , aor full model including all risk factors as shown in table 1 association between bacterial carriage in the first year of life and otitis media in the second year of life or univariate model , aor full model including all risk factors as shown in table 1 possible interaction between otitis media in the first year of life and bacterial carriage was tested . we present the associations between otitis media in the first year of life ( 612 months ) and otitis media in the second year of life stratified for different bacterial carriage states in the first year of life . bacterial carriage did not significantly change the increased risk for otitis media in the second year of life in case previous otitis media ( 612 months ) was registered.table 3association between otitis media in the first year of life ( 612 months ) and otitis media in the second year of life stratified by carriage state of different bacteriaotitis media ( 1224 months of age)n ( % ) no ( n = 308 ) ( % ) yes ( n = 217 ) ( % ) oraornever s. pneumoniaeotitis media 612 months of ageno176 ( 71.5)81.956.9referencereferenceyes70 ( 28.5)18.143.13.44 ( 1.936.14)2.81 ( 1.146.92)once or more s. pneumoniaeotitis media 612 months of ageno192 ( 68.8)78.055.7referencereferenceyes82 ( 31.2)22.044.32.83 ( 1.684.77)2.81 ( 1.276.24)never m. catarrhalisotitis media 612 months of ageno194 ( 70.3)79.556.4referencereferenceyes82 ( 29.7)20.543.63.01 ( 1.765.12)2.26 ( 1.005.12)once or more m. catarrhalisotitis media 612 months of ageno174 ( 69.9)80.356.1referencereferenceyes75 ( 30.1)19.743.93.19 ( 1.825.60)3.19 ( 1.317.74)never h. influenzaeotitis media 612 months of ageno197 ( 68.2)77.354.7referencereferenceyes92 ( 31.8)22.745.32.82 ( 1.704.70)2.90 ( 1.346.28)once or more h. influenzaeotitis media 612 months of ageno171 ( 72.5)83.158.0referencereferenceyes65 ( 27.5)16.942.03.56 ( 1.966.48)3.25 ( 1.308.12)never airway pathogenotitis media 612 months of ageno77 ( 72.6)84.155.8referencereferenceyes29 ( 27.4)15.944.24.20 ( 1.7010.37)4.20 ( 1.7010.37)once or more airway pathogenotitis media 612 months of ageno291 ( 69.5)78.856.3referencereferenceyes128 ( 30.5)78.843.72.88 ( 1.884.42)2.76 ( 1.505.09)or univariate model , aor full model including all risk factors as shown in table 1 association between otitis media in the first year of life ( 612 months ) and otitis media in the second year of life stratified by carriage state of different bacteria or univariate model , aor full model including all risk factors as shown in table 1 we observed a positive association between having siblings and otitis media . also , we observed a tendency that low education of the mother might attribute to the risk for otitis media , while breast - feeding might be a protective factor . no associations were observed between birth weight , gestational age , gender , maternal smoking , day care attendance or pacifier use and otitis media in the second year of life . in a meta - analysis of risk factors for acute otitis media by uhari et al . day care attendance was shown to be the most significant risk factor . furthermore , similar to the trend we observed , uhari et al . showed a positive association between siblings and otitis media and a negative association between breast feeding and otitis media . this study , in contrast to our results , also showed that parental smoking and pacifier use was associated with otitis media . we observed that otitis media in the first year of life is a risk factor for otitis media in the second year of life . corbeel has stated that the first period of otitis media determines the risk for recurrence because inflammation causes dysfunction of the eustachian tube in young children due to the small caliber and the horizontal direction and is , therefore , predisposing for a high risk for recurrent of otitis media . next , we analyzed in the focus cohort the association between bacterial carriage and otitis media in the second year of life , and otitis - proneness . no associations were observed between frequency of bacterial carriage , age of first bacterial carriage or the different species of bacterial carriage and otitis media or otitis - proneness . bacterial carriage in the first year of life is not associated with otitis media later on . in other studies the frequency of carriage with different bacteria was shown to be associated with otitis media [ 4 , 5 , 7 , 8 ] . faden et al . have shown that carriage of s. pneumoniae , h. influenzae and m. catarrhalis was associated with otitis - proneness in 17 otitis prone children versus 17 non - otitis prone children , during healthy periods and during periods of otitis media , in children aged 036 months in . the association in healthy periods was only significant for h. influenzae carriage but not for the other two bacteria studied . in another study in 157 infants , an association between carriage of h. influenzae , measured at 13 routine visits in the first year of life , and otitis - proneness was shown . in another study in 306 children aged 024 months , an association between m. catarrhalis and otitis - proneness was observed . in this population they also showed an association between the frequency of carriage of the three airway pathogens and the frequency of otitis media episodes . prellner et al . did not show an increased risk for otitis media when s. pneumoniae , h. influenzae or m. catarrhalis were present . early age of first colonization was also shown to be associated with the risk for otitis media [ 4 , 8 , 9 ] . have shown in their study on the temporal association between pneumococcal carriage and otitis media that not preceding pneumococcal carriage state , but newly acquired carriage is associated with otitis media . their data support the hypothesis that viral respiratory infection might enhance the acquisition of pneumococci . recently , revai et al . have shown an association between presence of pathogenic bacteria in the nasopharynx during upper respiratory tract infections and the risk for otitis media following the upper respiratory tract infection . taken together , it appears that the presence of newly acquired pathogenic bacteria in combination with ( viral ) upper respiratory tract infections increases the risk for otitis media , rather than the presence of pathogenic bacteria itself increases the risk for otitis media . to appreciate the results some limitations of our study had to be considered . in contrast to other studies , our definition of otitis media was based on parental reported questionnaire data , we do not have a doctor verified diagnosis of otitis media . we observed that 49 ( 7.2% ) of the children had at least one period of otitis media in their first 6 months of life , 247 ( 31.3% ) had otitis media in their second 6 months of life and 379 ( 43.3% ) of the children had at least one period of otitis media in their second year of life . 511 ( 71.8% ) of the children had at least one period of otitis media by the age of 24 months . this might be an overestimation of the number of children experiencing otitis media , resulting in less contrast between children with and without otitis media . on the other hand , children with fever and earache were only classified as otitis yes if they visited a doctor . to our opinion in our study , otitis media in the first year of life is an independent risk factor for otitis media in the second year of life . surprisingly , bacterial carriage in the first year of life did not add to this risk . moreover , no association was observed between bacterial carriage in the first year of life and otitis in the second year of life .

acute otitis media is the most frequent diagnosis in children visiting physicians offices . risk factors for otitis media have been widely studied . yet , the correlation between bacterial carriage and the development of otitis media is not entirely clear . our aim was to study in a population - based prospective cohort the risk factors for otitis media in the second year of life with special emphasis on the role of colonization with streptococcus pneumoniae , haemophilus influenzae and moraxella catarrhalis . the study was embedded in the generation r study . data on risk factors and doctor - diagnosed otitis media were obtained by midwives , hospital registries and postal questionnaires in the whole cohort ( n = 7,295 ) . nasopharyngeal swabs were obtained at the age of 1.5 , 6 and 14 months in the focus cohort ( n = 1,079 ) . of these children , 2,515 ( 47.2% ) suffered at least one period of otitis media in their second year of life . the occurrence of otitis media during the follow - up period in the first 6 months of life and between 6 and 12 months of age was associated with the risk of otitis media in the second year of life ( aor , 1.83 95% ci 1.242.71 and aor 2.72 , 95% ci 2.183.38 , respectively ) . having siblings was associated with an increased risk for otitis media in the second year of life ( aor 1.42 , 95% ci 1.131.79 ) . no associations were found between bacterial carriage in the first year of life and otitis media in the second year of life . in our study , otitis media in the first year of life is an independent risk factor for otitis media in the second year of life . surprisingly , bacterial carriage in the first year of life did not add to this risk . moreover , no association was observed between bacterial carriage in the first year of life and otitis in the second year of life .

Introduction Patients and methods Design Measurements and outcome Analyses Results Discussion Conclusion

most children experience at least one episode of otitis media ; in 5085% of the children acute otitis media is diagnosed at least once in the first 3 years of life [ 2 , 3 ] . frequency of colonization with bacterial airway pathogens was shown to be associated with otitis media in the first years of life [ 48 ] . streptococcus pneumoniae , haemophilus influenzae and moraxella catarrhalis are the most frequently cultured pathogens in acute otitis media . early age of colonization seems to increases the risk of otitis media [ 4 , 8 , 9 ] . our aim was to study in a population - based prospective cohort the risk factors for otitis media in children with special emphasis on the role of colonization with s. pneumoniae , h. influenzae and m. catarrhalis in the first year of life . this study was embedded in the generation r study , a population - based prospective cohort study from fetal life until young adulthood . information about maternal smoking , siblings , educational level of the mother , day care attendance , breast - feeding , pacifier use and otitis media in the first year of life was obtained by postal questionnaires at the infants age of 6 , 12 and 24 months . the diagnosis otitis media in the period from 0 to 6 months of age and the period from 6 to 12 months of age was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the 6 months before reaching the age of respectively 6 and 12 months . likewise , the diagnosis of otitis media in the second year of life was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the year before reaching the age of 24 months . risk factors for otitis media in the second year of life were studied in the whole cohort . in the focus cohort , samples were taken at the infants age of 1.5 , 6 , and 14 months to detect nasopharyngeal bacterial carriage . in the whole cohort associations of birth weight , gestational age , gender , maternal smoking , siblings , educational level of mother , day care attendance , duration of breast - feeding , pacifier use and otitis media in the first year of life with otitis media in the second year of life were assessed by logistic regression models resulting in odds ratios ( or ) with their 95% confidence interval ( ci ) . additionally , a multivariate logistic regression model was performed to assess the association of each risk factor separately with otitis media in the second year of life was adjusted for all the other risk factors resulting in adjusted odds ratios ( aor ) . in the focus cohort association of bacterial carriage in the first year of life with otitis media in the second year of life was assessed for the airway pathogens s. pneumoniae , m. catarrhalis and h. influenzae . the analyses were performed using frequency of specific bacterial carriage in the first year of life , and at ages of first bacterial carriage . furthermore , possible interaction between otitis media in the first year of life and bacterial carriage and the risk of otitis media in the second year of life was tested . 15)14.9%15.4%1.04 ( 0.871.25)1.16 ( 0.861.55)siblings no ( ref)2,159 ( 58.9)62.9%54.1% yes1,508 ( 41.1)37.1%45.9%1.44 * * * ( 1.261.65)1.42 * ( 1.131.79)educational level mother low292 ( 5.8)4.7%7.0% middle1,911 ( 37.8)37.3%38.4%0.69 * * ( 0.540.88)0.84 ( 0.361.95 ) high2,851 ( 56.4)58.1%54.5%0.62 * * * ( 0.490.80)1.03 ( 0.442.40)day care no ( ref)730 ( 22.3)21.3%23.5% yes2,543 ( 77.7)78.7%76.5%0.88 ( 0.751.04)0.86 ( 0.661.13)breastfeeding never ( ref)450 ( 19.0)17.7%20.4% less than 3 months1,031 ( 43.5)46.3%40.3%0.76 * ( 0.610.95)0.86 ( 0.631.16 ) more than 3 months890 ( 37.5)36.0%39.3%0.95 ( 0.761.19)1.14 ( 0.831.55)pacifier use no ( ref)1,242 ( 34.4)34.5%34.4% yes2,364 ( 65.6)65.5%65.6%1.01 ( 0.881.16)0.95 ( 0.751.20)otitis from 0 to 6 months no ( ref)3,309 ( 91.9)94.7%88.5% yes292 ( 8.1)5.3%11.5%2.34 * * * ( 1.823.00)1.83 * * ( 1.242.71)otitis from 6 to 12 months no ( ref)2,808 ( 52.8)67.4%45.8% yes2,515 ( 47.2)32.6%54.2%2.44 * * * ( 2.162.76)2.72 * * * ( 2.183.38)values of birth weight is mean ( standard deviation ) , gestational age is median ( range ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 differences of infant characteristics between infants with and without data on otitis media in the second year of life were assessed by the independent sample t - test for continuous normal distributed variables , non - parametric mann whitney test for continuous non - normal distributed variables and the chi - square test for categorical variables . differences of frequency of otitis media in the second year of life between groups with missing risk factors and complete data were assessed in the same way . this study was embedded in the generation r study , a population - based prospective cohort study from fetal life until young adulthood . information about maternal smoking , siblings , educational level of the mother , day care attendance , breast - feeding , pacifier use and otitis media in the first year of life was obtained by postal questionnaires at the infants age of 6 , 12 and 24 months . the diagnosis otitis media in the period from 0 to 6 months of age and the period from 6 to 12 months of age was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the 6 months before reaching the age of respectively 6 and 12 months . likewise , the diagnosis of otitis media in the second year of life was defined as having at least one period of fever accompanied by earache for which a doctor was visited during any part of the year before reaching the age of 24 months . risk factors for otitis media in the second year of life were studied in the whole cohort . in the focus cohort , samples were taken at the infants age of 1.5 , 6 , and 14 months to detect nasopharyngeal bacterial carriage . in the whole cohort associations of birth weight , gestational age , gender , maternal smoking , siblings , educational level of mother , day care attendance , duration of breast - feeding , pacifier use and otitis media in the first year of life with otitis media in the second year of life were assessed by logistic regression models resulting in odds ratios ( or ) with their 95% confidence interval ( ci ) . additionally , a multivariate logistic regression model was performed to assess the association of each risk factor separately with otitis media in the second year of life was adjusted for all the other risk factors resulting in adjusted odds ratios ( aor ) . in the focus cohort association of bacterial carriage in the first year of life with otitis media in the second year of life was assessed for the airway pathogens s. pneumoniae , m. catarrhalis and h. influenzae . the analyses were performed using frequency of specific bacterial carriage in the first year of life , and at ages of first bacterial carriage . furthermore , possible interaction between otitis media in the first year of life and bacterial carriage and the risk of otitis media in the second year of life was tested . data were missing on birth weight ( n = 8) , gestational age ( n = 1 ) , maternal smoking ( n = 1,659 ) , siblings ( n = 1,656 ) , educational level of mother ( n = 269 ) , day care attendance ( n = 2,050 ) , breast feeding ( n = 2,952 ) , pacifier use ( n = 1,717 ) , otitis from 0 to 6 months ( n = 1,722 ) and otitis from 6 to 12 months ( n = 815 ) . data were missing on birth weight ( n = 8) , gestational age ( n = 1 ) , maternal smoking ( n = 1,659 ) , siblings ( n = 1,656 ) , educational level of mother ( n = 269 ) , day care attendance ( n = 2,050 ) , breast feeding ( n = 2,952 ) , pacifier use ( n = 1,717 ) , otitis from 0 to 6 months ( n = 1,722 ) and otitis from 6 to 12 months ( n = 815 ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 differences of infant characteristics between infants with and without data on otitis media in the second year of life were assessed by the independent sample t - test for continuous normal distributed variables , non - parametric mann whitney test for continuous non - normal distributed variables and the chi - square test for categorical variables . differences of frequency of otitis media in the second year of life between groups with missing risk factors and complete data were assessed in the same way . data on otitis media in the second year of life was available in 5,323 ( response rate 73% ) children . characteristics of the children of the whole cohort and their association with otitis media in the second year of life are presented in table 1 . having siblings is associated with an increased risk of otitis media in the second year of life ( aor 1.42 , ci 1.131.79 ) . furthermore , the occurrence of otitis media during the follow - up period between 0 and 6 months and the occurrence of otitis media during the follow - up period between 6 and 12 months of age was associated with the occurrence of otitis media in the second year of life ( respectively , aor 1.83 , ci 1.242.71 and aor 2.72 , ci 2.183.38 ) . the children were planned to visit the generation r focus study research centre at the age of 1.5 month ( response rate 81.8% ) , 6 months ( response rate 81.6% ) and 14 months ( response rate 80.0% ) . of the infants 379 ( 43.3% ) had at least one episode of otitis media in the second year of life . in table 2 the associations between the frequency of bacterial carriage in the first year of life and otitis media in the second year of life are presented . we analysed the association between bacterial carriage in the first year of life and otitis media in the second year of life in two different ways . in addition to this , we assessed whether bacterial carriage in the first year of life was associated with otitis - proneness . again , no associations were observed ( data not shown).table 2association between bacterial carriage in the first year of life and otitis media in the second year of lifeotitis media 1224 months of agen ( % ) no ( n = 384 ) ( % ) yes ( n = 287 ) ( % ) oraorstreptococcus pneumoniae never ( reference)316 ( 47.1)47.147.0referencereference once241 ( 35.9)35.436.61.04 ( 0.741.45)0.64 ( 0.351.17 ) 2 or more114 ( 17.0)17.416.40.94 ( 0.611.45)0.72 ( 0.331.57)moraxella catarrhalis never ( reference)354 ( 52.8)54.750.2referencereference once240 ( 35.8)33.139.41.30 ( 0.931.81)1.57 ( 0.882.80 ) 2 or more77 ( 11.5)12.210.50.93 ( 0.561.54)0.91 ( 0.382.15)haemophilus influenzae never ( reference)381 ( 56.8)58.354.7referencereference 1224 ( 33.4)31.535.91.22 ( 0.871.69)1.08 ( 0.601.96 ) once66 ( 9.8)10.29.40.99 ( 0.581.68)1.16 ( 0.443.08)airway pathogen never ( reference)136 ( 20.3)20.619.9referencereference once163 ( 24.3)25.323.00.94 ( 0.591.50)0.58 ( 0.251.35 ) 2 or more372 ( 55.4)54.257.11.09 ( 0.731.63)0.86 ( 0.401.84)or univariate model , aor full model including all risk factors as shown in table 1 association between bacterial carriage in the first year of life and otitis media in the second year of life or univariate model , aor full model including all risk factors as shown in table 1 possible interaction between otitis media in the first year of life and bacterial carriage was tested . we present the associations between otitis media in the first year of life ( 612 months ) and otitis media in the second year of life stratified for different bacterial carriage states in the first year of life . bacterial carriage did not significantly change the increased risk for otitis media in the second year of life in case previous otitis media ( 612 months ) was registered.table 3association between otitis media in the first year of life ( 612 months ) and otitis media in the second year of life stratified by carriage state of different bacteriaotitis media ( 1224 months of age)n ( % ) no ( n = 308 ) ( % ) yes ( n = 217 ) ( % ) oraornever s. pneumoniaeotitis media 612 months of ageno176 ( 71.5)81.956.9referencereferenceyes70 ( 28.5)18.143.13.44 ( 1.936.14)2.81 ( 1.146.92)once or more s. pneumoniaeotitis media 612 months of ageno192 ( 68.8)78.055.7referencereferenceyes82 ( 31.2)22.044.32.83 ( 1.684.77)2.81 ( 1.276.24)never m. catarrhalisotitis media 612 months of ageno194 ( 70.3)79.556.4referencereferenceyes82 ( 29.7)20.543.63.01 ( 1.765.12)2.26 ( 1.005.12)once or more m. catarrhalisotitis media 612 months of ageno174 ( 69.9)80.356.1referencereferenceyes75 ( 30.1)19.743.93.19 ( 1.825.60)3.19 ( 1.317.74)never h. influenzaeotitis media 612 months of ageno197 ( 68.2)77.354.7referencereferenceyes92 ( 31.8)22.745.32.82 ( 1.704.70)2.90 ( 1.346.28)once or more h. influenzaeotitis media 612 months of ageno171 ( 72.5)83.158.0referencereferenceyes65 ( 27.5)16.942.03.56 ( 1.966.48)3.25 ( 1.308.12)never airway pathogenotitis media 612 months of ageno77 ( 72.6)84.155.8referencereferenceyes29 ( 27.4)15.944.24.20 ( 1.7010.37)4.20 ( 1.7010.37)once or more airway pathogenotitis media 612 months of ageno291 ( 69.5)78.856.3referencereferenceyes128 ( 30.5)78.843.72.88 ( 1.884.42)2.76 ( 1.505.09)or univariate model , aor full model including all risk factors as shown in table 1 association between otitis media in the first year of life ( 612 months ) and otitis media in the second year of life stratified by carriage state of different bacteria or univariate model , aor full model including all risk factors as shown in table 1 we observed a positive association between having siblings and otitis media . no associations were observed between birth weight , gestational age , gender , maternal smoking , day care attendance or pacifier use and otitis media in the second year of life . we observed that otitis media in the first year of life is a risk factor for otitis media in the second year of life . corbeel has stated that the first period of otitis media determines the risk for recurrence because inflammation causes dysfunction of the eustachian tube in young children due to the small caliber and the horizontal direction and is , therefore , predisposing for a high risk for recurrent of otitis media . next , we analyzed in the focus cohort the association between bacterial carriage and otitis media in the second year of life , and otitis - proneness . no associations were observed between frequency of bacterial carriage , age of first bacterial carriage or the different species of bacterial carriage and otitis media or otitis - proneness . bacterial carriage in the first year of life is not associated with otitis media later on . have shown that carriage of s. pneumoniae , h. influenzae and m. catarrhalis was associated with otitis - proneness in 17 otitis prone children versus 17 non - otitis prone children , during healthy periods and during periods of otitis media , in children aged 036 months in . in another study in 157 infants , an association between carriage of h. influenzae , measured at 13 routine visits in the first year of life , and otitis - proneness was shown . did not show an increased risk for otitis media when s. pneumoniae , h. influenzae or m. catarrhalis were present . early age of first colonization was also shown to be associated with the risk for otitis media [ 4 , 8 , 9 ] . have shown an association between presence of pathogenic bacteria in the nasopharynx during upper respiratory tract infections and the risk for otitis media following the upper respiratory tract infection . we observed that 49 ( 7.2% ) of the children had at least one period of otitis media in their first 6 months of life , 247 ( 31.3% ) had otitis media in their second 6 months of life and 379 ( 43.3% ) of the children had at least one period of otitis media in their second year of life . 511 ( 71.8% ) of the children had at least one period of otitis media by the age of 24 months . to our opinion in our study , otitis media in the first year of life is an independent risk factor for otitis media in the second year of life . surprisingly , bacterial carriage in the first year of life did not add to this risk . moreover , no association was observed between bacterial carriage in the first year of life and otitis in the second year of life .

type 2 diabetes mellitus ( t2 dm ) is a major global chronic disease with immense and increasing healthcare costs and a high disease burden . diabetes and diabetes - related complications considerably decrease health - related quality of life ( hrql ) . there is an indispensable need to study how diabetes care can further be improved to limit the disease burden and costs . health care systems attempt to deal with chronic diseases by standardizing treatment and clinical practice . important policy strategies include the development of national clinical practice guidelines and indicators for the quality of care [ 47 ] . a review of observational studies examining the relationship between guideline - defined diabetes processes and health outcomes notes that effects are often surprisingly small and inconsistent . health care planners are optimistic about the capacity of patients to take responsibility for their health and the management of their chronic illness [ 9 , 10 ] . the promotion of patients ' involvement in the management of their chronic disease has become a popular concept and is delineated in the chronic care model and the patient empowerment approach [ 11 , 12 ] . a patient - centered approach to diabetes care , endorsed as a central aim by the american diabetes association and the european association for the study of diabetes , includes patient education but goes beyond this to embrace multiprofessional care , care management activities , and a proactive , respectful communication with patients . others argue for a broader inclusion of patient - centered measures in studies of quality of diabetes care [ 1416 ] . studies have shown that diabetes care provided along these lines enhances metabolic control and clinical outcomes , in particular , when patients engage actively in their self - management [ 1720 ] . accordingly , we would argue that care delivery based on a partnership model between health professionals and patients should improve patient commitment to their health behaviour , self - management , and adherence , which can be conceptualised as patient outcomes . striving towards the use of patient - oriented quality of care indicators , the main limitation is a lack of empirical data . in fact , clinical trials may not be very suitable for determining the impact of these indicators when implemented in actual practice . population - based observational studies can offer both a view close to health care provision in practice and data on a wide range of structure , process , and outcome indicators of diabetes care . drawing on data from two population - based survey studies that were conducted in a region in southern germany from 2003 to 2008 we performed a post hoc analysis to test whether patients well supported by health professionals ( as indicated by higher levels of care processes such as medical examinations , diabetes education , and multiprofessional care as well as high treatment satisfaction and good quality of the relationship between patients and physicians ) attain higher levels of health behaviour and self - management and better adherence as well as less disease - related burden and higher hrql . the study population consisted of 486 participants with t2 dm who took part in either of two follow - up survey studies conducted in 200305 ( f3 ) and 200608 ( f4 ) . f3 was a follow - up cohort study ( n = 3184 ) to a 1994 - 1995 population - based monica survey ( s3 ) with a response rate of 76% . f4 was a follow - up cohort study ( n = 3080 ) of the kora s4 survey conducted in 19992001 with a response rate of 80% . the kora research platform ( cooperative research in the region of augsburg ) is a population - based data pool established in 1996 to continue and expand the monica project in augsburg , a region with a mixed urban and rural population of about 600,000 inhabitants in the southern part of germany , about 70 km west of munich . four cross - sectional health surveys ( s1 to s4 ) have been performed in the city of augsburg and its two surrounding counties at five - year intervals from 1984/85 onwards , each drawn as an independent random sample . the four cross - sectional surveys serve as cohorts for long term follow - up studies like f3 and f4 . both studies utilize the sampling in the original surveys , although data may not be fully representative due to death and loss to follow - up . study designs , sampling methods , and data collection have been described in detail elsewhere . both surveys included a personal interview , several self - administered questionnaires ( including the sf-12 questionnaire on hrql ) , medical examinations , physical measurements , computer assisted documentation of medication during the last 7 days ( idom ) , and laboratory tests . the analysed data is based mainly on items from a questionnaire filled in by participants with diabetes but includes information collected in the interview ( social - demographic data ; type and duration of diabetes ; cardiovascular comorbidity , bmi , physical activity , smoking , and alcohol ) and sf-12 questionnaire data . for the present analysis , only questions with identical wording and answer choice order were included to ascertain the validity of the joint analysis of both studies . both studies were approved by the ethics committee of the bavarian medical association and by the bavarian commissioner for data protection and privacy . only study participants with type 2 diabetes in f3 and f4 were included in our analysis . diabetes status and type of diabetes were assessed by a question in the interview ( self - report of a physician - confirmed diagnosis of diabetes ) . negative self - reports were checked with documentation of respective antidiabetes medication ( atc codes : a10a and a10b ) and corrected , if necessary . in case of doubt , the primary physician was contacted to confirm the diagnosis . of the n = 3184 participants of f3 , n = 259 were classified as participants with type 2 diabetes , respectively , n = 227 of the n = 3080 participants of f4 . we excluded n = 11 ( f3 ) respective n = 8 ( f4 ) participants with self - reported type 1 diabetes to improve sample homogeneity . the sample for analysis consists of 486 participants in total , combining the data of those two surveys to increase sample size for the analysis . participants over 75 years of age filled in a shortened version of the sf-12 in the f3 survey , leading to a higher number of missing values for hrql . diabetes care processes are defined in this study as elements of diabetes care that result from direct interactions between health professionals and patients . processes of diabetes care were assessed with the following variables : ( 1 ) medical examinations and advice , ( 2 ) diabetes education , ( 3 ) treatment satisfaction , ( 4 ) patient - perceived quality of patient / physician relationship , and ( 5 ) multiprofessional care . five types of medical examinations ( eye exam , foot exam , hba1c lab , blood pressure , and protein / urine testing ) and two types of advice given by the physician on diet and physical exercise were assessed by the percentage of participants who reported this activity as having been performed during the last 12 months ( yes / no ) . treatment satisfaction was assessed with a single item : in total , how satisfied are you with the diabetes treatment ( including treatment with insulin , tablets and/or diet ) you have been receiving in the past weeks ? responses from 1 ( very dissatisfied ) to 7 ( very satisfied ) were analyzed across the entire range . the quality of the patient / physician relationship was assessed by items relating to four different quality aspects : comprehensibility of information , opportunity to ask questions , shared decision - making , and psychological support . responses for these variables were dichotomized as excellent , good versus other to ascertain good discriminatory power . multiprofessional care was assessed by two items asking whether dieticians and/or podiatrists were involved in the diabetes care ( yes / no ) during the past 12 months . to prepare process parameters for the multivariate analysis , we added up answers to respective questions in each group ( medical examinations ( 7 items ) , quality of patient / physician relationship ( 4 items ) , and multiprofessional care ( 2 items ) ) , which were identical in wording and answer format ( see table 5 ) . diabetes education ( number of classes attended ) and treatment satisfaction ( range 17 ) were already available as single values . missing values were set to 0 except for missings with regard to treatment satisfaction , where the sample mean value of 5 was imputed . we excluded participants with > 3 missings in the variables relating to process parameters to minimize the need for such imputations in this dataset with its many missing values ( see table 5 ) but to retain a workable sample size for the multivariate analyses . in this study we perceive self - management , adherence , and health behaviour as intermediate patient outcomes , that is , activities that may result from care processes providing effective support of patients . complications and health - related quality of life are considered as medium and long term outcome indicators . taken together , patient outcomes were assessed with the following variables : ( 1 ) patient self - management , ( 2 ) adherence , ( 3 ) health behaviour , ( 4 ) diabetes complications , and ( 5 ) health - related quality of life . participants were asked whether and how frequently they had self - monitored feet , weight , blood glucose , and blood pressure within the past 6 months . each item was dichotomized into 1 for frequencies of once a month or more and 0 for less . these activities are included in a validated scale of diabetes self - care activities ( summary of diabetes self care activities , sdsca ) , although this scale uses a different design to assess frequency . adherence was assessed with self - reported information on whether participants were following medical advice regarding medication , physical activity , diet , and foot care ( i ) without difficulties , ( ii ) with some difficulties , or ( iii ) with major difficulties . each item was dichotomized into 1 for a report of no difficulties and 0 for some or major difficulties . for descriptive analysis the respective answer category reads does not apply to me ( table 5 ) . for health behaviour , we used self - reported information on physical activity , smoking , and alcohol consumption and coded them in accordance with guidelines [ 2527 ] . ( 2 hr / week regularly in winter and summer ) versus 0 ( < 2 hr / week regularly in winter , summer , or both ) . smoking is dichotomized into 1 ( never - smokers and ex - smokers ) and 0 ( regular and occasional smokers ) . alcohol intake is transformed by using cut - off values of weekly consumption in gram ( 0 = men > 20 g , women > 10 g ; 1 = equal or less ) . diabetes - related burden was assessed with regard to common diabetes complications and the occurrence of hyper- and hypoglycaemia . diabetic complications refer to the clinical diagnosis ( ever versus never ) of retinopathy , poor blood circulation in legs , peripheral neuropathy , and microalbuminuria . two items ask to recall the occurrence of hypoglycemic and/or hyperglycemic episodes during the past 6 months ( yes versus no ) . hrql is assessed with the sf-12 , a validated questionnaire and short form of the sf-36 . values are expressed as two scores , the physical health score and mental health score ( pcs and mcs ) . all of the above variables are described in table 5 with regard to their wording , the number of missing values , and the coding used for all analyses . covariates included in the regression analysis are time of survey , patient age , sex , educational level ( basic education versus higher ) , diabetes duration , diabetes treatment type , and comorbidities ( history of stroke / myocardial infarction : yes / no ) . treatment type refers to ( 1 ) no antidiabetic medication , ( 2 ) oral hypoglycemic agents ( oha ) , and ( 3 ) insulin treatment . age and duration of diabetes are entered as continuous variables . for sample description we added information on the bmi ( body mass in kilograms divided by the square of height in units of meters ( kg / m2 ) ) , health insurance status ( % statutory ) , and enrolment in a diabetes type 2 disease management program ( t2dm - dmp ) . descriptive statistics ( percentages , means ( sd ) ) were generated for sample characteristics and process and outcome parameters . differences between the populations of the two survey periods with time of survey entered as dichotomous variables ( f3 , f4 ) were tested by student 's t - test for continuous variables and -test for categorical variables . multiple logistic regression models were used to examine the association between care processes with patient outcomes , controlling for age , sex , education , treatment type , cardiovascular comorbidities ( previous myocardial infarction or stroke ) , and time of survey participation as covariates . odds ratios ( or ) were calculated for binary variables . for continuous variables , such as quality of life scores , p values less than 0.05 are considered as statistically significant but used in an explanatory sense to retrieve further hypotheses . the sociodemographic characteristics of the total sample and of groups divided by treatment type , ( 1 ) no antidiabetic medication , ( 2 ) oral hypoglycemic agents ( oha ) , and ( 3 ) insulin treatment , are shown in table 1 . about half of the sample was treated with oral hypoglycemic agents ( oha ) ( n = 259 , 53% ) , with the remainder equally divided between those on insulin only and insulin combination treatment ( n = 111 , 23% ) and those without antidiabetic medication ( n = 116 , 24% ) . average diabetes duration varies across the groups with different treatment types from 5.6 years in the group without antidiabetic medication to 16.2 years in the group with insulin treatment . overall t2 dm disease management - enrolment was 20% ; it was lowest in the group with no medication . there were no differences between the survey periods regarding baseline variables entered in the regression analyses ( table 4 ) . blood pressure testing was the most frequent ( 95% ) and feet exams the least frequent ( 50% ) of medical examinations and advice . of the seven types of medical examinations and advice , participants received a mean of 4.2 ( 2.2 sd ) . half of the participants had attended at least one patient education class ( 51% ) . across all participants , the mean value for treatment satisfaction was 5.3 ( 1.6 sd ) on a rating scale from 1 ( very dissatisfied ) to 7 ( very satisfied ) . ( 78% ) , compared with the comprehensibility of information ( 87% ) , the opportunity to ask questions ( 90% ) , and shared decision - making ( 85% ) . of the four items , participants rated an average of 2.9 ( sd 1.5 ) items as good or excellent . participants reported low use of multiprofessional care : only 24% had used a podiatrist during the last 12 months , 17% a dietician . patient self - management , that is , monthly or more self - monitoring of blood - pressure , weight , feet , and blood - glucose , was reported by 6580% of participants with the highest percentage for monitoring weight ( 81% ) . differences between high adherence with respect to medication and feet care ( > 80% ) and much lower adherence with regard to physical activity and diet ( ca . only 14% of participants reported to engage in regular physical activity of more than 2 hours per week . however , more than 88% reported not smoking and 73% moderate / no alcohol consumption . disease - related burden was increased for 9% of participants with diabetic retinopathy , 12% with microalbuminuria , 21% with poor circulation in legs , and 29% with peripheral neuropathy . more than half of the participants stated to have experienced episodes of hyper- and hypoglycemia in the last 6 months . mean hrql was 41.5 in the physical component score ( pcs-12 ) and 49.8 in the mental component score ( mcs-12 ) . we excluded n = 70 participants ( 14% of the total sample ) because of > 3 missing values in variables pertaining to care processes ( f3 : n = 49 ; f4 : n = 21 ) . most of them were participants who did not fill in the diabetes - specific questionnaire ( f3 : n = 34 of n = 259 ; f4 : n = 11 of n = 227 ) . associations with patient self - management , health behaviour , disease - related burden , and health - related quality of life were strongest for medical examinations and advice , diabetes education , and quality of relationship with physician and less strong for treatment satisfaction and multiprofessional care . a higher number of medical examinations and advice were associated with self - monitoring of blood pressure ( or 1.18 ; ci : 1.03 , 1.35 ) , feet ( or 1.24 ; ci : 1.09 , 1.42 ) , and blood glucose ( or 1.23 ; ci : 1.07 , 1.42 ) . however , participants with more medical examinations and advice also reported less frequently that they had no difficulties with adherence to diet ( or 0.82 ; ci 0.70 ; 0.95 ) . participation in diabetes education was positively associated with self - monitoring feet ( or 1.30 ; ci 1.06 , 1.60 ) and blood glucose ( or 1.50 ; ci 1.18 , 1.90 ) . patient - perceived positive quality of relationship with the physician was most strongly associated with adherence to medication ( or 1.92 ; ci : 1.39 , 2.64 ) and the mental component score of hrql ( mean score difference 2.04 ci : 0.95 , 3.13 ) . it was less strongly , but still significantly , associated with adherence to recommendations regarding diet ( or 1.33 ; ci : 1.04 , 1.71 ) and feet care ( or 1.33 ; ci : 1.02 ; 1.73 ) as well as self - monitoring of weight ( or 1.26 , ci : 1.00 ; 1.58 ) . multiprofessional care was positively associated with self - monitoring of blood glucose ( or 1.63 , ci : 1.02 , 2.62 ) . contrary to our hypothesis , a number of associations indicated that participants reporting a higher level of care processes were more likely to report diabetes - related complications , episodes of hyperglycemia , and lower scores in the mental component of hrql . participants who receive multiprofessional care are less likely to be free of poor blood circulation in legs ( or 0.54 ; ci : 0.35 , 0.83 ) , peripheral neuropathy ( or 0.68 ; ci : 0.47 , 0.99 ) , and hyperglycemia ( or 0.61 ; ci : 0.40 ; 0.91 ) . the risk of no retinopathy decreases with more diabetes education ( or 0.75 ; ci : 0.63 ; 0.90 ) , and the physical component hrql score is lower ( mean score difference : 1.01 ; ci : 1.76 ; 0.26 ) . the risk of no microalbuminuria decreases with a higher mean score in medical examination and advice ( or 0.79 ; ci : 0.63 ; 0.99 ) . finally , the only association found for treatment satisfaction indicates that participants with more treatment satisfaction are less likely to not have been diagnosed with retinopathy ( or 0.71 ; ci : 0.51 ; 0.98 ) . to investigate the robustness of our results , sensitivity analyses were performed . models were calculated when ( 1 ) adjusting for diabetes duration instead of treatment type , ( 2 ) diabetes treatment in four ( insulin only ; insulin and oha ; oha ; no medication ) versus three groups ( insulin only or insulin and oha ; oha ; no medication ) , and ( 3 ) controlling for t2dm - dmp enrolment . in each case measuring quality of care requires process and outcome measures that go beyond specific clinical interventions to integrate components that are guided by the patients themselves [ 1416 , 32 ] . we have tried to capture quality of care broadly by relating care processes to patient outcomes , expanding the set of indicators to include patients ' activities and experiences . following our hypotheses , frequent medical examinations increase the likelihood of self - monitoring activities . patient - experienced positive relationship with physician is associated with higher adherence to medical recommendations , for example , medication and the sf-12 mental component score . contrary to our expectation , higher levels of medical examinations and multiprofessional care did not preclude disease - related burden but were associated with it . this may indicate that patients ' support by health professionals is being initiated too late and is only intensified once complications are present . this study , however , used a cross - sectional design , which introduces problems with confounding . we have adjusted for cardiovascular comorbidity and treatment type , but these adjustments may not be enough to control for the intensified health care needs of sicker patients ( i.e. , those with more complications ) . further research with prospective studies is needed to reevaluate these findings . on a descriptive level , our results provide evidence that the frequency of care processes still falls short in germany . with the exception of blood pressure , all medical examinations required to be performed yearly are reported only from 50%75% of participants . for example , annual eye exams are recommended for all patients with t2 dm , but in our data only about two thirds of the patients reported receiving one in the past 12 months . only 5060% of participants reported receiving advice on diet or on physical exercise or ever participating in patient diabetes education . guidelines recommend that all patients should receive these types of examinations and advice as well as patient education . patient self - monitoring levels as reported in this study must also be regarded as relatively low level , considering that 2030% of patients do not carry out these activities at least on a monthly basis . however , in the light of diabetes treatment guidelines which only very generally recommend that physicians should review health behaviour with each patient on a regular basis ( i.e. , at least annually ) this may be explainable [ 7 , 34 ] . although self - monitoring of weight was reported relatively more often compared to the other types of self - monitoring , adherence to medical recommendations concerning physical activities and diet was difficult for the majority of participants . whilst adherence to most types of medical recommendations , except recommendations concerning physical activity , is higher when patients perceive the quality of patient - physician relationship to be good , patients ' self - monitoring is associated with more frequent medical examinations , advice , and diabetes education . in the guidelines , self - monitoring of blood glucose reimbursement of glucose test strips has been cut for oha - treated patients with diabetes in 2011 by the federal regulatory bodies . care processes were not associated with any of the three types of health behaviour which we assessed , that is , physical activity , smoking , and alcohol consumption . only about 15% of participants report regular physical exercise . participants do better managing smoking and alcohol consumption , but approximately 30% report a higher than recommended intake of alcohol and approximately 10% are smoking . these figures are consistent with data from a nationwide german health survey , carried out between 2008 and 2011 . . only half of the participants in our study state that they have attended diabetes education classes at least once . ( 2008 ) conclude that patient education must have a clear program at the outset and be reinforced at additional points of contact and should be delivered by a team of educators . there need to be more efforts to monitor age , sex , and socioeconomic differences in health behaviour to target interventions and evaluate these kinds of complex interventions . in our study , we found that a higher patient - perceived quality of patient - physician relationship is associated with a higher score for the mental component of health - related quality of life ( mcs-12 ) , roughly equalling the difference in mcs-12 scores between women with and without diabetes . this indicates the potential benefits of intensified patient - oriented care processes for patient outcomes . the strength of this study is its population - based approach , providing data regardless of contact to medical care providers or membership in a particular sickness insurance fund . baseline characteristics of our sample , such as the distribution across the treatment groups ( no medication , oha , and insulin ) , are similar to what has been found for patients with t2 dm in other study samples in germany . we have used multiple logistic regression models and adjusted for important covariables such as education , cardiovascular comorbidity , and duration of diabetes . models proved to be very robust when sensitivity analyses were performed , that is , when adjusting for diabetes duration instead of treatment type . the questionnaire used in this study did not contain validated scales for the assessment of care processes or self - management , such as the patient assessment of chronic illness care ( pacic ) , diabetes management self - efficacy scale ( dmses ) , diabetes treatment satisfaction questionnaire ( dtsq ) , or sdsca scale , most of which are not available in german or have only recently been translated [ 24 , 43 ] . we combined the items on medical examinations , quality of patient - physician relationship , and multiprofessional care to reduce complexity of the analysis . this must be regarded as exploratory and calls for further validation or the use of validated scales in future studies . with regard to measuring hrql , the use of disease - specific quality of life questionnaires for type 2 diabetes in addition to generic questionnaires such as the sf-12 is important to capture the full spectrum of experiences with diabetes , including the psychological burden of the disease . thus the analysis was run on a smaller sample , removing participants with > 3 missing values in process or outcome variables . the assessment of comorbidities and diabetic complications based on patient - reports may be particularly susceptible to information and recall bias . however , studies that use insurance data or review physicians ' charts have found comparable rates of comorbidities and diabetic complications in germany [ 45 , 46 ] . a comparison with data from the largest statutory health insurance fund in germany shows good agreement with regard to hba1c testing ( in our data 64% , health insurance data 69% ) but a higher frequency of self - reported eye exams ( in our data 69% , health insurance data 35% ) . other types of medical examinations studied here are not accounted for in administrative data in way that would be comparable to our data . our data only captured some aspects of quality of care while others may be added by other studies . also , classifying the elements as done in this study is one way to conceptualise possible associations and there may be others which are equally valuable . for example , treatment satisfaction can be regarded as patient outcome , when factors not under control of the health care system or the health professionals are considered equally important . following this , self - monitoring , adherence , and health behaviours can be treated as care processes when focusing on the interaction between health professional and patient to initiate , adopt , and maintain actions . in this study however , we perceive self - management , adherence , and health behaviour as intermediate outcomes , that is , activities that may develop when care processes work well beforehand . generalizations from this rather small regional study with mainly self - reported data should be done carefully and only in the light of additional data . this study relied mainly on self - reported questionnaire data with its limitations in validity and reliability . however , the data offer rich insights on patients ' perceptions of quality of diabetes care , which are rare and valuable for expanding our understanding of patient - oriented processes and outcomes in diabetes care . efforts to improve diabetes care need to go beyond guidelines to standardizing treatment and clinical practice and integrate indicators pertaining to higher levels of patient - oriented care processes and outcomes . our study underlines the importance of monitoring and evaluating diabetes care by drawing on patient - reported indicators for both processes and outcomes as an indispensable part of clinical practice . our results stress the importance of finding more effective strategies to support patients to change health behaviour , in particular with regard to physical activity . attention should be paid to fostering the patient - perceived quality of patient - physician relationship . diabetes education must broaden its reach and scope , for example , in the field of health behaviour . rather than programs delivered just once per patient , it must be remodelled into providing long - term support to maintain patient engagement [ 47 , 48 ] . further research is warranted to consider how diabetes self - management is associated with patients ' prioritization of health outcomes and quality of life , caregiver support as well as costs .

objective . this study aims to examine the relationship of diabetes care processes and patient outcomes with an expanded set of indicators regarding patient - oriented care delivery , such as treatment satisfaction , the quality of patient - physician relationship , and a wider range of patient outcomes such as self - management , health behaviour , disease - related burden , and health - related quality of life ( hrql ) . methods . the study population consisted of 486 participants with type 2 diabetes in two population - based follow - up surveys , conducted in 2003 to 2005 and 2006 to 2008 in southern germany . data were self - reported and questionnaire - based , including the sf-12 for hrql . multiple regression models were used to identify associations between care processes and outcomes with adjustment for confounders . results . frequent medical examinations increased the likelihood of self - monitoring activities , such as foot care . a positive patient experienced relationship with their physician is associated with higher adherence to medical recommendations , such as medication intake , and the score of the sf-12 mental component . participants with diabetes - related complications reported higher levels of medical examinations and multiprofessional care . conclusions . indicators of patient - oriented care should become an indispensable part of diabetes clinical practice guidelines with the aim of striving for more effective support of patients .

1. Introduction 2. Methods 3. Results 4. Discussion and Conclusion

type 2 diabetes mellitus ( t2 dm ) is a major global chronic disease with immense and increasing healthcare costs and a high disease burden . diabetes and diabetes - related complications considerably decrease health - related quality of life ( hrql ) . there is an indispensable need to study how diabetes care can further be improved to limit the disease burden and costs . important policy strategies include the development of national clinical practice guidelines and indicators for the quality of care [ 47 ] . a review of observational studies examining the relationship between guideline - defined diabetes processes and health outcomes notes that effects are often surprisingly small and inconsistent . health care planners are optimistic about the capacity of patients to take responsibility for their health and the management of their chronic illness [ 9 , 10 ] . the promotion of patients ' involvement in the management of their chronic disease has become a popular concept and is delineated in the chronic care model and the patient empowerment approach [ 11 , 12 ] . a patient - centered approach to diabetes care , endorsed as a central aim by the american diabetes association and the european association for the study of diabetes , includes patient education but goes beyond this to embrace multiprofessional care , care management activities , and a proactive , respectful communication with patients . others argue for a broader inclusion of patient - centered measures in studies of quality of diabetes care [ 1416 ] . studies have shown that diabetes care provided along these lines enhances metabolic control and clinical outcomes , in particular , when patients engage actively in their self - management [ 1720 ] . accordingly , we would argue that care delivery based on a partnership model between health professionals and patients should improve patient commitment to their health behaviour , self - management , and adherence , which can be conceptualised as patient outcomes . striving towards the use of patient - oriented quality of care indicators , the main limitation is a lack of empirical data . population - based observational studies can offer both a view close to health care provision in practice and data on a wide range of structure , process , and outcome indicators of diabetes care . drawing on data from two population - based survey studies that were conducted in a region in southern germany from 2003 to 2008 we performed a post hoc analysis to test whether patients well supported by health professionals ( as indicated by higher levels of care processes such as medical examinations , diabetes education , and multiprofessional care as well as high treatment satisfaction and good quality of the relationship between patients and physicians ) attain higher levels of health behaviour and self - management and better adherence as well as less disease - related burden and higher hrql . the study population consisted of 486 participants with t2 dm who took part in either of two follow - up survey studies conducted in 200305 ( f3 ) and 200608 ( f4 ) . f3 was a follow - up cohort study ( n = 3184 ) to a 1994 - 1995 population - based monica survey ( s3 ) with a response rate of 76% . f4 was a follow - up cohort study ( n = 3080 ) of the kora s4 survey conducted in 19992001 with a response rate of 80% . the kora research platform ( cooperative research in the region of augsburg ) is a population - based data pool established in 1996 to continue and expand the monica project in augsburg , a region with a mixed urban and rural population of about 600,000 inhabitants in the southern part of germany , about 70 km west of munich . the four cross - sectional surveys serve as cohorts for long term follow - up studies like f3 and f4 . both studies utilize the sampling in the original surveys , although data may not be fully representative due to death and loss to follow - up . both surveys included a personal interview , several self - administered questionnaires ( including the sf-12 questionnaire on hrql ) , medical examinations , physical measurements , computer assisted documentation of medication during the last 7 days ( idom ) , and laboratory tests . the analysed data is based mainly on items from a questionnaire filled in by participants with diabetes but includes information collected in the interview ( social - demographic data ; type and duration of diabetes ; cardiovascular comorbidity , bmi , physical activity , smoking , and alcohol ) and sf-12 questionnaire data . only study participants with type 2 diabetes in f3 and f4 were included in our analysis . diabetes status and type of diabetes were assessed by a question in the interview ( self - report of a physician - confirmed diagnosis of diabetes ) . of the n = 3184 participants of f3 , n = 259 were classified as participants with type 2 diabetes , respectively , n = 227 of the n = 3080 participants of f4 . we excluded n = 11 ( f3 ) respective n = 8 ( f4 ) participants with self - reported type 1 diabetes to improve sample homogeneity . the sample for analysis consists of 486 participants in total , combining the data of those two surveys to increase sample size for the analysis . participants over 75 years of age filled in a shortened version of the sf-12 in the f3 survey , leading to a higher number of missing values for hrql . diabetes care processes are defined in this study as elements of diabetes care that result from direct interactions between health professionals and patients . processes of diabetes care were assessed with the following variables : ( 1 ) medical examinations and advice , ( 2 ) diabetes education , ( 3 ) treatment satisfaction , ( 4 ) patient - perceived quality of patient / physician relationship , and ( 5 ) multiprofessional care . five types of medical examinations ( eye exam , foot exam , hba1c lab , blood pressure , and protein / urine testing ) and two types of advice given by the physician on diet and physical exercise were assessed by the percentage of participants who reported this activity as having been performed during the last 12 months ( yes / no ) . treatment satisfaction was assessed with a single item : in total , how satisfied are you with the diabetes treatment ( including treatment with insulin , tablets and/or diet ) you have been receiving in the past weeks ? the quality of the patient / physician relationship was assessed by items relating to four different quality aspects : comprehensibility of information , opportunity to ask questions , shared decision - making , and psychological support . multiprofessional care was assessed by two items asking whether dieticians and/or podiatrists were involved in the diabetes care ( yes / no ) during the past 12 months . to prepare process parameters for the multivariate analysis , we added up answers to respective questions in each group ( medical examinations ( 7 items ) , quality of patient / physician relationship ( 4 items ) , and multiprofessional care ( 2 items ) ) , which were identical in wording and answer format ( see table 5 ) . missing values were set to 0 except for missings with regard to treatment satisfaction , where the sample mean value of 5 was imputed . in this study we perceive self - management , adherence , and health behaviour as intermediate patient outcomes , that is , activities that may result from care processes providing effective support of patients . complications and health - related quality of life are considered as medium and long term outcome indicators . taken together , patient outcomes were assessed with the following variables : ( 1 ) patient self - management , ( 2 ) adherence , ( 3 ) health behaviour , ( 4 ) diabetes complications , and ( 5 ) health - related quality of life . participants were asked whether and how frequently they had self - monitored feet , weight , blood glucose , and blood pressure within the past 6 months . these activities are included in a validated scale of diabetes self - care activities ( summary of diabetes self care activities , sdsca ) , although this scale uses a different design to assess frequency . adherence was assessed with self - reported information on whether participants were following medical advice regarding medication , physical activity , diet , and foot care ( i ) without difficulties , ( ii ) with some difficulties , or ( iii ) with major difficulties . for health behaviour , we used self - reported information on physical activity , smoking , and alcohol consumption and coded them in accordance with guidelines [ 2527 ] . diabetes - related burden was assessed with regard to common diabetes complications and the occurrence of hyper- and hypoglycaemia . hrql is assessed with the sf-12 , a validated questionnaire and short form of the sf-36 . all of the above variables are described in table 5 with regard to their wording , the number of missing values , and the coding used for all analyses . for sample description we added information on the bmi ( body mass in kilograms divided by the square of height in units of meters ( kg / m2 ) ) , health insurance status ( % statutory ) , and enrolment in a diabetes type 2 disease management program ( t2dm - dmp ) . multiple logistic regression models were used to examine the association between care processes with patient outcomes , controlling for age , sex , education , treatment type , cardiovascular comorbidities ( previous myocardial infarction or stroke ) , and time of survey participation as covariates . for continuous variables , such as quality of life scores , p values less than 0.05 are considered as statistically significant but used in an explanatory sense to retrieve further hypotheses . the sociodemographic characteristics of the total sample and of groups divided by treatment type , ( 1 ) no antidiabetic medication , ( 2 ) oral hypoglycemic agents ( oha ) , and ( 3 ) insulin treatment , are shown in table 1 . about half of the sample was treated with oral hypoglycemic agents ( oha ) ( n = 259 , 53% ) , with the remainder equally divided between those on insulin only and insulin combination treatment ( n = 111 , 23% ) and those without antidiabetic medication ( n = 116 , 24% ) . blood pressure testing was the most frequent ( 95% ) and feet exams the least frequent ( 50% ) of medical examinations and advice . of the seven types of medical examinations and advice , participants received a mean of 4.2 ( 2.2 sd ) . across all participants , the mean value for treatment satisfaction was 5.3 ( 1.6 sd ) on a rating scale from 1 ( very dissatisfied ) to 7 ( very satisfied ) . ( 78% ) , compared with the comprehensibility of information ( 87% ) , the opportunity to ask questions ( 90% ) , and shared decision - making ( 85% ) . patient self - management , that is , monthly or more self - monitoring of blood - pressure , weight , feet , and blood - glucose , was reported by 6580% of participants with the highest percentage for monitoring weight ( 81% ) . disease - related burden was increased for 9% of participants with diabetic retinopathy , 12% with microalbuminuria , 21% with poor circulation in legs , and 29% with peripheral neuropathy . we excluded n = 70 participants ( 14% of the total sample ) because of > 3 missing values in variables pertaining to care processes ( f3 : n = 49 ; f4 : n = 21 ) . associations with patient self - management , health behaviour , disease - related burden , and health - related quality of life were strongest for medical examinations and advice , diabetes education , and quality of relationship with physician and less strong for treatment satisfaction and multiprofessional care . a higher number of medical examinations and advice were associated with self - monitoring of blood pressure ( or 1.18 ; ci : 1.03 , 1.35 ) , feet ( or 1.24 ; ci : 1.09 , 1.42 ) , and blood glucose ( or 1.23 ; ci : 1.07 , 1.42 ) . however , participants with more medical examinations and advice also reported less frequently that they had no difficulties with adherence to diet ( or 0.82 ; ci 0.70 ; 0.95 ) . participation in diabetes education was positively associated with self - monitoring feet ( or 1.30 ; ci 1.06 , 1.60 ) and blood glucose ( or 1.50 ; ci 1.18 , 1.90 ) . patient - perceived positive quality of relationship with the physician was most strongly associated with adherence to medication ( or 1.92 ; ci : 1.39 , 2.64 ) and the mental component score of hrql ( mean score difference 2.04 ci : 0.95 , 3.13 ) . it was less strongly , but still significantly , associated with adherence to recommendations regarding diet ( or 1.33 ; ci : 1.04 , 1.71 ) and feet care ( or 1.33 ; ci : 1.02 ; 1.73 ) as well as self - monitoring of weight ( or 1.26 , ci : 1.00 ; 1.58 ) . multiprofessional care was positively associated with self - monitoring of blood glucose ( or 1.63 , ci : 1.02 , 2.62 ) . contrary to our hypothesis , a number of associations indicated that participants reporting a higher level of care processes were more likely to report diabetes - related complications , episodes of hyperglycemia , and lower scores in the mental component of hrql . participants who receive multiprofessional care are less likely to be free of poor blood circulation in legs ( or 0.54 ; ci : 0.35 , 0.83 ) , peripheral neuropathy ( or 0.68 ; ci : 0.47 , 0.99 ) , and hyperglycemia ( or 0.61 ; ci : 0.40 ; 0.91 ) . the risk of no retinopathy decreases with more diabetes education ( or 0.75 ; ci : 0.63 ; 0.90 ) , and the physical component hrql score is lower ( mean score difference : 1.01 ; ci : 1.76 ; 0.26 ) . finally , the only association found for treatment satisfaction indicates that participants with more treatment satisfaction are less likely to not have been diagnosed with retinopathy ( or 0.71 ; ci : 0.51 ; 0.98 ) . models were calculated when ( 1 ) adjusting for diabetes duration instead of treatment type , ( 2 ) diabetes treatment in four ( insulin only ; insulin and oha ; oha ; no medication ) versus three groups ( insulin only or insulin and oha ; oha ; no medication ) , and ( 3 ) controlling for t2dm - dmp enrolment . we have tried to capture quality of care broadly by relating care processes to patient outcomes , expanding the set of indicators to include patients ' activities and experiences . following our hypotheses , frequent medical examinations increase the likelihood of self - monitoring activities . patient - experienced positive relationship with physician is associated with higher adherence to medical recommendations , for example , medication and the sf-12 mental component score . contrary to our expectation , higher levels of medical examinations and multiprofessional care did not preclude disease - related burden but were associated with it . with the exception of blood pressure , all medical examinations required to be performed yearly are reported only from 50%75% of participants . patient self - monitoring levels as reported in this study must also be regarded as relatively low level , considering that 2030% of patients do not carry out these activities at least on a monthly basis . although self - monitoring of weight was reported relatively more often compared to the other types of self - monitoring , adherence to medical recommendations concerning physical activities and diet was difficult for the majority of participants . whilst adherence to most types of medical recommendations , except recommendations concerning physical activity , is higher when patients perceive the quality of patient - physician relationship to be good , patients ' self - monitoring is associated with more frequent medical examinations , advice , and diabetes education . in the guidelines , self - monitoring of blood glucose reimbursement of glucose test strips has been cut for oha - treated patients with diabetes in 2011 by the federal regulatory bodies . care processes were not associated with any of the three types of health behaviour which we assessed , that is , physical activity , smoking , and alcohol consumption . there need to be more efforts to monitor age , sex , and socioeconomic differences in health behaviour to target interventions and evaluate these kinds of complex interventions . in our study , we found that a higher patient - perceived quality of patient - physician relationship is associated with a higher score for the mental component of health - related quality of life ( mcs-12 ) , roughly equalling the difference in mcs-12 scores between women with and without diabetes . this indicates the potential benefits of intensified patient - oriented care processes for patient outcomes . the strength of this study is its population - based approach , providing data regardless of contact to medical care providers or membership in a particular sickness insurance fund . baseline characteristics of our sample , such as the distribution across the treatment groups ( no medication , oha , and insulin ) , are similar to what has been found for patients with t2 dm in other study samples in germany . we have used multiple logistic regression models and adjusted for important covariables such as education , cardiovascular comorbidity , and duration of diabetes . the questionnaire used in this study did not contain validated scales for the assessment of care processes or self - management , such as the patient assessment of chronic illness care ( pacic ) , diabetes management self - efficacy scale ( dmses ) , diabetes treatment satisfaction questionnaire ( dtsq ) , or sdsca scale , most of which are not available in german or have only recently been translated [ 24 , 43 ] . we combined the items on medical examinations , quality of patient - physician relationship , and multiprofessional care to reduce complexity of the analysis . with regard to measuring hrql , the use of disease - specific quality of life questionnaires for type 2 diabetes in addition to generic questionnaires such as the sf-12 is important to capture the full spectrum of experiences with diabetes , including the psychological burden of the disease . a comparison with data from the largest statutory health insurance fund in germany shows good agreement with regard to hba1c testing ( in our data 64% , health insurance data 69% ) but a higher frequency of self - reported eye exams ( in our data 69% , health insurance data 35% ) . other types of medical examinations studied here are not accounted for in administrative data in way that would be comparable to our data . for example , treatment satisfaction can be regarded as patient outcome , when factors not under control of the health care system or the health professionals are considered equally important . following this , self - monitoring , adherence , and health behaviours can be treated as care processes when focusing on the interaction between health professional and patient to initiate , adopt , and maintain actions . in this study however , we perceive self - management , adherence , and health behaviour as intermediate outcomes , that is , activities that may develop when care processes work well beforehand . generalizations from this rather small regional study with mainly self - reported data should be done carefully and only in the light of additional data . this study relied mainly on self - reported questionnaire data with its limitations in validity and reliability . however , the data offer rich insights on patients ' perceptions of quality of diabetes care , which are rare and valuable for expanding our understanding of patient - oriented processes and outcomes in diabetes care . efforts to improve diabetes care need to go beyond guidelines to standardizing treatment and clinical practice and integrate indicators pertaining to higher levels of patient - oriented care processes and outcomes . our study underlines the importance of monitoring and evaluating diabetes care by drawing on patient - reported indicators for both processes and outcomes as an indispensable part of clinical practice . our results stress the importance of finding more effective strategies to support patients to change health behaviour , in particular with regard to physical activity . attention should be paid to fostering the patient - perceived quality of patient - physician relationship . further research is warranted to consider how diabetes self - management is associated with patients ' prioritization of health outcomes and quality of life , caregiver support as well as costs .

modern neuroimaging offers tremendous opportunities for gaining insights into the molecular basis of neuropsychiatric disorders . this contrasts with conventional diagnostic imaging procedures , which simply provide anatomical or structural pictures of organs and tissues . several techniques including functional magnetic resonance imaging ( fmri ) and nuclear medicine imaging can measure regional changes in cerebral activity ; however , nuclear medicine imaging is the only tool for direct measurements of neurotransmitter levels and the distribution , density , and activity of receptors or transporters . in contrast to fmri , nuclear medicine imaging involves the administration of radioactively labeled tracers , which decay over time by emitting gamma rays that can be detected by a positron emission tomography ( pet ) or single photon emission computed tomography ( spect ) scanner . the radioisotopes used in pet can reach a more stable configuration by the emission of positrons , which travel a short distance and collide with electrons . the annihilation of positron and electron produces 2 gamma rays , 511 kev each , which are emitted in opposite directions . in pet , annihilation coincidence detection is used in lieu of absorptive collimation to determine the directionality of the detected photons ; this partially explains the greater spatial resolution and sensitivity of pet . however , compared with spect , pet is more expensive and less readily available . the most commonly used radioisotopes for labeling pet radiopharmaceuticals are c and f. c has a short half - life of approximately 20 min , which allows multiple studies in the same day . in contrast , f has a half - life of nearly 2 h , which allows shipment of tracers over considerable distances to pet centers that do not have a cyclotron . spect radiotracers typically have longer physical half - lives than most pet tracers ; this may partially compensate for their disadvantages , particularly when measurements over several hours are required to eliminate nonspecific binding and reach equilibrium . a major advantage of nuclear medicine imaging is the extraordinarily high sensitivity ( 10 to 10 m ) , many orders of magnitude greater than the sensitivities available with mri ( 10 m ) . because many molecules relevant to neuropsychiatric disorders are present at concentrations below 10 m , nuclear medicine imaging is currently the only available in vivo method capable of quantifying subtle cerebral pathophysiological changes that might occur before neurostructural abnormalities take place . pet and spect can measure biological processes , like glucose consumption and regional cerebral blood flow ( rcbf ) , which may change in various neuropsychiatric disorders . in pet imaging , active neurons have higher metabolic rates and higher glucose uptake rates than less active neurons . similarly , active brain regions have higher regional cerebral blood flow ( rcbf ) compared to less active brain regions . with pet , intravenous o - h2o can be administered to measure rcbf . in spect imaging , tc - hexamethylpropyleneamine oxime ( tc - hmpao ) is the most commonly used radiopharmaceutical . radioligands must fulfill several criteria for successful pet or spect imaging , including stability of labeling , sufficient affinity and high selectivity for the specific receptor , combined with low nonspecific binding to brain tissues that do not contain the receptor of interest , and rapid permeation through the blood - brain barrier to permit tracers access to receptors . selective radioligands are available for various transmitter systems ; these enable the visualization of receptor distributions in the normal brain and the detection of changes in receptor binding during various physiologic activities or under pathologic conditions . quantitative imaging has gained clinical importance for measuring the activities of several receptors / transporters and molecular targets , for example , quantification of dopamine transporters for detecting loss of functional dopaminergic neuron terminals in the striatum , quantification of dopamine d2 receptors for studies of movement disorders and for assessments of receptor occupancy by neuroleptic drugs , quantification of serotonin ( 5-hydroxytryptamine , or 5-ht ) receptors in affective disorders , quantification of serotonin and norepinephrine transporters for assessment of occupancy of antidepressants , and quantification of -amyloid and acetylcholinesterase as markers of cognitive and memory impairments . neuroprotective and disease - modifying drug research has intensified , and results rely heavily on accurate , early diagnoses . new advances in neuroimaging offer the promise of more personalized treatment for individuals with neuropsychiatric disorders . molecular imaging can provide patient - specific information that allows treatment to be tailored to the specific biological attributes of both the disease and the patient . the eventual success of experimental therapies rests on understanding the mechanisms of the various neuropsychiatric disorders , early and accurate diagnoses , and noninvasive approaches for monitoring the progression of disease and the response to treatment . although nuclear imaging is a promising technique , several barriers must be addressed to facilitate its successful application to neuropsychiatric disorders . the major barrier to nuclear medicine imaging of molecular targets may be the difficulty in developing radiotracers . another barrier is the lack of current evidence to support the use of molecular imaging as a diagnostic tool in clinical practice . the radiotracers used as biomarkers in neuropsychiatric disorders must have both biologic relevance and a strong linkage to the clinical outcome of interest . despite the development of a large number of radioactive ligands for receptors , most do not fulfill the criteria for a good label , and only a few have been applied in clinical nuclear medicine . however , recent developments in improved detector crystals and three - dimensional image acquisition have markedly enhanced both sensitivity and resolution . radiotracer imaging with pet and , to a lesser extent , with spect is ideally suited for in vivo applications , due to its superior anatomic resolution . parkinson 's disease ( pd ) , the second most common neurodegenerative disorder , is characterized by severe loss of nigrostriatal neurons , which results in a deficiency of the neurotransmitter , dopamine . clinical diagnosis of pd relies on the presence of characteristic motor symptoms , including bradykinesia , rigidity , and resting tremors . in addition , nonmotor features have been increasingly recognized as early symptoms [ 4 , 5 ] . nonetheless , an early differential diagnosis can be difficult , particularly because the initial presentation may include overlapping clinical features , like essential tremor , vascular parkinsonism , drug - induced parkinsonism , and atypical parkinsonian syndrome ( i.e. , multiple system atrophy and progressive supranuclear palsy ) . often , the clinical diagnosis of pd is supported by a positive response to dopaminergic drugs , particularly levodopa . however , some patients with pathologically confirmed pd respond poorly to levodopa ; conversely , other patients with early multiple system atrophy or progressive supranuclear palsy respond well to drug treatment . previously , the rate of misdiagnosis of idiopathic pd was as high as 24% [ 6 , 7 ] . the evolution of neuroimaging techniques over the past several years has yielded unprecedented information about the degenerative processes in pd and other movement disorders . pet and spect techniques have been successfully employed in identifying dopaminergic dysfunction in pd by detecting changes in brain levodopa , glucose metabolism , and dopamine transporter binding . first , dopa decarboxylase activity and dopamine turnover can both be measured with f - fdopa pet . second , the availability of presynaptic dopamine transporters ( dats ) can be assessed with various tracers , which are typically tropane based . third , vesicle monoamine density in dopamine terminals can be evaluated with c - dihydrotetrabenazine pet [ 8 , 9 ] . the uptake of f - fdopa reflects both the density of the axonal terminal plexus and the activity of the striatal aromatic amino acid decarboxylase ( aadc ) , the enzyme responsible for the conversion of f - fdopa to f - dopamine [ 3 , 10 ] . therefore , particularly in the early stages of disease , f - fdopa pet may underestimate the degenerative process , due to the compensatory upregulation of aadc in the remaining terminals . moreover , because aadc is present in the terminals of all monoaminergic neurons , measurements of f - fdopa uptake into extrastriatal areas provide an index of the density of the serotonergic , noradrenergic , and dopaminergic terminals [ 3 , 10 ] . dat is a protein complex located in presynaptic dopaminergic nerve terminals , which serves as the primary means for removing monoaminergic neurotransmitters from the synaptic cleft . several pet ligands ( c - cft , f - cft , f - fp - cit , and c - pe2i ) and spect tracers ( i--cit , i - fp - cit , i - altropane , i - pe2i , and tc - trodat-1 ) are now available to measure dat availability [ 10 , 11 ] . the technetium - based tc - trodat-1 has the advantage of being relatively inexpensive and available in kit form . however , its specific signal is lower than the i - based spect tracers . in general , all the dat markers have shown efficacy in pd similar to that achieved with f - fdopa pet ; all are able to differentiate patients with early pd from healthy subjects with a sensitivity and specificity of approximately 90% [ 1315 ] . a multicenter , phase iii trial conducted at institute of nuclear energy research ( iner ) indicated that patients with pd were easily distinguished from healthy volunteers with the tc - trodat-1 spect , which had a sensitivity of 97.2% and a specificity of 92.6% ( unpublished data ) . figure 1 depicts the typical dopamine transporter images in 4 patients with pd for different stage and a healthy control . the differentiation between normal and parkinson 's disease is primarily based on shape , which reflects differences of uptake intensity . the high sensitivity and specificity of dat images make them useful in both the clinical diagnosis of pd and preclinical screening for asymptomatic individuals . to date , only datscan ( i - fp - cit ) and tc - trodat-1 are available on the market and licensed for detecting loss of functional dopaminergic neuron terminals in the striatum . in contrast to f - fdopa , the striatal uptake of dat ligands in early pd may overestimate the reduction in terminal density due to the relative downregulation of dat in the remaining neurons as a response to nigral neuron loss ; this is a compensatory mechanism that acts to maintain synaptic dopamine levels . dat binding falls with age in healthy subjects , but striatal f - fdopa uptake does not appear to be age dependent [ 3 , 12 , 16 ] . dopaminergic neurotransmission plays an important role in regulating several aspects of basic brain function , including motor behavior , motivation , and working memory . dopaminergic systems are also a central element in the brain reward system that controls learning . there are five subtypes of dopamine receptors , d1 , d2 , d3 , d4 , and d5 . d1 and d5 receptors are members of the d1-like family of dopamine receptors , and d2 , d3 , and d4 receptors are members of the d2-like family . the assessment of d1-like receptors has not achieved clinical significance ; therefore , in the past , many investigations have focused on the d2-like receptor system . positron - emitting ligands , like c - raclopride , f - spiperone , and f - methyl - benperidol have been developed for the visualization of dopamine d2 receptors in vivo with pet ; alternatively , single - proton emitting ligands , like i - iodobenzamide ( i - ibzm ) , i - epidrpride , and i - ibf have been developed for spect imaging . c - raclopride , a derivative of benzamide , is currently the gold standard pet tracer for d2 receptors . it is important to discriminate between idiopathic parkinson 's disease ( ipd ) and other neurodegenerative parkinsonian syndromes ( non - ips ) , because there are marked differences in the prognoses and therapies . dat imaging with tc - trodat-1 spect has proven successful in the differential diagnosis between pd and vascular parkinsonism ; patients with pd displayed a significant decrease in striatal tc - trodat-1 uptake compared to patients with vascular parkinsonism ( p < .01 ) . higher diagnostic accuracy in the differential diagnosis of parkinsonism may be achieved by combining pre- and postsynaptic quantitative information about the dopaminergic system . previous imaging studies with the most commonly used dopamine d2 receptor tracers , c - raclopride and i - ibzm , have consistently shown that dats , which represent the integrity of the nigrostriatal neurons , are downregulated in patients with early ipd , but d2 receptors are normal or even upregulated . in contrast , patients with multiple system atrophy ( msa ) typically show reductions in both dat and d2 binding [ 19 , 20 ] . however , with progression of pd , striatal d2 receptor activity returns to normal or may fall below normal levels , and the small differences in d2 binding make it difficult to distinguish between pd , ipd , and healthy control groups ( unpublished data ) . gradually , we have attained a broader understanding of the genetic linkages among different aspects of parkinsonism . several genes , including pink1 , snca , parkin , uchl1 , dj1 , and lrrk2 , have been associated with familial parkinsonism and/or sporadic early onset parkinson 's disease ( eopd ) . a tc - trodat-1 scan showed that patients with the pink1 mutation displayed a rather even , symmetrical reduction of dopamine uptake ; in contrast , patients with late - onset parkinson 's disease ( lopd ) displayed a dominant decline in dopamine uptake in the putamen . in addition , tc - trodat-1 spect revealed that the dopamine transporter concentration was significantly reduced in patients with machado - joseph disease and in asymptomatic gene carriers compared to healthy volunteers ( p < .001 ) . this indicated that the brain spect was capable of detecting early alterations in dopamine neurons of the striatal region . molecular imaging is also a major tool for the evaluation of new experimental therapeutic strategies in pd , particularly for those that aim to restore or protect striatal dopaminergic innervation . neuroprotective and disease - modifying drug research has intensified , and the results rely heavily on accurate early diagnosis . several teams of investigators have reported the results from double - blind , placebo - controlled trials of human embryonic dopaminergic tissue transplantation for the treatment of pd . evaluations with f - fdopa scans have shown that significant declines in the motor scores over time after transplantation ( p < .001 ) , based on the unified parkinson disease rating scale ( updrs ) , were associated with increases in putamen f - fdopa uptake at 4-year posttransplantation followups ( p < .001 ) . furthermore , posttransplantation changes in putamen pet signals and clinical outcomes were significantly intercorrelated ( p current imaging biomarkers may be a valuable adjunct to clinical data for assessing both the mechanism and efficacy of neuroprotective agents . to date , functional imaging studies in these trials have failed to demonstrate a clear - cut neuroprotective effect on nigrostriatal degeneration . in addition , discordance between clinical and imaging outcomes has been reported in studies that compared a dopamine agonist versus levodopa [ 10 , 27 ] . gene therapy may be potentially useful for ameliorating the motor symptoms of pd and the motor complications of pd treatments . several gene therapy studies in humans investigated transductions ( with various viral vectors ) of glial - derived neurotrophic factor ( gdnf ) , neurturin ( ntn ) , aadc , or glutamic acid decarboxylase ( gad ) . brain imaging with f - fdoda pet , f - ffmt , or f - fdg pet was used to evaluate clinical outcomes adjunct to the updrs scores [ 2933 ] . data suggest that the number of patients with ad worldwide will increase from 26.6 million in 2006 to 106.8 million in 2050 [ 35 , 36 ] . beta amyloid ( a 142 ) is considered a primary cause of ad , but there are numerous other ongoing processes , including oxidative stress , inflammatory reactions , microglial activation , tau phosphorylation , and neurotransmitter impairments , that can lead to cognitive impairments [ 36 , 37 ] . some methods have shown promise as diagnostic tools for ad , including mri measurements of medial temporal lobe atrophy , pet imaging of glucose metabolism and a deposits , and cerebral spinal fluid ( csf ) biomarkers . a substantial number of studies have shown that mri measurements of hippocampal atrophy can distinguish between subjects with ad and older subjects with normal cognition with 8090% accuracy [ 34 , 38 ] . pet tracers have been developed for studies of functional activity , neurotransmitter activity , and pathologic processes in different dementia disorders . pet imaging with f - fdg has shown that ad was associated with metabolic deficits in the neocortical association areas , but normal activity was observed in the basal ganglia , thalamus , cerebellum , primary sensory motor cortex , and visual cortex . during disease progression , glucose metabolism continues to decline , and this is associated with declining scores on cognitive tests . a large multicenter study showed 93% sensitivity and specificity for distinguishing between individuals with ad and normal individuals . this indicated that f - fdg pet can serve as a biomarker in clinical trials . imaging techniques that include radiolabeled pet tracers that can bind to aggregated a peptides in a plaques have the potential of directly assessing relative brain a plaque pathology . several research groups have applied the small - molecule approach to the development of tracers suitable for amyloid imaging ; for example , derivatives of congo red , thioflavin , stilbene , chrysamine - g , and acridine were developed for pet and spect imaging . this opens up new possibilities for early diagnosis and provides new tools for monitoring antiamyloid therapy in ad [ 36 , 4144 ] . f - fddnp was the first pet tracer used in vivo for detecting cerebral amyloid plaques . increased f - fddnp binding was observed in the temporal , parietal , and frontal regions of the ad brain , compared with brains from older control subjects with no cognitive impairments . pittsburgh compound b ( c - pib ) , a derivative of thioflavin - t , binds with high affinity and high specificity to neuritic a plaques , but it shows no binding to diffuse plaques or neurofibrillary tangles . c - pib pet is presently the most used amyloid pet ligand ; it has been studied in over 2000 subjects [ 39 , 40 ] . imaging withc - pib pet showed significantly higher retention in the frontal , temporal , parietal , and occipital cortices and the striatum of patients with ad compared to healthy controls ( 1.9- to 1.5-fold differences ) . interestingly , the increase in c - pib retention observed in mild ad patients relative to age - matched healthy controls was larger than the decrease in f - fdg uptake in ad patients relative to controls . figure 2 illustrates cerebral glucose metabolism as assessed by f - fdg pet and amyloid plaque burden as assessed by c - pib pet in two ad patients and a healthy control . the memory predominant subtype , amnestic mild cognitive impairment ( mci ) , has been suggested to constitute a transitional stage between normal aging and ad . clinical studies suggested that about 12% of patients with the amnestic form of mci progressed to ad within one year and up to 80% progressed to ad after 6 y . glucose metabolism is a sensitive measure of change in cognition and functional ability , both in ad and in mci ; therefore , accurate detection of changes in glucose metabolism has value in predicting future cognitive decline . clinical follow - up studies have shown that patients with mci who converted to ad showed significantly higher c - pib retention than those with mci that did not convert ; this suggested the possibility of identifying prodromal ad with -amyloid imaging [ 36 , 50 ] . to date , c - pib pet is the most widely used imaging approach for the visualization of a. although c - pib can be used for academic studies , the 20-minute half - life of c and limited manufacturing access makes the molecule unsuitable for widespread use as a routine diagnostic agent . f , with a half - life of 110 minutes , offers much better potential for manufacturing and distribution . in addition to f - fddnp , several f - labeled a tracers have been successfully investigated in clinical trials , including f - pib ( f - flutemetamol ) , f - av-1 ( f - bay94 - 9172 ) , and f - av-45 ( florbetapir f-18 ) . f - av-45 , a derivative of stilbene , has demonstrated high binding to the a aggregates in patients with ad . in a pilot study , patients with ad and healthy controls displayed averages of 1.67 0.18 and 1.25 0.18 , respectively , in standard uptake value ratios ( suvrs ) of f - av-45 for the cortical area relative to the cerebellum . the interim results of a phase iii study demonstrated that the f - av-45 pet images correlated strongly with the postmortem histopathology findings . the pet images correctly identified subjects that had -amyloid deposits and showed where in the brain the deposits had accumulated . recently , a marketing application for florbetapir was submitted to the fda . in the regions of the highest retention , the specific signal from f - fddnp was only 0.3 times that of the reference tissue ; this contrasts with the 1.52.0-fold specific signal with respect to the reference tissue that was associated with the c - pib and c - sb13 tracers . visual assessment of c - pib pet images demonstrated promise as a supportive diagnostic marker for ad with both sensitivity and specificity values of 0.95 . additionally , f - pib showed promise as an ad biomarker with both sensitivity and specificity values of 0.93 ; in contrast , f - fddnp pet images showed only moderate accuracy , with a sensitivity of 0.81 and a specificity of 0.72 [ 53 , 54 ] . development of amyloid probes applicable to both pet and spect in ad may provide a cost - effective approach , particularly when effective antiamyloid therapy becomes available . accurate and early diagnoses of dementias will become increasingly important as new therapies are introduced . the presently available imaging ligands will be valuable for monitoring any reduction in a plaques . ongoing studies with antiamyloid therapies , including a vaccination , an a fibrillization inhibitor , or a secretase modulator , have described some difficulties in the assessment of drug effects at autopsy . assessments of the efficacy of antiamyloid therapy could be greatly facilitated with the visualization of amyloid plaques in the brains of living patients with ad . one hypothesis holds that there is a cholinergic mechanism underlying ad ; this hypothesis proposes that degeneration of cholinergic neurons in the basal forebrain nuclei causes disturbances in presynaptic cholinergic terminals in the hippocampus and neocortex , which are important for memory disturbances and other cognitive symptoms . the most important degrading enzyme for acetylcholine in the human cortex is acetylcholinesterase , which is present in cholinergic axons . the pet tracers , c - pmp and c - mp4a , have been used to measure acetylcholinesterase activity . these tracers showed a decline in acetylcholinesterase activity in patients with ad compared to healthy controls . reduction in acetylcholinesterase activity has also been reported in patients with mci , particularly in those that later converted to ad . the inhibition of acetylcholinesterase with specific therapeutic drugs , like donepezil , can also be measured with these tracers . serotonin ( 5-hydroxy - tryptamine , 5-ht ) is a modulatory neurotransmitter in the human brain that regulates mood , anger , reward , aggression , and appetite . serotonergic neurotransmission is altered in many neuropsychiatric disorders , including depression , anxiety disorders , biopolar disorders ( bd ) , compulsive disorders , autism , schizophrenia , ad , and pd [ 2 , 59 ] . there is broad heterogeneity in the postsynaptic serotonin receptors ; however , the only suitable radioligands available are for 5-ht1a and 5-ht2a receptors . for visualization of brain 5-ht1a receptor activity , the c - way-100635 ( way ) and f - labeled analogs of way ( f - trans - fcway ) exhibited high affinity . 5-ht1a receptors are present in high density in the hippocampus , septum , amygdala , hypothalamus , and neocortex of the human brain . pet imaging with f - fcway demonstrated that abnormalities of 5-ht1a receptors were present in affective disorders , particularly in depression and panic disorder [ 6062 ] . however , there were only small differences between the study population and the healthy control groups ; this may indicate considerable overlap between groups , which would impede a clear diagnostic cutoff . 5-ht2a receptors are present in all neocortical regions , with lower densities in the hippocampus , basal ganglia , and thalamus . 5-ht2a receptors have been quantified with f - altanserin , f - setoperone , and i-2-ketanserin . f - setoperone showed specific binding in the cortex , reversibility , and a favorable ratio of specific binding to nonspecific binding and unbound label . pet studies with f - setoperone showed significantly decreased 5-ht2a receptor densities in neuroleptic - nave patients with schizophrenia and in patients with ad [ 64 , 65 ] . the serotonin transporter ( sert , 5-htt ) sert is the primary target for selective serotonin reuptake inhibitors ( ssris ) , which are commonly used for treatment of psychiatric disorders , and as the first - line treatment for major depression and anxiety disorders . i--cit was the first sert marker to be applied ; however , it binds all the monoamine transporters , including sert , dat , and norepinephrine transporters ( nets ) . the first pet radiotracer that was selective for brain sert was c - mcn5652 [ 67 , 68 ] . subsequently , two excellent pet tracers were developed for visualization of sert in human brains ; these are known as c - dasb and c - madam , and both exhibit high affinity and selectivity for sert . alternatively , a promising spect tracer was also developed for visualization of sert in humans ; this i - adam , a derivative of 403u76 , displays high affinity and selectivity for sert . i - adam was efficiently taken up in the hypothalamus , brain stem , pons , and medial temporal lobe . compared with previous radioligands , c - dasb offers both high selectivity and a favorable ratio of specific binding relative to free and nonspecific binding . therefore , c - dasb pet imaging is considered as the state - of - the - art method for quantifying sert in humans . in vivo imaging studies of sert have generally been conducted in patients with acute depression ; however , the results have been somewhat inconsistent . those patients had lower sert binding potential in the amygdala and midbrain region compared to healthy subjects . in a study with c - dasb pet , brain sert density was investigated in subjects with a history of major depressive episodes ( mdes ) . those patients displayed no regional differences in sert binding potential compared to healthy subjects [ 73 , 74 ] . newberg et al . used i - adam spect and found reduced sert binding potential in the midbrain regions of patients with depression compared to healthy volunteers ; however another study with i - adam spect found no difference between similar groups . bipolar i disorder ( bd i ) is defined by a full - blown episode of mania , whereas bipolar ii disorder ( bd ii ) is defined by hypomanic episodes and depressive episodes . assessment of sert binding in the midbrain with i - adam spect demonstrated no differences in brain sert binding between healthy controls and patients with bd that were in a euthymic state . however , the sert binding was lower in patients with bd i compared to those with bd ii . furthermore , the decreased specific uptake ratios ( surs ) in bd i patients were well correlated with the duration of the illness ( r = 0.742 , p = .014 ) . in addition to the serotonin system , the dat gene was also shown to play a role in the etiology of both adult and pediatric bd . using tc - trodat-1 spect , chang et al . found that striatal dat availability was significantly higher in patients with euthymic bd than in healthy volunteers . central serotoninergic function has been linked to both normal and pathological aggressiveness . because hostility shares some similarities with aggression , it is possible that central serotonin activity is also related to hostility . i - adam spect imaging indicates that central serotoninergic activities may play a role in hostility . the hostile attribution subscore was negatively correlated with sert availability ( r = 0.71 , p < .05 ) . although imaging of the serotonin system has many potential clinical uses , currently , serotonin imaging is not used for the routine diagnosis of any neuropsychiatric diseases . perhaps one of the most valuable current uses of molecular imaging technology is the determination of the in vivo brain occupancy of a putative pharmaceutical at the target of interest when developing a pharmaceutical treatment for depression . currently , there is no other method available for in vivo determinations of receptor occupancy in humans . drug concentrations in plasma may vary by over tenfold when identical doses are administered to different patients . therapeutic drug monitoring can reduce the number of nonresponders and decrease the risk for developing side effects by identifying patients with either insufficient or excess serum concentrations of the drug . the first ssri occupancy study with c - dasb pet found 80% striatal occupancy in multiple regions after 4 weeks of antidepressant treatment , with doses of paroxetine and citalopram known to have clinical effects distinct from placebo . thereafter , occupancy measurements of citalopram , fluoxetine , sertraline , paroxetine , or venlafaxine with c - dasb pet suggested that 80% occupancy of the sert was a necessary minimum for ssri efficacy for depressive episodes . moreover , occupancy increased as the dose or plasma level increased , and occupancy reached a plateau at saturating doses . figure 3(a ) illustrates a typical occupancy study of citalopram on the serotonin transporters with c - dasb pet scan in a depressed subject . figure 3(b ) summaries the striatal sert occupancy in depressed subjects after 4 weeks of treatment at minimum therapeutic doses of five ssris . given the association between the clinically relevant dose and sert occupancy for all ssris , it is now generally believed that an 80% sert occupancy with an ssri is therapeutically useful . this practical approach can be applied in phase i trials to assess whether potential new antidepressant drugs can adequately penetrate the brain and in phase ii clinical trials to guide dosing selections . nets are responsible for the reuptake of norepinephrine into presynaptic nerves , and they are a primary target of antidepressants . with the development of net tracers , like f - fmener - d2 and c - mrb , it is currently possible to investigate net occupancy for antidepressants that are selective norepinephrine reuptake inhibitors ( snris ) . in an investigation with f - fmener - d2 , the net occupancy of nortriptyline was 16% to 41% , which corresponded to a dose of 1075 mg . in addition to net , antidepressants also target dat . bupropion is thought to exert its antidepressive effect by blocking the dat and net with an efficacy comparable to that of ssris . with bupropion treatment , the average dat occupancy was 20.84% , measured by tc - trodat-1 spect . to achieve snri efficacy , the optimal and minimal occupancies of net remain to be determined . for antipsychotic drugs , it was shown that the occupancy of dopamine d2-like receptors was better correlated to plasma concentrations than to the doses administered . in association with the analysis of clinical effects , farde et al . established the concept that at least 6070% dopamine receptor occupancy was necessary for treating positive symptoms of schizophrenia . however , occupancies above 80% were associated with extrapyramidal side effects [ 82 , 86 ] . with the appropriate radioligands , molecular imaging enables the visualization of presynaptic and postsynaptic sites in the dopaminergic system . this can provide crucial information for the assessment and monitoring of neuroprotective agents , gene therapies , and stem cell strategies for the treatment of pd . future studies are needed in the development of new radiotracers to target nondopaminergic brain pathways and the glial reaction to disease . accurate and early differentiation of dementias will become increasingly important as new therapies are introduced . recently , reliable pet tracers for assessing the a burden in the brain have been introduced ; these tracers may be valuable for early diagnosis of the presymptomatic stages of ad . the measurement of -amyloid in vivo will greatly facilitate our understanding of the underlying pathophysiological mechanisms of ad ; in addition , these measurements will promote the testing of new antiamyloid drugs and therapies . although early studies were performed with c - pib , several groups have developed fluorinated compounds for widespread and routine diagnostic uses . although serotonin imaging has many potential clinical applications , currently , serotonin imaging is not used for the routine diagnosis of any neuropsychiatric diseases . perhaps one of the most valuable current uses of molecular imaging technology is the determination of in vivo brain occupancy of a putative pharmaceutical agent when developing a treatment for depression . finally , to achieve snri efficacy , the optimal and minimal occupancies of net remain to be determined . in the future , enhancements in image resolution and specific molecular tags will permit accurate diagnoses of a wide range of diseases , based on both structural and molecular changes in the brain . for widespread application , advances in molecular imaging should include the characterization of new radiotracers , application of modeling techniques , standardization and automation of image - processing techniques , and appropriate clinical settings in large multicenter trials . the growing field of molecular imaging is helping nuclear medicine physicians identify pathways into personalized patient care .

neuropsychiatric disorders are becoming a major socioeconomic burden to modern society . in recent years , a dramatic expansion of tools has facilitated the study of the molecular basis of neuropsychiatric disorders . molecular imaging has enabled the noninvasive characterization and quantification of biological processes at the cellular , tissue , and organism levels in intact living subjects . this technology has revolutionized the practice of medicine and has become critical to quality health care . new advances in research on molecular imaging hold promise for personalized medicine in neuropsychiatric disorders , with adjusted therapeutic doses , predictable responses , reduced adverse drug reactions , early diagnosis , and personal health planning . in this paper , we discuss the development of radiotracers for imaging dopaminergic , serotonergic , and noradrenergic systems and -amyloid plaques . we will underline the role of molecular imaging technologies in various neuropsychiatric disorders , describe their unique strengths and limitations , and suggest future directions in the diagnosis and management of neuropsychiatric disorders .

1. Introduction 2. Parkinson's Disease and Related Disorders 3. Alzheimer's Disease 4. Affective Disorders 5. Conclusions and Future Directions

modern neuroimaging offers tremendous opportunities for gaining insights into the molecular basis of neuropsychiatric disorders . several techniques including functional magnetic resonance imaging ( fmri ) and nuclear medicine imaging can measure regional changes in cerebral activity ; however , nuclear medicine imaging is the only tool for direct measurements of neurotransmitter levels and the distribution , density , and activity of receptors or transporters . in pet , annihilation coincidence detection is used in lieu of absorptive collimation to determine the directionality of the detected photons ; this partially explains the greater spatial resolution and sensitivity of pet . the most commonly used radioisotopes for labeling pet radiopharmaceuticals are c and f. c has a short half - life of approximately 20 min , which allows multiple studies in the same day . a major advantage of nuclear medicine imaging is the extraordinarily high sensitivity ( 10 to 10 m ) , many orders of magnitude greater than the sensitivities available with mri ( 10 m ) . because many molecules relevant to neuropsychiatric disorders are present at concentrations below 10 m , nuclear medicine imaging is currently the only available in vivo method capable of quantifying subtle cerebral pathophysiological changes that might occur before neurostructural abnormalities take place . pet and spect can measure biological processes , like glucose consumption and regional cerebral blood flow ( rcbf ) , which may change in various neuropsychiatric disorders . radioligands must fulfill several criteria for successful pet or spect imaging , including stability of labeling , sufficient affinity and high selectivity for the specific receptor , combined with low nonspecific binding to brain tissues that do not contain the receptor of interest , and rapid permeation through the blood - brain barrier to permit tracers access to receptors . selective radioligands are available for various transmitter systems ; these enable the visualization of receptor distributions in the normal brain and the detection of changes in receptor binding during various physiologic activities or under pathologic conditions . quantitative imaging has gained clinical importance for measuring the activities of several receptors / transporters and molecular targets , for example , quantification of dopamine transporters for detecting loss of functional dopaminergic neuron terminals in the striatum , quantification of dopamine d2 receptors for studies of movement disorders and for assessments of receptor occupancy by neuroleptic drugs , quantification of serotonin ( 5-hydroxytryptamine , or 5-ht ) receptors in affective disorders , quantification of serotonin and norepinephrine transporters for assessment of occupancy of antidepressants , and quantification of -amyloid and acetylcholinesterase as markers of cognitive and memory impairments . neuroprotective and disease - modifying drug research has intensified , and results rely heavily on accurate , early diagnoses . new advances in neuroimaging offer the promise of more personalized treatment for individuals with neuropsychiatric disorders . molecular imaging can provide patient - specific information that allows treatment to be tailored to the specific biological attributes of both the disease and the patient . the eventual success of experimental therapies rests on understanding the mechanisms of the various neuropsychiatric disorders , early and accurate diagnoses , and noninvasive approaches for monitoring the progression of disease and the response to treatment . the major barrier to nuclear medicine imaging of molecular targets may be the difficulty in developing radiotracers . another barrier is the lack of current evidence to support the use of molecular imaging as a diagnostic tool in clinical practice . the radiotracers used as biomarkers in neuropsychiatric disorders must have both biologic relevance and a strong linkage to the clinical outcome of interest . despite the development of a large number of radioactive ligands for receptors , most do not fulfill the criteria for a good label , and only a few have been applied in clinical nuclear medicine . radiotracer imaging with pet and , to a lesser extent , with spect is ideally suited for in vivo applications , due to its superior anatomic resolution . parkinson 's disease ( pd ) , the second most common neurodegenerative disorder , is characterized by severe loss of nigrostriatal neurons , which results in a deficiency of the neurotransmitter , dopamine . clinical diagnosis of pd relies on the presence of characteristic motor symptoms , including bradykinesia , rigidity , and resting tremors . nonetheless , an early differential diagnosis can be difficult , particularly because the initial presentation may include overlapping clinical features , like essential tremor , vascular parkinsonism , drug - induced parkinsonism , and atypical parkinsonian syndrome ( i.e. pet and spect techniques have been successfully employed in identifying dopaminergic dysfunction in pd by detecting changes in brain levodopa , glucose metabolism , and dopamine transporter binding . therefore , particularly in the early stages of disease , f - fdopa pet may underestimate the degenerative process , due to the compensatory upregulation of aadc in the remaining terminals . moreover , because aadc is present in the terminals of all monoaminergic neurons , measurements of f - fdopa uptake into extrastriatal areas provide an index of the density of the serotonergic , noradrenergic , and dopaminergic terminals [ 3 , 10 ] . several pet ligands ( c - cft , f - cft , f - fp - cit , and c - pe2i ) and spect tracers ( i--cit , i - fp - cit , i - altropane , i - pe2i , and tc - trodat-1 ) are now available to measure dat availability [ 10 , 11 ] . to date , only datscan ( i - fp - cit ) and tc - trodat-1 are available on the market and licensed for detecting loss of functional dopaminergic neuron terminals in the striatum . in contrast to f - fdopa , the striatal uptake of dat ligands in early pd may overestimate the reduction in terminal density due to the relative downregulation of dat in the remaining neurons as a response to nigral neuron loss ; this is a compensatory mechanism that acts to maintain synaptic dopamine levels . dopaminergic neurotransmission plays an important role in regulating several aspects of basic brain function , including motor behavior , motivation , and working memory . dopaminergic systems are also a central element in the brain reward system that controls learning . d1 and d5 receptors are members of the d1-like family of dopamine receptors , and d2 , d3 , and d4 receptors are members of the d2-like family . the assessment of d1-like receptors has not achieved clinical significance ; therefore , in the past , many investigations have focused on the d2-like receptor system . positron - emitting ligands , like c - raclopride , f - spiperone , and f - methyl - benperidol have been developed for the visualization of dopamine d2 receptors in vivo with pet ; alternatively , single - proton emitting ligands , like i - iodobenzamide ( i - ibzm ) , i - epidrpride , and i - ibf have been developed for spect imaging . it is important to discriminate between idiopathic parkinson 's disease ( ipd ) and other neurodegenerative parkinsonian syndromes ( non - ips ) , because there are marked differences in the prognoses and therapies . dat imaging with tc - trodat-1 spect has proven successful in the differential diagnosis between pd and vascular parkinsonism ; patients with pd displayed a significant decrease in striatal tc - trodat-1 uptake compared to patients with vascular parkinsonism ( p < .01 ) . higher diagnostic accuracy in the differential diagnosis of parkinsonism may be achieved by combining pre- and postsynaptic quantitative information about the dopaminergic system . previous imaging studies with the most commonly used dopamine d2 receptor tracers , c - raclopride and i - ibzm , have consistently shown that dats , which represent the integrity of the nigrostriatal neurons , are downregulated in patients with early ipd , but d2 receptors are normal or even upregulated . however , with progression of pd , striatal d2 receptor activity returns to normal or may fall below normal levels , and the small differences in d2 binding make it difficult to distinguish between pd , ipd , and healthy control groups ( unpublished data ) . gradually , we have attained a broader understanding of the genetic linkages among different aspects of parkinsonism . several genes , including pink1 , snca , parkin , uchl1 , dj1 , and lrrk2 , have been associated with familial parkinsonism and/or sporadic early onset parkinson 's disease ( eopd ) . a tc - trodat-1 scan showed that patients with the pink1 mutation displayed a rather even , symmetrical reduction of dopamine uptake ; in contrast , patients with late - onset parkinson 's disease ( lopd ) displayed a dominant decline in dopamine uptake in the putamen . this indicated that the brain spect was capable of detecting early alterations in dopamine neurons of the striatal region . molecular imaging is also a major tool for the evaluation of new experimental therapeutic strategies in pd , particularly for those that aim to restore or protect striatal dopaminergic innervation . neuroprotective and disease - modifying drug research has intensified , and the results rely heavily on accurate early diagnosis . evaluations with f - fdopa scans have shown that significant declines in the motor scores over time after transplantation ( p < .001 ) , based on the unified parkinson disease rating scale ( updrs ) , were associated with increases in putamen f - fdopa uptake at 4-year posttransplantation followups ( p < .001 ) . beta amyloid ( a 142 ) is considered a primary cause of ad , but there are numerous other ongoing processes , including oxidative stress , inflammatory reactions , microglial activation , tau phosphorylation , and neurotransmitter impairments , that can lead to cognitive impairments [ 36 , 37 ] . pet tracers have been developed for studies of functional activity , neurotransmitter activity , and pathologic processes in different dementia disorders . pet imaging with f - fdg has shown that ad was associated with metabolic deficits in the neocortical association areas , but normal activity was observed in the basal ganglia , thalamus , cerebellum , primary sensory motor cortex , and visual cortex . during disease progression , glucose metabolism continues to decline , and this is associated with declining scores on cognitive tests . several research groups have applied the small - molecule approach to the development of tracers suitable for amyloid imaging ; for example , derivatives of congo red , thioflavin , stilbene , chrysamine - g , and acridine were developed for pet and spect imaging . this opens up new possibilities for early diagnosis and provides new tools for monitoring antiamyloid therapy in ad [ 36 , 4144 ] . increased f - fddnp binding was observed in the temporal , parietal , and frontal regions of the ad brain , compared with brains from older control subjects with no cognitive impairments . pittsburgh compound b ( c - pib ) , a derivative of thioflavin - t , binds with high affinity and high specificity to neuritic a plaques , but it shows no binding to diffuse plaques or neurofibrillary tangles . imaging withc - pib pet showed significantly higher retention in the frontal , temporal , parietal , and occipital cortices and the striatum of patients with ad compared to healthy controls ( 1.9- to 1.5-fold differences ) . f , with a half - life of 110 minutes , offers much better potential for manufacturing and distribution . f - av-45 , a derivative of stilbene , has demonstrated high binding to the a aggregates in patients with ad . the pet images correctly identified subjects that had -amyloid deposits and showed where in the brain the deposits had accumulated . in the regions of the highest retention , the specific signal from f - fddnp was only 0.3 times that of the reference tissue ; this contrasts with the 1.52.0-fold specific signal with respect to the reference tissue that was associated with the c - pib and c - sb13 tracers . additionally , f - pib showed promise as an ad biomarker with both sensitivity and specificity values of 0.93 ; in contrast , f - fddnp pet images showed only moderate accuracy , with a sensitivity of 0.81 and a specificity of 0.72 [ 53 , 54 ] . development of amyloid probes applicable to both pet and spect in ad may provide a cost - effective approach , particularly when effective antiamyloid therapy becomes available . ongoing studies with antiamyloid therapies , including a vaccination , an a fibrillization inhibitor , or a secretase modulator , have described some difficulties in the assessment of drug effects at autopsy . assessments of the efficacy of antiamyloid therapy could be greatly facilitated with the visualization of amyloid plaques in the brains of living patients with ad . one hypothesis holds that there is a cholinergic mechanism underlying ad ; this hypothesis proposes that degeneration of cholinergic neurons in the basal forebrain nuclei causes disturbances in presynaptic cholinergic terminals in the hippocampus and neocortex , which are important for memory disturbances and other cognitive symptoms . the most important degrading enzyme for acetylcholine in the human cortex is acetylcholinesterase , which is present in cholinergic axons . serotonin ( 5-hydroxy - tryptamine , 5-ht ) is a modulatory neurotransmitter in the human brain that regulates mood , anger , reward , aggression , and appetite . serotonergic neurotransmission is altered in many neuropsychiatric disorders , including depression , anxiety disorders , biopolar disorders ( bd ) , compulsive disorders , autism , schizophrenia , ad , and pd [ 2 , 59 ] . there is broad heterogeneity in the postsynaptic serotonin receptors ; however , the only suitable radioligands available are for 5-ht1a and 5-ht2a receptors . 5-ht1a receptors are present in high density in the hippocampus , septum , amygdala , hypothalamus , and neocortex of the human brain . however , there were only small differences between the study population and the healthy control groups ; this may indicate considerable overlap between groups , which would impede a clear diagnostic cutoff . 5-ht2a receptors are present in all neocortical regions , with lower densities in the hippocampus , basal ganglia , and thalamus . 5-ht2a receptors have been quantified with f - altanserin , f - setoperone , and i-2-ketanserin . f - setoperone showed specific binding in the cortex , reversibility , and a favorable ratio of specific binding to nonspecific binding and unbound label . the serotonin transporter ( sert , 5-htt ) sert is the primary target for selective serotonin reuptake inhibitors ( ssris ) , which are commonly used for treatment of psychiatric disorders , and as the first - line treatment for major depression and anxiety disorders . i--cit was the first sert marker to be applied ; however , it binds all the monoamine transporters , including sert , dat , and norepinephrine transporters ( nets ) . subsequently , two excellent pet tracers were developed for visualization of sert in human brains ; these are known as c - dasb and c - madam , and both exhibit high affinity and selectivity for sert . alternatively , a promising spect tracer was also developed for visualization of sert in humans ; this i - adam , a derivative of 403u76 , displays high affinity and selectivity for sert . i - adam was efficiently taken up in the hypothalamus , brain stem , pons , and medial temporal lobe . those patients had lower sert binding potential in the amygdala and midbrain region compared to healthy subjects . used i - adam spect and found reduced sert binding potential in the midbrain regions of patients with depression compared to healthy volunteers ; however another study with i - adam spect found no difference between similar groups . assessment of sert binding in the midbrain with i - adam spect demonstrated no differences in brain sert binding between healthy controls and patients with bd that were in a euthymic state . furthermore , the decreased specific uptake ratios ( surs ) in bd i patients were well correlated with the duration of the illness ( r = 0.742 , p = .014 ) . in addition to the serotonin system , the dat gene was also shown to play a role in the etiology of both adult and pediatric bd . although imaging of the serotonin system has many potential clinical uses , currently , serotonin imaging is not used for the routine diagnosis of any neuropsychiatric diseases . perhaps one of the most valuable current uses of molecular imaging technology is the determination of the in vivo brain occupancy of a putative pharmaceutical at the target of interest when developing a pharmaceutical treatment for depression . therapeutic drug monitoring can reduce the number of nonresponders and decrease the risk for developing side effects by identifying patients with either insufficient or excess serum concentrations of the drug . the first ssri occupancy study with c - dasb pet found 80% striatal occupancy in multiple regions after 4 weeks of antidepressant treatment , with doses of paroxetine and citalopram known to have clinical effects distinct from placebo . thereafter , occupancy measurements of citalopram , fluoxetine , sertraline , paroxetine , or venlafaxine with c - dasb pet suggested that 80% occupancy of the sert was a necessary minimum for ssri efficacy for depressive episodes . moreover , occupancy increased as the dose or plasma level increased , and occupancy reached a plateau at saturating doses . figure 3(a ) illustrates a typical occupancy study of citalopram on the serotonin transporters with c - dasb pet scan in a depressed subject . figure 3(b ) summaries the striatal sert occupancy in depressed subjects after 4 weeks of treatment at minimum therapeutic doses of five ssris . nets are responsible for the reuptake of norepinephrine into presynaptic nerves , and they are a primary target of antidepressants . with the development of net tracers , like f - fmener - d2 and c - mrb , it is currently possible to investigate net occupancy for antidepressants that are selective norepinephrine reuptake inhibitors ( snris ) . with the appropriate radioligands , molecular imaging enables the visualization of presynaptic and postsynaptic sites in the dopaminergic system . this can provide crucial information for the assessment and monitoring of neuroprotective agents , gene therapies , and stem cell strategies for the treatment of pd . future studies are needed in the development of new radiotracers to target nondopaminergic brain pathways and the glial reaction to disease . recently , reliable pet tracers for assessing the a burden in the brain have been introduced ; these tracers may be valuable for early diagnosis of the presymptomatic stages of ad . although serotonin imaging has many potential clinical applications , currently , serotonin imaging is not used for the routine diagnosis of any neuropsychiatric diseases . perhaps one of the most valuable current uses of molecular imaging technology is the determination of in vivo brain occupancy of a putative pharmaceutical agent when developing a treatment for depression . in the future , enhancements in image resolution and specific molecular tags will permit accurate diagnoses of a wide range of diseases , based on both structural and molecular changes in the brain . for widespread application , advances in molecular imaging should include the characterization of new radiotracers , application of modeling techniques , standardization and automation of image - processing techniques , and appropriate clinical settings in large multicenter trials . the growing field of molecular imaging is helping nuclear medicine physicians identify pathways into personalized patient care .

in the field of artificial photosynthesis , the coupling of photocatalytic and enzymatic processes has recently gained increasing attention . one possible merging point is the combination of photocatalytic cofactor reduction steps with enzymatic carbon dioxide reduction . here , recycling of two - electron - reduced compounds , such as the reduced form of nicotinamide adenine dinucleotide ( nadh ) or flavin cofactors , is a widely applied strategy that saves valuable resources . in the presence of electron - donating substrates , nadh regeneration can be achieved either biochemically , using different oxidoreductase enzymes , or via chemical catalysis and artificial photosynthetic reaction centers . in response to increasing interest in sustainable co2 fixation based on renewable energy research , visible - light - driven cofactor conversion systems have been suggested for photoenzymatic co2 reduction . the goal of such processes is to couple the photocatalyzed recycling of nicotinamide adenine dinucleotide ( nad ) to biocatalytic transformations of co2 or carbonates with a cascade of enzymes producing formates , formaldehyde , and/or methanol , which can be considered as carbon - neutral solar fuels . photoenzymatic reactions require light - harvesting chromophores efficiently interacting with the functional components responsible for catalysis . in this context , metal - binding and visible - light - absorbing polymeric compounds with a bis-5,5-(phenylene - ethynylene)-2,2-bipyridylene backbone represent quite interesting building blocks for the development of novel photocatalytic systems , as they provide the possibility of tuning the optical properties of the polymer - based ligand as well as the catalytic and optical properties of the coordinated metal complexes . different rhenium bis-5,5-(phenylene - ethynylene)-2,2-bipyridylene complexes , for example , were thoroughly investigated by schanze and co - workers . ruthenium - containing polymers incorporating 4,4- or 5,5-(phenylene - ethynylene)-2,2-bipyridylene moieties as ligands have been introduced and widely characterized by klemm et al . as well as other groups . in earlier studies we investigated both spectroscopic and catalytic properties of bis-5,5-(phenylene - ethynylene)-2,2-bipyridylene - based oligomeric and polymeric rhenium and rhodium compounds . the rhodium -cyclopentadienyl half - sandwich complex of bis-5,5-(phenyl - ethynylene)-2,2-bipyridine was successfully applied on a redox - active electrode for the regeneration of nad to nadh . it is well established that organometallic rhodium complexes of the type [ rhcp*(1,2-diimine)h2o ] ( with 1,2-diimine being derivatives of 2,2-bipyridine ( bpy ) or 1,10-phenanthroline ; cp * = pentamethyl - cyclopentadienyl ) accelerate the formation of nadh in neutral to slightly basic aqueous solutions . they have been investigated for chemical , photochemical , and electrocatalytic cofactor reduction of nadh and model compounds . these rhodium diimine complexes exhibit favorable catalytic properties , e.g. , good selectivity in the hydrogenation of nad to form the enzymatically active 1,4-dihydronicotinamide isomer of nadh in addition to high reaction rates for the reduction reaction . work in this direction has been published by cosnier et al . , who electropolymerized a pyrrole - substituted bipyridyl - rhodium complex and applied this electrode to electrochemical cofactor reduction . light - driven regeneration reactions of the nadh cofactor in various systems are also , with few exceptions , often based on [ rhcp*(bpy)h2o ] as hydride - transfer catalyst . they are combined with different molecular and heterogeneous photosensitizers , e.g. , rhodamine dyes , semiconducting particles and quantum dots , metalloporphyrins , and , quite recently , graphene - derived photosensitizers . in the field of heterogeneous photosensitizing agents , structured carbon nitride materials have recently shown promising performance as photosensitizers for [ rhcp*(bpy)h2o ] in selective 1,4-nadh regeneration . these surface - engineered carbon nitride materials can also reduce nad without use of a redox mediator ; however , this leads also to the formation of enzymatically inactive 1,6-dihydronicotinamide isomer . it seems thus advantageous to keep the [ rhcp*(diimine)h2o ] moiety inside the photochemical nadh regeneration system . separation of the products and the regenerating catalyst for photoenzymatic processes was found to be an unsolved issue . as the rhodium catalyst is the costly part of most described regioselective photochemical regeneration methods for nadh , it would be beneficial to separate and reuse the hydride - transfer catalyst also in photochemical systems . furthermore , [ rhcp*(bpy)h2o ] can inactivate certain redox enzymes that are used to reduce various substrates of interest . this can be the case for co2 reduction with formate dehydrogenase and for numerous more specialized and stereoselective enzymatic reactions . schematic energy diagram and reaction pathway of the photochemical reduction of nadh with coupled enzymatic processes : d , donor ; ps , photosensitizer ; cat . , hydrogenation catalyst - oxidized form ; cat .- h , hydrogenation catalyst - active hydride species ; s , substrate ; p , product . in the present work , a new bipyridine - containing poly(arylene - ethynylene)-alt - poly(arylene - vinylene ) copolymer with a redox - active rhodium cyclopentadienyl complex as the active site for catalytic substrate conversion is presented . its application for the chemoenzymatic and photochemical regeneration of nadh is introduced and described in detail . when transferred on solid substrates , such as glass beads , this active polymer - based hydride - transfer catalyst can be easily separated from the reaction solution and furthermore be used repeatedly under different aqueous reaction conditions because of its high stability . the polymer compound reduces redox cofactors with triethanolamine ( teoa ) as electron donor . it works as both immobilizing agent and photosensitizer and thus , in contrast to similar systems with free rhodium complexes in solution , could minimize the contact of the catalyst compound with the nadh - binding site of enzymes applied in photoenzymatic redox systems . a prerequisite for obtaining the polymer - based rhodium complex rh - bipye - pvab is the prior synthesis of the metal - free polymer bipye - pvab , as depicted in scheme 1 . 5,5-bis(4-formyl-2,5-dioctyloxyphenylethynyl)-2,2-bipyridine ( 3 ) was obtained in 55% yield through sonogashira pd - catalyzed cross - coupling reaction of 4-formyl-2,5-dioctyloxyphenylacetylene ( 1 ) with 5,5-dibromo-2,2-bipyridine ( 2 ) and chromatographic workup . h nmr scan ( 200 mhz , cdcl3 ) of compound 3 . all peaks can be readily assigned to the expected chemical structure . emmons olefination reaction of 3 with 2,5-bis(2-ethylhexyloxy)-p - xylylene - bis(diethylphosphonate ) ( 4 ) using potassium tert - butoxide in excess as base led to the desired polymer . bipye - pvab was obtained as a red product in 84% yield after soxhlet extraction of the crude polymer with a refluxing methanol / diethyl ether mixture . the combination of linear octyl- and branched 2-ethylhexyloxy side chains enables good solubility in common organic solvents and good film - forming ability . the chemical structure of bipye - pvab was confirmed by h and c nmr spectroscopy . the broadness of the h nmr peaks is a clear indication for the polymeric nature of the material . calibrated with polystyrene standards , it provided a number - average molecular weight , mn , of 10 900 g mol and a weight - average molecular weight , mw , of 38 500 g mol , leading to a polydispersity index , pdi , of 3.5 and an average degree of polymerization , pn , of 9 . the newly synthesized materials were designed to work as light - harvesting photosensitizers in the presence of substrate - containing solvents . therefore , at first the photophysical characterization was performed in solution , and the maxima of absorption ( a ) and emission ( f ) , as well as fluorescence quantum yield ( f ) values were determined . the uv vis spectrum of bipye - pvab shows a strong red shift due to higher conjugation length compared to the initial bipyridyl - containing dialdehyde 3 . a further red shift occurs after complexation with rhodium at the bipyridyl moiety upon formation of rh - bipye - pvab , which has also been reported for the corresponding monomeric rhodium(iii ) complexes of such 5,5-bis(phenylethynyl)-2,2-bipyridine ligands . the luminescence maximum lies at 443 nm , with a quantum yield of = 0.5 ( excitation at 402 nm , calibration against quinine sulfate ) . it is important to note that the intensity of the blue luminescence usually observed for this class of polypyridyl derivatives at room temperature in solution is known to be very sensitive to slight ph variations and solvent purity effects . in chloroform solution , the polymeric bipyridine - containing material bipye - pvab also shows two main absorption bands , with peaks at 336 and 491 nm . upon excitation at 347 nm , the emitted light shows maximum intensity at 526 nm . using two different luminescence standards ( quinine sulfate and sodium fluorescein ) , a quantum yield of = 0.50 the blue - green luminescence of the polymer bipye - pvab , with a maximum around 526 nm , is almost completely quenched upon complexation of the polymer with rhodium to give rh - bipye - pvab . such behavior was also found in earlier studies with the corresponding monomeric model substances . the metal - containing polymer rh - bipye - pvab shows only very weak background emission . additionally , the optical absorbance is significantly red - shifted by about 50 nm compared to that of the parent polymer upon metalation of the bipyridine moiety . optical absorption and emission spectra of the bipye - pvab polymer and the rh - bipye - pvab system in chloroform absorption ( solid lines ) and emission spectra ( dashed lines ) of bipye - pvab and rh - bipye - pvab ( rh - polymer ) in spectroscopic dichloromethane ; the optical absorbance of the initial bipyridyl polymer bipye - pvab in chloroform is red - shifted upon complex formation with rhodium by about 50 nm . the emission of bipye - pvab , with a maximum at 657 nm , is quenched upon complexation of the bipyridyl moiety . the rh - bipye - pvab polymer shows a weak emission , with a maximum at 571 nm . the optical properties of the bipye - pvab polymer as a thin film were determined using spectroscopic ellipsometry ( se ) . using this technique , information about the material s dielectric measured se and functions were fitted using the three oscillator model , and the values of the real and imaginary part of the dielectric function were obtained . the resulting plots are presented in figure 4 . for better visibility of the absorption features , the plot of the imaginary part ( in black ) , which describes the absorption of the material , shows two main absorption maxima at 460 nm ( 2.7 ev ) and 348 nm ( 3.5 ev ) , respectively . an additional broad absorption band can be found at 218 nm ( 5.7 ev ) . the plotted absorptions are connected with the oscillations , which are described by the real part of the dielectric function . for all described absorption peaks , the related oscillators could be found and are kramers the optical band gap of the film material was found to be 590 nm ( 2.1 ev ) . the solid - state absorption features of the material determined by this method , with maxima at 348 and 460 nm , are relatively similar to the data measured for the nonmetalated polymer bipye - pvab in chloroform solution , where maxima were found at 336 and 459 nm , respectively . the calculated real part ( plotted in red ) and the imaginary part ( plotted in black ) of the dielectric function . to characterize the effects of possible redox processes involving the polymer backbone , a detailed characterization using atr - ftir was performed . upon chemical oxidation with iodine , positively charged defects ( holes ) are formed within the polymer chain . due to this oxidation ( doping ) process , the conductivity of the polymer increases and strong changes in the vibronic structure of the material occur , which can be monitored spectroscopically . in the present case , upon doping of the polymer with iodine , characteristic peaks of high oscillator strength arise in the ftir spectrum of the material , which are connected with new infrared - active vibrational ( irav ) modes . moreover , since the positive charge induces distortions in the polymer chain , a polaron band will be visible in the ir spectrum as a broad absorption feature . while the appearance of these new bands in the ir spectrum can be described by many theories , in the present paper the girlando painelli the resulting difference spectrum is presented in the supporting information ( see figure s1 ) . when the material gets doped , two polaronic features appear at 5500 and 3500 cm upon increasing degree of oxidation . after doping , a broad absorption feature with a maximum at 5200 together with the formation of the polaron , the appearance of new irav bands with maxima at 2100 and 1503 cm as well as a broader peak at 1188 cm can be observed . the presence of these bands suggests that , after doping , due to an increased distortion , previously symmetric vibrations became asymmetric and thus observable by ir . moreover , their rather high sharpness suggests that the free charge present in the polymer chain is rather localized . usually , such localization in a conjugated polymer suggests rather poor charge transport properties . however , for polymers containing a triple bond , the usual theories for conjugated polymers can not directly describe the conducting properties of the polymer . such polymers are usually characterized by strongly bound charges ( between the triple bonds ) that hinder intramolecular charge transport . on the other hand , the presence of this type of bonds can greatly improve the intermolecular charge transport ( increase the film conductivity ) due to the linear orientation of parts of the molecule . both the polaron absorption at 5200 cm and the irav bands at lower energy show stronger intensity only with increasing doping level , and no significant shift of the bands themselves can be observed , which was also found to be the case when other triple - bond - containing polymers were studied . the electrochemistry of the bipye - pvab polymer was investigated in order to obtain more detailed information about the redox properties of the system . as the total amount of sample was quite small , the oxidation of bipye - pvab was characterized using photoelectrochemical scanning droplet cell microscopy , which allows us to perform a full electrochemical characterization using small amounts of the studied material . due to the slow kinetics of the electrochemical process monitored , the measurement was performed with a polarization speed of 1 mv s. the resulting curve is presented in figure 6a . electrochemical ( a ) and photoelectrochemical ( b ) characterization of the bipye - pvab polymer . as can be seen , for potentials up to 1.10 v vs she ( standard hydrogen electrode ) , no electrochemical processes occur , and only a background current can be measured . above 1.10 v vs she , an increase in the current density can be noticed with a maximum at 1.22 v vs she . this increase is a result of the electrochemical oxidation of the bipye - pvab polymer . at higher potentials ( above 1.3 v vs she ) , an additional increase in the current density however , the nature of this current density increase is mostly connected with degradation of the electrochemical system . measuring in the backward direction , only background current was measured , suggesting the irreversibility of the electrochemical oxidation process . since the polymer was developed to serve as a photosensitizer , additional photoelectrochemical characterization was performed . in this measurement , the polymer layer was kept at constant potential of 0 v vs she , and the change in the current upon illumination was measured . the sample was illuminated with a laser diode ( 532 nm , 153 mw cm ) fitting to the absorbance maximum of the polymer ( as presented in figure 4 ) . the measurement was performed in 10 s dark / illumination sequences using a manually controlled shutter . as can be noticed , during the first 10 s when the measurement was performed in the dark , only a background current could be measured in the range of na cm . after the sample was exposed to light , a strong change of the current was observed , originating from the photogeneration of separate charges . within 10 s of illumination , a continuous decrease in current value can be observed , which results from shifted ground state and doped state absorption . after the shutter was closed , again only a background current could be measured . further illumination sequences show a constant decrease of the photocurrent , which probably results from irreversible photo - oxidation of the investigated material . this photodegradation could also be enhanced by nonefficient charge extraction from the working electrode . in order to show that the rhodium site of the metalated polymer material rh - bipye - pvab is accessible for the nad substrate and active in aqueous solution , catalyzed chemical reduction of nad to nadh in the presence of sodium formate was performed . the related rhodium 2,2-bipyridyl complex [ rh(bpy)cp*cl]cl readily hydrolyzes once in contact with water , forming either the hydroxo or the aquo complex when the initial chloride ligand is lost . the ph used in this study was around 7.4 for chemical reduction with formate and photoenzymatic reactions with glutamate dehydrogenase ( gdh ) and 8.9 for the photochemical reduction of nad using teoa as a donor . the [ rh(diimine)cp*h2o ] derivative of rh - bipye - pvab is assumed to be the actual species in the former and [ rh(diimine)cp*(oh ) ] in the latter case , as the pka of the water bound to the rhodium metal center of [ rh(bpy)cp*h2o ] used for comparison is around 8.2 . clean simple glass beads ( diameter : 5 mm ) were coated with rh - bipye - pvab by mixing them into a solution of rh - bipye - pvab in dichloromethane and leaving them to dry . the beads were transferred to an aqueous buffer solution containing both nad and sodium formate . the reaction solution was purged with ar . the setup and the reaction scheme for this experiment formate can donate h to the rhodium reaction center , resulting in formation of co2 gas as an easily removable side product . the formation of nadh due to the catalyzed chemical reduction of nad in this dark experiment was followed by recording uv vis spectra of the sample solution at regular time intervals of 2 h ( figure 8) . ( b ) scheme of the surface reaction in the chemical reduction of nad to nadh with sodium formate as hydride donor to the polymer - bound rhodium catalyst reaction center . the formation of nadh can be observed with increasing absorbance at 340 nm . spectra were recorded at regular time intervals of 2 h for a total time of 80 h. the reaction solution was not stirred . no formation of a similar absorbance band could be observed in the reference sample without nad . formation of the reduction product nadh was further confirmed via measurement of the fluorescence spectrum of the sample , as can be seen in figure 9 . upon excitation of the sample at 340 nm , the luminescence spectrum of the nadh formed in the solution had a maximum at 470 nm . both these value are well in accordance with literature data on the reduced cofactor nadh . the experiment was also repeated with the same batch of rh - bipye - pvab - coated beads after they were rinsed with distilled water and put into a fresh solution containing nad and formate . luminescence and optical excitation spectrum of a dark sample containing the rh - polymer beads and nad after 80 h of reaction without stirring . the specific luminescence with a maximum at 470 nm upon excitation with a wavelength of 340 nm indicates the formation of nadh . to investigate the applicability of the synthesized rhodium polymer further , a photolysis experiment was performed . teoa was used as a sacrificial donor in a 0.1 m aqueous buffered sodium phosphate solution to enhance the stability of both the nad and the nadh present in the sample , which was necessary due to the relatively long irradiation time . one advantage of the system used here is the fact that the uv vis spectrum of the solution can be measured separately from the that of the photocatalyst , as the cuvette was not completely filled with the catalyst - coated glass beads ( similar to the reaction depicted in figure 7 ) . rh - bipye - pvab - coated glass beads were used in an experiment with 3 ml solution of 15 w / v% teoa in 0.1 m sodium phosphate buffer with a ph value of 8.9 , and 1.2 mg of nad , stirred and irradiated at 390 nm for 26 h. the uv vis spectra recorded can be seen in figure 10 . blue lines show the uv vis absorbance and emission data of the dark sample ; similar data for the irradiated sample are shown in red . after 26 h , no spectral changes occurred in the dark sample , whereas for the sample that had been irradiated at 390 nm , a distinct absorbance band with a maximum at 340 nm was formed , which indicates the successful photocatalytic formation of nadh catalyzed by the rh - bipye - pvab polymer . the reduction product nadh was further confirmed by measuring the fluorescence spectrum ( figure 10 ) of the sample upon excitation at 340 nm , and the excitation spectrum was recorded at 470 nm . the amount of product formed in the photocatalytic system was estimated from the difference in uv vis absorbance maximum at 340 nm applying lambert the conversion to nadh after 26 h compared to the initial amount of nad was more than 21% ( 8.6 mg this results , using the surface area of the glass beads , in a turnover number ( ton ) of 480 mol cm and a turnover frequency ( tof ) of 1.8 mol cm h. the rh - bipye - pvab polymer - coated glass beads were used in a second photolysis experiment after being rinsed carefully with ultrapure water . in a solution with ph 8 , the quantum yield of the rh - polymer film was measured to be 1.010 ( 0.110 ) when using a 370 nm low - pass filter . it was calculated according to the formula applied in the literature as the ratio between 2 times the amount of nadh formed and the amount of absorbed photons . the amount of photons absorbed by the rh - bipye - pvab film was obtained by integration of the difference in transmitted light through the sample with and without a polymer film drop - cast on the inside of the cuvette over the spectral range up to 650 nm , which is the low - energy absorbance edge of rh - bipye - pvab ( see figure 3 ) . vis absorption and emission spectrum of an irradiated sample ( 390 nm ) with rh - bipye - pvab glass beads , 15 w / v% teoa as sacrificial donor , and nad as substrate . after 26 h , an increased absorbance at 340 nm indicates the photochemical formation of nadh . photoluminescence measurements of the sample with exc : 340 nm also indicates the successful synthesis of nadh , which shows a specific emission at 470 nm ; after 26 h , the conversion of nadh was estimated to be 21% , compared to the initial amount of nad added from the uv vis spectrum applying lambert in order to show that the nadh formed by the photoreaction of nad on rh - bipye - pvab is enzymatically active , enzymatic reduction of -ketoglutarate to l - glutamate was coupled to the photochemical regeneration system described before . optimized conditions for the performance of gdh type 1 from bovine liver were applied , similar to described literature procedures.rh-bipye-pvab ( 0.08 mg ) was drop - cast from dichloromethane solution on a simple glass sheet . the reaction buffer ( ph 7.4 ) containing 0.1 m phosphate , 0.1 m ammonium sulfite , and 15 w / v% teoa with 0.7 mm nad , 13 mm -ketoglutarate , and 5 u gdh was first stirred in the dark for 1 h. after 5 h of irradiation with a 370 nm cutoff filter and after 22 h of irradiation , 6.9 and 24.3 ppm of l - glutamic acid were detected , respectively , in the photolysis solution by hplc - ms . no l - glutamic acid was detected after the solution was stirred without light irradiation . the luminescence of the bipye - pvab polymer is almost completely quenched after complexation with the rhodium metal ( to form rh - bipye - pvab ) . the catalytically active sites the rhodium metal centers of the polymer have been shown to be readily accessible to both a hydride donor and the nadh substrate , making the polymer applicable in the catalyzed chemical nad reduction with formate as a hydride source . the polymer material can also be applied in a photocatalytic system with teoa as electron donor . concerning the mechanism of this photoreaction , there are several different possibilities , which at the present stage of our investigations can not be fully distinguished . moreover , the question of how such a polymer works as a photocatalyst for nad hydrogenation can not be described with a classical theory about molecular catalysis alone . as the iodine doping experiments show , the bipye - pvab material is dopable to become a p - type organic semiconductor , and thus it can also show electrical conductivity . the polymer does not behave completely like a conjugated conducting polymer because the triple bond structure favors intermolecular conductivity over intramolecular conductivity . thus , the theoretical concepts for ( conducting ) polymer chemistry , as applied in organic electronics with a classical moving polaron approach , can not be used to describe the system . classical photochemistry , as it would be assumed to take place in dilute solution , does not seem to be sufficient either . schematic representation of two possible catalytic pathways proposed for the photochemical reduction of nad to nadh by the rhodium polymer rh - bipye - pvab : ( i ) the donor teoa reductively quenches the excited state of the rh - complex . this would have to happen twice before a hydride can be formed by protonation and then be transferred to nad . ( ii ) the rhodium complex oxidatively quenches the excited state of the polymer backbone material bipye - pvab , which is regenerated by teoa from the aqueous solution . the two most probable catalytic pathways tentatively proposed for the reaction are shown in figure 11 . the first possibility ( i ) would involve direct excitation of the rhodium subunit or energy transfer from the irradiated polymer bipye - pvab to the rh center in rh - bipye - pvab and reductive quenching of the excited state by teoa . this would have to occur twice at the same rh site for later reduction of nad to nadh . the second possible pathway ( ii ) involves ( oxidative ) quenching of the photoexcited polymer chain by a rhodium center . this is accompanied by electron transfer to the rhodium active site , possibly also involving a different polymer chain . especially considering the facts that iodine vapor , with an estimated redox potential of 0.35 0.1 v , is sufficient to oxidize the bipye - pvab polymer backbone and the bandgap measured by ellipsometry is 2.1 ev , this pathway is the more probable one . the ground state of the polymer chain is presumably recovered by teoa as a sacrificial electron donor . after two electron - transfer steps to the rhodium center , a proton can be taken up from the solution , and the active rh - hydride complex is formed at the active bipyridyl coordination site of the rhodium polymer . the hydride is then transferred regioselectively to the additionally coordinated nad to form 1,4-nadh . the amount of l - glutamate formed per repetition unit of the rh - bipye - pvab polymer ( m = 1560 g mol ) can be used to roughly estimate a ton per rhodium center of 3.2 ( after 22 h ) . however , as this is a surface reaction on a randomly drop - cast film , it is most likely the case that not all rhodium centers that are stoichiometrically present in the structure are similarly accessible to drive the photoreaction . nevertheless , it shows that the nadh regeneration in the enzymatically coupled photoreaction is catalytic with respect to the rh - bipye - pvab polymer photosensitizer . the tof for rh - bipye - pvab is 0.145 h , comparable to those of other reported systems applying heterogeneous photosensitizers ( cdse , 0.168 h ; cds , 0.120 h ; p - doped tio2 , 0.003 h ; and w2fe4ta2o17 , 0.002 h. the photochemical quantum yield , 1.010 ( 0.110 ) , and the amount of nadh formed per unit area and time , 1.8 mol cm h , are comparable measures for this heterogeneous polymer catalyst system . the rh - bipye - pvab polymer can be used as very stable , separable , and reusable photocatalyst for visible - light - powered cofactor reduction with electron donors present in the solution as well as for chemical cofactor reduction with hydride donor molecules such as formate . the precious component of the otherwise very effective rhodium systems for nadh regeneration can be separated and reused in different experimental environments , and interference with the following reactions as well as possible deactivation of the active side of redox - active enzymes which are coupled to the system by soluble redox mediators can be minimized . the concept probably can be extended to photoelectrochemical pathways of nad reduction by modification of the polymer backbone to enhance intramolecular charge carrier mobility . the polymers could be modified to provide more active sites per repetition unit , and the optical properties of the polymer can be tuned by using different copolymerization agents . a method for more uniform film formation on the glass substrates has to be found , and the use and investigation of the catalytic film on glass substrates are areas of interest for upcoming research . further work on electrocatalysis and photoelectrocatalysis using this conjugated polymer is also underway .

a 2,2-bipyridyl - containing poly(arylene - ethynylene)-alt - poly(arylene - vinylene ) polymer , acting as a light - harvesting ligand system , was synthesized and coupled to an organometallic rhodium complex designed for photocatalytic nad+/nadh reduction . the material , which absorbs over a wide spectral range , was characterized by using various analytical techniques , confirming its chemical structure and properties . the dielectric function of the material was determined from spectroscopic ellipsometry measurements . photocatalytic reduction of nucleotide redox cofactors under visible light irradiation ( 390650 nm ) was performed and is discussed in detail . the new metal - containing polymer can be used to cover large surface areas ( e.g. glass beads ) and , due to this immobilization step , can be easily separated from the reaction solution after photolysis . because of its high stability , the polymer - based catalyst system can be repeatedly used under different reaction conditions for ( photo)chemical reduction of nad+ . with this concept , enzymatic , photo - biocatalytic systems for solar energy conversion can be facilitated , and the precious metal catalyst can be recycled .

Introduction Results and Discussion Conclusions

photoenzymatic reactions require light - harvesting chromophores efficiently interacting with the functional components responsible for catalysis . in this context , metal - binding and visible - light - absorbing polymeric compounds with a bis-5,5-(phenylene - ethynylene)-2,2-bipyridylene backbone represent quite interesting building blocks for the development of novel photocatalytic systems , as they provide the possibility of tuning the optical properties of the polymer - based ligand as well as the catalytic and optical properties of the coordinated metal complexes . ruthenium - containing polymers incorporating 4,4- or 5,5-(phenylene - ethynylene)-2,2-bipyridylene moieties as ligands have been introduced and widely characterized by klemm et al . it is well established that organometallic rhodium complexes of the type [ rhcp*(1,2-diimine)h2o ] ( with 1,2-diimine being derivatives of 2,2-bipyridine ( bpy ) or 1,10-phenanthroline ; cp * = pentamethyl - cyclopentadienyl ) accelerate the formation of nadh in neutral to slightly basic aqueous solutions . they have been investigated for chemical , photochemical , and electrocatalytic cofactor reduction of nadh and model compounds . light - driven regeneration reactions of the nadh cofactor in various systems are also , with few exceptions , often based on [ rhcp*(bpy)h2o ] as hydride - transfer catalyst . , rhodamine dyes , semiconducting particles and quantum dots , metalloporphyrins , and , quite recently , graphene - derived photosensitizers . in the present work , a new bipyridine - containing poly(arylene - ethynylene)-alt - poly(arylene - vinylene ) copolymer with a redox - active rhodium cyclopentadienyl complex as the active site for catalytic substrate conversion is presented . when transferred on solid substrates , such as glass beads , this active polymer - based hydride - transfer catalyst can be easily separated from the reaction solution and furthermore be used repeatedly under different aqueous reaction conditions because of its high stability . a prerequisite for obtaining the polymer - based rhodium complex rh - bipye - pvab is the prior synthesis of the metal - free polymer bipye - pvab , as depicted in scheme 1 . 5,5-bis(4-formyl-2,5-dioctyloxyphenylethynyl)-2,2-bipyridine ( 3 ) was obtained in 55% yield through sonogashira pd - catalyzed cross - coupling reaction of 4-formyl-2,5-dioctyloxyphenylacetylene ( 1 ) with 5,5-dibromo-2,2-bipyridine ( 2 ) and chromatographic workup . all peaks can be readily assigned to the expected chemical structure . bipye - pvab was obtained as a red product in 84% yield after soxhlet extraction of the crude polymer with a refluxing methanol / diethyl ether mixture . the broadness of the h nmr peaks is a clear indication for the polymeric nature of the material . the newly synthesized materials were designed to work as light - harvesting photosensitizers in the presence of substrate - containing solvents . therefore , at first the photophysical characterization was performed in solution , and the maxima of absorption ( a ) and emission ( f ) , as well as fluorescence quantum yield ( f ) values were determined . the uv vis spectrum of bipye - pvab shows a strong red shift due to higher conjugation length compared to the initial bipyridyl - containing dialdehyde 3 . in chloroform solution , the polymeric bipyridine - containing material bipye - pvab also shows two main absorption bands , with peaks at 336 and 491 nm . the metal - containing polymer rh - bipye - pvab shows only very weak background emission . additionally , the optical absorbance is significantly red - shifted by about 50 nm compared to that of the parent polymer upon metalation of the bipyridine moiety . optical absorption and emission spectra of the bipye - pvab polymer and the rh - bipye - pvab system in chloroform absorption ( solid lines ) and emission spectra ( dashed lines ) of bipye - pvab and rh - bipye - pvab ( rh - polymer ) in spectroscopic dichloromethane ; the optical absorbance of the initial bipyridyl polymer bipye - pvab in chloroform is red - shifted upon complex formation with rhodium by about 50 nm . the optical properties of the bipye - pvab polymer as a thin film were determined using spectroscopic ellipsometry ( se ) . using this technique , information about the material s dielectric measured se and functions were fitted using the three oscillator model , and the values of the real and imaginary part of the dielectric function were obtained . for better visibility of the absorption features , the plot of the imaginary part ( in black ) , which describes the absorption of the material , shows two main absorption maxima at 460 nm ( 2.7 ev ) and 348 nm ( 3.5 ev ) , respectively . the plotted absorptions are connected with the oscillations , which are described by the real part of the dielectric function . for all described absorption peaks , the related oscillators could be found and are kramers the optical band gap of the film material was found to be 590 nm ( 2.1 ev ) . the solid - state absorption features of the material determined by this method , with maxima at 348 and 460 nm , are relatively similar to the data measured for the nonmetalated polymer bipye - pvab in chloroform solution , where maxima were found at 336 and 459 nm , respectively . the calculated real part ( plotted in red ) and the imaginary part ( plotted in black ) of the dielectric function . to characterize the effects of possible redox processes involving the polymer backbone , a detailed characterization using atr - ftir was performed . due to this oxidation ( doping ) process , the conductivity of the polymer increases and strong changes in the vibronic structure of the material occur , which can be monitored spectroscopically . in the present case , upon doping of the polymer with iodine , characteristic peaks of high oscillator strength arise in the ftir spectrum of the material , which are connected with new infrared - active vibrational ( irav ) modes . after doping , a broad absorption feature with a maximum at 5200 together with the formation of the polaron , the appearance of new irav bands with maxima at 2100 and 1503 cm as well as a broader peak at 1188 cm can be observed . the presence of these bands suggests that , after doping , due to an increased distortion , previously symmetric vibrations became asymmetric and thus observable by ir . however , for polymers containing a triple bond , the usual theories for conjugated polymers can not directly describe the conducting properties of the polymer . on the other hand , the presence of this type of bonds can greatly improve the intermolecular charge transport ( increase the film conductivity ) due to the linear orientation of parts of the molecule . both the polaron absorption at 5200 cm and the irav bands at lower energy show stronger intensity only with increasing doping level , and no significant shift of the bands themselves can be observed , which was also found to be the case when other triple - bond - containing polymers were studied . as the total amount of sample was quite small , the oxidation of bipye - pvab was characterized using photoelectrochemical scanning droplet cell microscopy , which allows us to perform a full electrochemical characterization using small amounts of the studied material . due to the slow kinetics of the electrochemical process monitored , the measurement was performed with a polarization speed of 1 mv s. the resulting curve is presented in figure 6a . as can be seen , for potentials up to 1.10 v vs she ( standard hydrogen electrode ) , no electrochemical processes occur , and only a background current can be measured . since the polymer was developed to serve as a photosensitizer , additional photoelectrochemical characterization was performed . in this measurement , the polymer layer was kept at constant potential of 0 v vs she , and the change in the current upon illumination was measured . the sample was illuminated with a laser diode ( 532 nm , 153 mw cm ) fitting to the absorbance maximum of the polymer ( as presented in figure 4 ) . as can be noticed , during the first 10 s when the measurement was performed in the dark , only a background current could be measured in the range of na cm . further illumination sequences show a constant decrease of the photocurrent , which probably results from irreversible photo - oxidation of the investigated material . in order to show that the rhodium site of the metalated polymer material rh - bipye - pvab is accessible for the nad substrate and active in aqueous solution , catalyzed chemical reduction of nad to nadh in the presence of sodium formate was performed . the ph used in this study was around 7.4 for chemical reduction with formate and photoenzymatic reactions with glutamate dehydrogenase ( gdh ) and 8.9 for the photochemical reduction of nad using teoa as a donor . the reaction solution was purged with ar . the setup and the reaction scheme for this experiment formate can donate h to the rhodium reaction center , resulting in formation of co2 gas as an easily removable side product . the formation of nadh due to the catalyzed chemical reduction of nad in this dark experiment was followed by recording uv vis spectra of the sample solution at regular time intervals of 2 h ( figure 8) . ( b ) scheme of the surface reaction in the chemical reduction of nad to nadh with sodium formate as hydride donor to the polymer - bound rhodium catalyst reaction center . formation of the reduction product nadh was further confirmed via measurement of the fluorescence spectrum of the sample , as can be seen in figure 9 . teoa was used as a sacrificial donor in a 0.1 m aqueous buffered sodium phosphate solution to enhance the stability of both the nad and the nadh present in the sample , which was necessary due to the relatively long irradiation time . one advantage of the system used here is the fact that the uv vis spectrum of the solution can be measured separately from the that of the photocatalyst , as the cuvette was not completely filled with the catalyst - coated glass beads ( similar to the reaction depicted in figure 7 ) . rh - bipye - pvab - coated glass beads were used in an experiment with 3 ml solution of 15 w / v% teoa in 0.1 m sodium phosphate buffer with a ph value of 8.9 , and 1.2 mg of nad , stirred and irradiated at 390 nm for 26 h. the uv vis spectra recorded can be seen in figure 10 . the reduction product nadh was further confirmed by measuring the fluorescence spectrum ( figure 10 ) of the sample upon excitation at 340 nm , and the excitation spectrum was recorded at 470 nm . the amount of product formed in the photocatalytic system was estimated from the difference in uv vis absorbance maximum at 340 nm applying lambert the conversion to nadh after 26 h compared to the initial amount of nad was more than 21% ( 8.6 mg this results , using the surface area of the glass beads , in a turnover number ( ton ) of 480 mol cm and a turnover frequency ( tof ) of 1.8 mol cm h. the rh - bipye - pvab polymer - coated glass beads were used in a second photolysis experiment after being rinsed carefully with ultrapure water . in a solution with ph 8 , the quantum yield of the rh - polymer film was measured to be 1.010 ( 0.110 ) when using a 370 nm low - pass filter . the amount of photons absorbed by the rh - bipye - pvab film was obtained by integration of the difference in transmitted light through the sample with and without a polymer film drop - cast on the inside of the cuvette over the spectral range up to 650 nm , which is the low - energy absorbance edge of rh - bipye - pvab ( see figure 3 ) . vis absorption and emission spectrum of an irradiated sample ( 390 nm ) with rh - bipye - pvab glass beads , 15 w / v% teoa as sacrificial donor , and nad as substrate . photoluminescence measurements of the sample with exc : 340 nm also indicates the successful synthesis of nadh , which shows a specific emission at 470 nm ; after 26 h , the conversion of nadh was estimated to be 21% , compared to the initial amount of nad added from the uv vis spectrum applying lambert in order to show that the nadh formed by the photoreaction of nad on rh - bipye - pvab is enzymatically active , enzymatic reduction of -ketoglutarate to l - glutamate was coupled to the photochemical regeneration system described before . optimized conditions for the performance of gdh type 1 from bovine liver were applied , similar to described literature procedures.rh-bipye-pvab ( 0.08 mg ) was drop - cast from dichloromethane solution on a simple glass sheet . the reaction buffer ( ph 7.4 ) containing 0.1 m phosphate , 0.1 m ammonium sulfite , and 15 w / v% teoa with 0.7 mm nad , 13 mm -ketoglutarate , and 5 u gdh was first stirred in the dark for 1 h. after 5 h of irradiation with a 370 nm cutoff filter and after 22 h of irradiation , 6.9 and 24.3 ppm of l - glutamic acid were detected , respectively , in the photolysis solution by hplc - ms . the catalytically active sites the rhodium metal centers of the polymer have been shown to be readily accessible to both a hydride donor and the nadh substrate , making the polymer applicable in the catalyzed chemical nad reduction with formate as a hydride source . moreover , the question of how such a polymer works as a photocatalyst for nad hydrogenation can not be described with a classical theory about molecular catalysis alone . as the iodine doping experiments show , the bipye - pvab material is dopable to become a p - type organic semiconductor , and thus it can also show electrical conductivity . thus , the theoretical concepts for ( conducting ) polymer chemistry , as applied in organic electronics with a classical moving polaron approach , can not be used to describe the system . ( ii ) the rhodium complex oxidatively quenches the excited state of the polymer backbone material bipye - pvab , which is regenerated by teoa from the aqueous solution . the ground state of the polymer chain is presumably recovered by teoa as a sacrificial electron donor . after two electron - transfer steps to the rhodium center , a proton can be taken up from the solution , and the active rh - hydride complex is formed at the active bipyridyl coordination site of the rhodium polymer . the amount of l - glutamate formed per repetition unit of the rh - bipye - pvab polymer ( m = 1560 g mol ) can be used to roughly estimate a ton per rhodium center of 3.2 ( after 22 h ) . the tof for rh - bipye - pvab is 0.145 h , comparable to those of other reported systems applying heterogeneous photosensitizers ( cdse , 0.168 h ; cds , 0.120 h ; p - doped tio2 , 0.003 h ; and w2fe4ta2o17 , 0.002 h. the photochemical quantum yield , 1.010 ( 0.110 ) , and the amount of nadh formed per unit area and time , 1.8 mol cm h , are comparable measures for this heterogeneous polymer catalyst system . the rh - bipye - pvab polymer can be used as very stable , separable , and reusable photocatalyst for visible - light - powered cofactor reduction with electron donors present in the solution as well as for chemical cofactor reduction with hydride donor molecules such as formate . the precious component of the otherwise very effective rhodium systems for nadh regeneration can be separated and reused in different experimental environments , and interference with the following reactions as well as possible deactivation of the active side of redox - active enzymes which are coupled to the system by soluble redox mediators can be minimized . the concept probably can be extended to photoelectrochemical pathways of nad reduction by modification of the polymer backbone to enhance intramolecular charge carrier mobility . the polymers could be modified to provide more active sites per repetition unit , and the optical properties of the polymer can be tuned by using different copolymerization agents . a method for more uniform film formation on the glass substrates has to be found , and the use and investigation of the catalytic film on glass substrates are areas of interest for upcoming research .

study population : sixteen clinically healthy cats ( 12 domestic shorthairs , 1 abyssinian , 1 norwegian forest cat and 2 russian blues ) were used in the present study as a control group . they were considered clinically normal when no evidence of cardiopulmonary and systemic disease that might influence their cardiac functions was noticed , based on a physical examination , an electrocardiogram ( standard limb lead ) , thoracic radiography and echocardiography , a complete blood count , a serum biochemical test , a urine specific gravity of>1.035 and a systemic blood pressure of < 170 mmhg in systole . the hcm group consisted of 17 client - owned cats with hcm diagnosed at the azabu university veterinary teaching hospital ( 8 american shorthairs , 3 domestic shorthairs , 3 scottish fold , 1 persian , 1 chinchilla and 1 russian blue ) . they were clinically diagnosed as hcm when : 1 ) the interventricular septum and/or lv free - wall thickness during end - diastoles ( ivsd and lvfwd ) were greater than 6 mm , and the lv end - diastolic internal diameter ( lvidd ) was less than 18 mm by m - mode imaging of the left ventricular short - axis obtained from the right parasternal view . 2 ) values of blood nitrogen urea and creatinine fell within the reference range in serum biochemical testing or they did not exhibit systemic hypertension , and 3 ) hyperthyroidism was excluded on the basis of clinical signs and biochemistry , including serum t4 and hepatic enzyme concentrations . when a patient was younger than 4 years , hyperthyroidism was automatically excluded [ 8 , 25 ] . hcm with a dynamic left ventricular outflow tract obstruction ( lvoto ) was included in the present study . lvoto was defined as a disturbed flow across the left ventricular outflow tract ( lvot ) with an estimated pressure gradient greater than 30 mmhg concurrent with a late systolic acceleration [ 1 , 27 ] . systemic arterial blood pressure measurement : systemic arterial blood pressure was measured indirectly on the tail or limb at a quiet and isolated place by the doppler method as previously described using an aneroid sphygmomanometer ( tycos tr-2 hand aneroid sphygmomanometer , welch allyn , tokyo , japan ) . echocardiographic examination : echocardiographic examinations were performed by two examiners ( yf and ht ) using one ultrasound machine ( vivid 7 dimension , ge medical system , tokyo , japan ) . ecg monitoring ( lead ii ) with a clear r wave recognition was recorded simultaneously using the same ultrasound machine used for the healthy cats . off - line ste measurements for main analysis were obtained by the same examiner ( ht ) , and those for analysis of interobserver variabilities were by another ( ti ) . b- , m- and doppler mode echocardiography : lv short - axis images at the chordae tendineae ( ct ) level between the mitral valve and papillary muscle for m - mode measurements ( ivsd , lvidd , lvpwd and fractional shortening [ fs ] ) and those at the aortic root level for b - mode measurements of the left atrium size ( diameters of left atrial diameter [ lad ] and aortic root , and the left atrium to aorta ratio [ la / ao ] ) were obtained on right lateral recumbency . left atrial enlargement was defined as la / ao > 1.5 . the segmental thickness of the interventricular septum below the aortic valve was measured at the end - diastole ( ivsb ) using long - axis images from a right - lateral recumbency view . apical 4 and 5 chamber images for doppler measurements ( velocity of transmitral flow during early and late diastole and their ratio [ tmf ; e wave , a wave , and e / a ratio ] , and that of a flow through lvot , respectively ) were obtained from a left lateral recumbency view . when ea fusion was seen , values of e and a waves of the cats were excluded from analysis ( the same way as with ste and tdi ) . segmental thickness of the lv wall at the end - diastole was measured in the hcm group using lv short - axis images of the chordae tendineae level . the images used to measure lv wall thickness were the same as those used to analyze ste . the lv was divided into 6 regions ( anteroseptum , anterior , lateral , posterior , inferior and septum ) , and those segments corresponded to the regions for the segmental strain and strain rate variables . tissue doppler echocardiography : the mitral annulus velocity ( mav ) was measured at the lateral side in apical 4 chamber views . peak values of mav during systole and early diastole ( sa and ea , respectively ) were obtained , and e / ea was derived from e wave divided by ea . speckle tracking echocardiography : right parasternal short - axis views at the ct were used to measure all strain and strain rate variables . images were acquired in cine loops triggered by the qrs complex , saved in a digital format and analyzed using off - line software ( echopac pc , ge medical system , tokyo , japan ) . the principle behind speckle tracking analysis has been reported in several previous studies [ 4 , 5 , 18 , 23 , 24 , 34,35,36 , 39 , 40 ] . the cardiac cycles used for analysis were determined by a simultaneous ecg ( from r to r wave ) . the peak systolic radial and circumferential strains ( sr and sc ) , and the peak radial and circumferential strain rates during the systole and early diastole ( srrs , srre , srcs and srce ) were measured at the level of ct ( fig . 1.images of radial strain rate of a healthy cat ( a ) and a cat with hypertrophic cardiomyopathy ( b ) . srrs , radial strain rate during systole ; srre , radial strain rate during early diastole ; srra , radial strain rate during late diastole . ) . the e/ srre and e/ srce all strain and strain rate variables were calculated by 6 segmental values ( anteroseptum , anterior , lateral , posterior , inferior and septum ) . images of radial strain rate of a healthy cat ( a ) and a cat with hypertrophic cardiomyopathy ( b ) . srrs , radial strain rate during systole ; srre , radial strain rate during early diastole ; srra , radial strain rate during late diastole . in the present study , simultaneous ecg monitoring was not recorded in some cats with hcm , who were nervous and excited during an examination . however , comparisons between ste variables that analyzed with simultaneous ecg monitoring and those without such monitoring showed good agreement ( statistics analysis was performed using bland - altman plotting [ table 1table 1.assessment of effects of ste variables with or without simultaneous ecg during analysis in healthy 10 catsparameterunitmean sdmean of the differencelimits of agreementrp valuewith simultaneous ecgwithout simultaneous ecgsr%50.68 8.0552.11 9.321.53 3.298.00 to 5.140.94<0.001srrs / sec3.79 0.573.94 0.700.15 0.410.96 to 0.670.810.0041srre / sec3.84 1.194.14 1.160.29 0.400.51 to 1.100.94<0.001sc%23.64 3.7524.23 4.760.59 1.662.73 to 3.910.95<0.001srcs / sec3.52 0.463.69 0.530.17 0.280.39 to 0.730.850.0017srce / sec4.29 1.324.44 1.470.15 0.360.87 to 0.570.97<0.001sd : standard deviation . 2.bland-altman agreement plot comparing strain and strain rate variables analyzed with and without ecg monitoring in 10 healthy cats . ] ) , and images unattached simultaneous ecg monitoring were allowed to include statistical analysis . in case images in the absence of simultaneous ecg monitoring were used for ste analysis , the frame at the time lv contracts the most right before lv distension was considered as proper timing of the end - diastole ( r wave ) . bland - altman agreement plot comparing strain and strain rate variables analyzed with and without ecg monitoring in 10 healthy cats . study protocol : to determine the feasibility of radial and circumferential strain analysis using ste in the healthy cats , the following 4 factors were assessed : ( 1 ) percentage of lv wall segments capable of successfully analyzing strain and strain rate variables ; ( 2 ) temporal resolution of images for ste analysis in cats ; ( 3 ) influence of sedation ; global strain and strain rate variables were obtained at the baseline and 15 min after sedation with acetylpromazine ( 0.01 mg / kg , sc ) and buprenorphine ( 0.0075 mg / kg , sc ) and then compared in 10 cats out of the healthy cats ; and ( 4 ) inter- and intraobserver variabilities in offline analysis were also assessed in 6 healthy cats . then , global strain and strain rate variables in cats with hcm were compared with those in the healthy cats . furthermore , segmental strain and strain rate variables were assessed for correlations with corresponding lv segmental hypertrophy . statistical analysis : statistical analysis between the 2 groups was performed using a student s or welch s t - test when the data showed a normal distribution . when the normality test failed , a mann - whitney u - test was applied . one way anova was applied for comparison of ste variables among the 6 segments of lv in the control groups . assessment of the influence of sedation was performed using a paired t - test . when the normality test failed , a wilcoxon signed - rank test was applied . for the comparisons between ste variables that analyzed with simultaneous ecg monitoring and those without such monitoring to assess agreement , bland - altman plotting was applied . intra- and interobserver variability was expressed as a mean of the absolute difference as a percentage of the mean of two absolute measurements . multivariate regression analysis was applied to evaluate the relationships between ste variables and patient s conditions . the relationships between segmental ste variables and corresponding lv segmental wall thickness were determined by linear regression analysis . the study population comprised the 16 healthy cats and the 17 cats with hcm . in the cats with hcm , atenolol ( n=2 ) , angiotensin - converting enzyme inhibitor ( n=3 ) and thromboprophylaxis ( n=2 ) one cat in the hcm group presented with congestive heart failure , though others had no clinical sign . although few cases had mitral regurgitation , they are judged as mild , because regions of their jet signals are limited . the case characteristics and echocardiographic parameters are documented in table 2table 2.characteristics of healthy cats and cats with hcmparameterunitcontrol grouphcm groupp valuegenderf / mf 8 / m 8(16)f 2 / m 15(17)bwkg4.14 1.05(16)4.39 1.00(17)0.49agemo40.56 40.11(16)71.88 50.24(17)<0.05hrbpm182.75 24.94(16)182.64 43.58(14)0.99sbpmmhg142.98 10.15(16)125.88 15.16(11)0.0017usg1.07 0.02(16)1.04 0.01(6)0.0011bunmg / dl27.94 4.85(16)30.72 17.53(16)>0.05cremg / dl1.61 0.36(16)1.85 1.15(16)>0.05ivsdcm0.38 0.05(16)0.64 0.11(17)<0.001lviddcm1.49 0.20(16)1.53 0.22(17)>0.05lvpwdcm0.40 0.05(16)0.55 0.16(17)0.0012fs%42.71 6.46(16)56.69 7.87(17)<0.001ivsbcm0.41 0.06(16)0.70 0.19(16)<0.001ladcm1.04 0.13(16)1.39 0.27(16)<0.001la / ao1.17 0.09(16)1.36 0.24(15)<0.05ecm / sec67.45 16.58(16)68.70 22.68(11)0.87acm / sec58.53 13.18(16)66.91 26.99(11)0.36e / a1.19 0.32(16)1.22 0.78(11)>0.05lvotvm / sec0.96 0.15(16)2.37 1.51(17)0.0015sam0 ( 0%)(16)5 ( 29%)(17)sacm / sec7.83 2.33(16)6.17 1.47(8)0.079eacm / sec9.50 ea : peak of mitral annulus velocity during early diastole .. bw : body weight . assessment of strain and strain rate variables using ste and feasibility of ste analysis in healthy cats : results of global strain and strain rate variables in the healthy cats are shown in table 3table 3.global strain and strain rate variables in healthy cats and cats with hcmparameterunitcontrol grouphcm groupp valueradialsr%47.13 13.18(16)42.68 17.29(17)0.41srrs / sec3.57 0.48(16)3.67 1.08(17)0.74srre / sec3.69 0.82(16)2.59 0.63(8)0.0030 e / srre18.8 5.27(16)29.31 9.07(8)0.0015 circumferentialsc%24.38 4.64(16)23.15 6.11(17)0.52srcs / sec3.43 0.55(16)3.57 1.06(17)0.62srce / sec4.52 1.37(13)3.24 0.78(6)0.049 e / srce16.4 6.3(13)23.42 6.06(6)0.035 a ) significant difference , sr : peak systolic radial strain . srce : peak circumferential strain rate during early diastole .. in comparison of segmental variables among lv 6 segments , a significant difference was seen in all circumferential variables ( sc ; p<0.001 , srcs ; p<0.001 and srce ; p=0.003 ) . however , no significant difference was seen in radial variables among the segments ( sr ; p=0.93 , srrs ; p=0.27 and srre ; p=0.76 ) ( fig . 3fig . 3.segmental strain and strain rate variables in left ventricular 6 segments of healthy cats.sr , radial strain ; srrs , radial strain rate during systole ; srre , radial strain rate during early diastole ; sc , circumferential strain ; srcs , circumferential strain rate during systole ; srce , circumferential strain rate during early diastole . ) . segmental strain and strain rate variables in left ventricular 6 segments of healthy cats.sr , radial strain ; srrs , radial strain rate during systole ; srre , radial strain rate during early diastole ; sc , circumferential strain ; srcs , circumferential strain rate during systole ; srce , circumferential strain rate during early diastole . ninety - seven point six percent of segments ( 281/288 ) were able to perform semi - auto tracking for an analysis of strain and strain rate variables . although a fusion of e and a waves of srr was not seen , one of src was observed in 3 cats out of the 16 healthy cats ( 18.8% ) . the frame rate of acquired images used for ste analysis was 146.9 13.1 fps ( 119167 fps ) , and the rate per cardiac cycle ( fpc ) was 48.8 5.9 frames . global strain and strain rate variables with and without sedation were compared ( table 4table 4.effect of sedation in healthy 10 catsparameterunitawakesedatedp valueheart ratebpm180 25179 190.44radialsr%50.7 8.0544.7 16.90.09srrs / sec3.79 0.573.85 0.710.58srre / sec3.84 1.193.89 0.840.88circumferentialsc%23.6 3.7523.3 4.70.62srcs / sec3.24 1.053.71 the intra- and inter - observer variability of each parameter is shown in table 5table 5.intra- and interobserver variability of each parameter in offline analysisparameterintraobserver ( % ) interobserver ( % ) radialsr5.548.45srrs4.18.09srre12.0715.64circumferentialsc4.935.56srcs6.754.75srce6.65.43sr : peak systolic radial strain . srce : peak circumferential strain rate during early diastole .. sr : peak systolic radial strain . assessment of strain and strain rate variables in cats with hcm : results of global strain and strain rate variables using ste in the cats with hcm are shown in table 3 . srre , e / srre , srce and e / srce exhibited significant differences between the healthy cats and those with hcm . however , multivariate regression analysis showed no significant relationships between ste variables and patient s condition , gender and age ( table 6table 6.multivariate regression analysis to evaluate relathionships between ste variables and patient s condition , gender and ageparameterp valuesrpatient s condition0.1370.507gender0.1090.577age0.3350.740srrspatient s condition0.0180.930gender0.1260.526age0.0380.846srrepatient s condition0.2910.172gender0.0070.970age0.2340.239scpatient s condition0.1090.597gender0.1240.527age0.1480.448srcspatient s srce : peak circumferential strain rate during early diastole . ) . a fusion of e and a in srr was observed in 9 cats and in src in 11 , respectively , out of the 17 cats with hcm ( 52.9 and 64.7% each ) . relatively dominant hypertrophy in intraventricular wall and lv free wall was seen in 10 and 3 cats , respectively , out of the cats with hcm ( 59 and 18% each ) . meanwhile , nearly homogeneous hypertrophy was seen in 4 cats out of the same ( 24% ) . comparison of segmental strain and strain rate variables between lv wall segments with hypertrophy ( thickness of lv wall during end - diastole is 6 mm or more ) and those without hypertrophy ( the same is less than 6 mm ) in the cats with hcm revealed significant differences in sr and srre ( table 7table 7.comparison of segmental radial strain and strain rate variables between left ventricular wall segments with and without hypertrophy in cats with hcmparameterunitnon - hypertrophic segmentshypertrophic segmentsp valuesegmental lv wall thickness in diastolemean sdmm4.86 0.59(53)6.86 0.90(49)<0.001rangemm3.65.96.09.3sr%47.27 17.15(53)37.72 17.68(49)0.007srrs / sec3.82 1.13(53)3.51 1.06(49)0.15srre / sec3.34 1.36(41)2.68 1.27(44)0.024sc%23.26 8.54(53)23.03 6.89(49)0.88srcs / sec3.69 1.22(53)3.45 1.18(49)0.32srce / sec3.41 segmental radial variables ( sr and srrs ) and corresponding lv segmental wall thickness also showed significant relations , though their correlations were weak ( sr , r= 0.23 p=0.018 ; srrs , r= 0.20 p=0.047 ; and srre , no significant relation was observed in segmental circumferential variables ( sc , r= 0.038 p=0.707 ; srcs , r= 0.081 p=0.418 ; and srce , in the present study , we assessed the feasibility of analysis for radial and circumferential strain variables using ste in cats . because of the relatively high heart rates of cats , it was anticipated that a somewhat inferior resolution in 2d images may have compromised the ste analysis . however , the ste proved both feasible and clinically acceptable in healthy cats . a high - frequency transducer allowed us to record an adequate quality of images and frame rates , even in cats with a small - sized heart . on the other hand , the fpc in cats appeared to be lower ( 48.8 5.9 fpc ) due to their high heart rates , when compared with those in dogs and humans ( 61.7 20.9 and 57 - 71fpc , respectively ) . therefore , it might prove less accurate to assess ste parameters using time intervals in cats , compared with those in dogs and humans . furthermore , ea fusion due to their high heart rates would often interfere with assessment of diastolic function in cats , although it also posed even in tdi analysis and tmf measurements . in the present study , ea fusion was frequently exhibited in the circumferential strain rate rather than in the radial one . keeping the cats as relaxed as possible or using sedatives to maintain low heart rate for them might help to diminish the prevalence of ea fusion when scanning an echocardiogram . it has been reported that decreased heart rate using drugs allows for assessment of e and a wave separately in cats . the inter- and intraobserver variabilities of strain analysis in cats appeared to be satisfactory in the present study . sedation with a combination of acepromazine and buprenorphine did not significantly influence strain and strain rate variables using ste in the present study . since increased heart rates due to excitement during scanning may lead to a poor quality of images and ea fusion , sedation should be used as needed . we evaluated whether or not it is possible to find significant difference of ste variables in cats with overt heart disease that may be detectable by morphological assessment using echocardiogram , in comparison with healthy cats . ste analysis in the present study could detect significant decrease of global radial and circumferential strain rate during early diastole in the cats with hcm , compared with healthy cats . in the present study , since almost cats with hcm did not have congestive heart failure and even left atrial enlargement , reduced global srre and srce may suggest the presence of impaired lv diastolic function due to lv hypertrophy and fibrosis in relatively early stage . however , since multivariate regression analysis did not show significant relationships between the ste variables and patient s condition , global ste variables in radial and circumferential directions could not detect impaired lv myocardial function . actually , in some studies in human patients with hcm , circumferential strain and strain variables have increased abnormally compared with controls in contrast to the studies described above and the present study [ 10 , 15 ] . values of circumferential strain also have been suggested to be different among layers of lv ( epicardial , mid - myocardial and endocardial layers ) in normal human subjects . furthermore , since endocardium is most susceptible to the deleterious effects of fibrosis and hypoperfusion , endocardial circumferential strain has been suggested to be able to identify regional ischemia accurately . these factors may be one of the causes to occur the discrepancy in results of studies when circumferential strain is analyzed in a whole lv segment . further assessments for circumferential strain and strain variables of the layers separately may be needed in cats . on the other hand , it has been suggested that circumferential strain variables increase or preserved appear to maintain to the normal lv ejection fraction as a compensation for longitudinal and radial strains [ 26 , 42 ] . because of different regional contributions of laminar mechanisms , regional systolic lv wall thickening is various . . further assessment of each change of strain variables in the 3 directions among different regions of lv in hcm of cats is needed . in the present study , systolic indices of global ste variables including sr , sc , srrs and srcs did not decrease significantly in cats with hcm compared with controls . however , a significant decrease in longitudinal strain using tdi in cats with hcm has been previously reported . since it has been reported that lv myocardial contraction and relaxation were first impaired in the longitudinal direction among radial , circumferential and longitudinal ones in subclinical human patients with cardiovascular risk factors , longitudinal strain parameters might be sensitive enough to detect myocardial dysfunction in cats as well as humans . a further study to assess longitudinal deformation in cats will be necessary . in the present study , a significant difference was seen in segmental radial strain and strain rate variables between hypertrophic and non - hypertrophic segments . furthermore , segmental radial strain and strain rate variables had significant correlation with segmental lv thickness . it has been reported that lv segmental fibrosis and end - diastolic wall thickness were significantly related to segmental longitudinal strain in human patients with hcm [ 12 , 33 ] . assessing lv functions segmentally using ste , local ventricular dysfunctions could be detected even in the radial direction in the present study . this potentiality of ste may be useful in detections of left ventricular dysfunction in its early stage and asymmetric or local lvh with no concern for angle dependence . in addition , segmental values of strain and strain rate variables in radial direction are more invariant than those in circumferential direction among the lv 6 segments of normal subjects in the present study . the results of previous report using normal adults support the results of our present study . first , diagnosis of lvh was based on morphological rather than pathological criteria . at the same time , we did not assess lv functions invasively , such as hemodynamic indices or severity of lv fibrosis , using mri . second , few cats with hcm were given medications , such as atenolol , at their examinations , and the fact that such medications may influence lv systolic and diastolic functions shall be taken into consideration [ 30 , 41 ] . third , in the present study , hcm cats with lvoto were included in our analysis . hcm with lvoto might have different lv systolic or diastolic myocardial functions compared with hcm which have no obstruction through lv outflow tract [ 10 , 38 ] . fourth , the male - female ratio was different between the control and hcm groups in the present study , because feline hcm is prevalent in male . lv functions have been reported to have gender - specific change in human studies . in healthy human adults , longitudinal and circumferential strains , except for a radial one , have showed a significant difference between genders . furthermore , a significant difference in age was also seen in the 2 groups of our study . age - specific change in lv function longitudinal strain and strain rate have been reported to decrease significantly in an older population compared with a young one in a human study , though no significant difference was seen in the radial and circumferential strain and strain rate in systole and early diastole . further studies to assess gender- and age - specific differences in strain and strain rate variables using ste are warranted even in cats . in conclusion , radial and circumferential strain analysis using ste was considered clinically feasible for assessing cardiac functions in cats . in lv segments of cats with hcm , reduced segmental radial strain and strain rate variables have possibility to have relations with lv segmental hypertrophy . in short axis image of lv in cats with hcm , radial strain and strain rate variables have possibility to detect myocardial dysfunctions locally and early .

the purpose of the present study is to investigate the feasibility of strain analysis using speckle tracking echocardiography ( ste ) in cats and to evaluate ste variables in cats with hypertrophic cardiomyopathy ( hcm ) . sixteen clinically healthy cats and 17 cats with hcm were used . radial and circumferential strain and strain rate variables in healthy cats were measured using ste to assess the feasibility . comparisons of global strain and strain variables between healthy cats and cats with hcm were performed . segmental assessments of left ventricle ( lv ) wall for strain and strain rate variables in cats with hcm were also performed . as a result , technically adequate images were obtained in 97.6% of the segments for ste analysis . sedation using buprenorphine and acepromazine did not affect any global strain nor strain rate variable . in lv segments of cats with hcm , reduced segmental radial strain and strain rate variables had significantly related with segmental lv hypertrophy . it is concluded that ste analysis using short axis images of lv appeared to be clinically feasible in cats , having the possibility to be useful for detecting myocardial dysfunctions in cats with diseased heart .

MATERIALS AND METHODS RESULTS DISCUSSION

study population : sixteen clinically healthy cats ( 12 domestic shorthairs , 1 abyssinian , 1 norwegian forest cat and 2 russian blues ) were used in the present study as a control group . b- , m- and doppler mode echocardiography : lv short - axis images at the chordae tendineae ( ct ) level between the mitral valve and papillary muscle for m - mode measurements ( ivsd , lvidd , lvpwd and fractional shortening [ fs ] ) and those at the aortic root level for b - mode measurements of the left atrium size ( diameters of left atrial diameter [ lad ] and aortic root , and the left atrium to aorta ratio [ la / ao ] ) were obtained on right lateral recumbency . the lv was divided into 6 regions ( anteroseptum , anterior , lateral , posterior , inferior and septum ) , and those segments corresponded to the regions for the segmental strain and strain rate variables . speckle tracking echocardiography : right parasternal short - axis views at the ct were used to measure all strain and strain rate variables . the peak systolic radial and circumferential strains ( sr and sc ) , and the peak radial and circumferential strain rates during the systole and early diastole ( srrs , srre , srcs and srce ) were measured at the level of ct ( fig . 1.images of radial strain rate of a healthy cat ( a ) and a cat with hypertrophic cardiomyopathy ( b ) . the e/ srre and e/ srce all strain and strain rate variables were calculated by 6 segmental values ( anteroseptum , anterior , lateral , posterior , inferior and septum ) . images of radial strain rate of a healthy cat ( a ) and a cat with hypertrophic cardiomyopathy ( b ) . in the present study , simultaneous ecg monitoring was not recorded in some cats with hcm , who were nervous and excited during an examination . however , comparisons between ste variables that analyzed with simultaneous ecg monitoring and those without such monitoring showed good agreement ( statistics analysis was performed using bland - altman plotting [ table 1table 1.assessment of effects of ste variables with or without simultaneous ecg during analysis in healthy 10 catsparameterunitmean sdmean of the differencelimits of agreementrp valuewith simultaneous ecgwithout simultaneous ecgsr%50.68 8.0552.11 9.321.53 3.298.00 to 5.140.94<0.001srrs / sec3.79 0.573.94 0.700.15 0.410.96 to 0.670.810.0041srre / sec3.84 1.194.14 1.160.29 0.400.51 to 1.100.94<0.001sc%23.64 3.7524.23 4.760.59 1.662.73 to 3.910.95<0.001srcs / sec3.52 0.463.69 0.530.17 0.280.39 to 0.730.850.0017srce / sec4.29 1.324.44 1.470.15 0.360.87 to 0.570.97<0.001sd : standard deviation . 2.bland-altman agreement plot comparing strain and strain rate variables analyzed with and without ecg monitoring in 10 healthy cats . ] in case images in the absence of simultaneous ecg monitoring were used for ste analysis , the frame at the time lv contracts the most right before lv distension was considered as proper timing of the end - diastole ( r wave ) . bland - altman agreement plot comparing strain and strain rate variables analyzed with and without ecg monitoring in 10 healthy cats . study protocol : to determine the feasibility of radial and circumferential strain analysis using ste in the healthy cats , the following 4 factors were assessed : ( 1 ) percentage of lv wall segments capable of successfully analyzing strain and strain rate variables ; ( 2 ) temporal resolution of images for ste analysis in cats ; ( 3 ) influence of sedation ; global strain and strain rate variables were obtained at the baseline and 15 min after sedation with acetylpromazine ( 0.01 mg / kg , sc ) and buprenorphine ( 0.0075 mg / kg , sc ) and then compared in 10 cats out of the healthy cats ; and ( 4 ) inter- and intraobserver variabilities in offline analysis were also assessed in 6 healthy cats . then , global strain and strain rate variables in cats with hcm were compared with those in the healthy cats . furthermore , segmental strain and strain rate variables were assessed for correlations with corresponding lv segmental hypertrophy . one way anova was applied for comparison of ste variables among the 6 segments of lv in the control groups . the study population comprised the 16 healthy cats and the 17 cats with hcm . in the cats with hcm , atenolol ( n=2 ) , angiotensin - converting enzyme inhibitor ( n=3 ) and thromboprophylaxis ( n=2 ) one cat in the hcm group presented with congestive heart failure , though others had no clinical sign . the case characteristics and echocardiographic parameters are documented in table 2table 2.characteristics of healthy cats and cats with hcmparameterunitcontrol grouphcm groupp valuegenderf / mf 8 / m 8(16)f 2 / m 15(17)bwkg4.14 1.05(16)4.39 1.00(17)0.49agemo40.56 40.11(16)71.88 50.24(17)<0.05hrbpm182.75 24.94(16)182.64 43.58(14)0.99sbpmmhg142.98 10.15(16)125.88 15.16(11)0.0017usg1.07 0.02(16)1.04 0.01(6)0.0011bunmg / dl27.94 4.85(16)30.72 17.53(16)>0.05cremg / dl1.61 0.36(16)1.85 1.15(16)>0.05ivsdcm0.38 0.05(16)0.64 0.11(17)<0.001lviddcm1.49 0.20(16)1.53 0.22(17)>0.05lvpwdcm0.40 0.05(16)0.55 0.16(17)0.0012fs%42.71 6.46(16)56.69 7.87(17)<0.001ivsbcm0.41 0.06(16)0.70 0.19(16)<0.001ladcm1.04 0.13(16)1.39 0.27(16)<0.001la / ao1.17 0.09(16)1.36 0.24(15)<0.05ecm / sec67.45 16.58(16)68.70 22.68(11)0.87acm / sec58.53 13.18(16)66.91 26.99(11)0.36e / a1.19 0.32(16)1.22 0.78(11)>0.05lvotvm / sec0.96 0.15(16)2.37 1.51(17)0.0015sam0 ( 0%)(16)5 ( 29%)(17)sacm / sec7.83 2.33(16)6.17 1.47(8)0.079eacm / sec9.50 ea : peak of mitral annulus velocity during early diastole .. bw : body weight . assessment of strain and strain rate variables using ste and feasibility of ste analysis in healthy cats : results of global strain and strain rate variables in the healthy cats are shown in table 3table 3.global strain and strain rate variables in healthy cats and cats with hcmparameterunitcontrol grouphcm groupp valueradialsr%47.13 13.18(16)42.68 17.29(17)0.41srrs / sec3.57 0.48(16)3.67 1.08(17)0.74srre / sec3.69 0.82(16)2.59 0.63(8)0.0030 e / srre18.8 5.27(16)29.31 9.07(8)0.0015 circumferentialsc%24.38 4.64(16)23.15 6.11(17)0.52srcs / sec3.43 0.55(16)3.57 1.06(17)0.62srce / sec4.52 1.37(13)3.24 0.78(6)0.049 e / srce16.4 6.3(13)23.42 6.06(6)0.035 a ) significant difference , sr : peak systolic radial strain . 3.segmental strain and strain rate variables in left ventricular 6 segments of healthy cats.sr , radial strain ; srrs , radial strain rate during systole ; srre , radial strain rate during early diastole ; sc , circumferential strain ; srcs , circumferential strain rate during systole ; srce , circumferential strain rate during early diastole . ) segmental strain and strain rate variables in left ventricular 6 segments of healthy cats.sr , radial strain ; srrs , radial strain rate during systole ; srre , radial strain rate during early diastole ; sc , circumferential strain ; srcs , circumferential strain rate during systole ; srce , circumferential strain rate during early diastole . ninety - seven point six percent of segments ( 281/288 ) were able to perform semi - auto tracking for an analysis of strain and strain rate variables . global strain and strain rate variables with and without sedation were compared ( table 4table 4.effect of sedation in healthy 10 catsparameterunitawakesedatedp valueheart ratebpm180 25179 190.44radialsr%50.7 8.0544.7 16.90.09srrs / sec3.79 0.573.85 0.710.58srre / sec3.84 1.193.89 0.840.88circumferentialsc%23.6 3.7523.3 4.70.62srcs / sec3.24 1.053.71 the intra- and inter - observer variability of each parameter is shown in table 5table 5.intra- and interobserver variability of each parameter in offline analysisparameterintraobserver ( % ) interobserver ( % ) radialsr5.548.45srrs4.18.09srre12.0715.64circumferentialsc4.935.56srcs6.754.75srce6.65.43sr : peak systolic radial strain . srce : peak circumferential strain rate during early diastole .. sr : peak systolic radial strain . assessment of strain and strain rate variables in cats with hcm : results of global strain and strain rate variables using ste in the cats with hcm are shown in table 3 . srre , e / srre , srce and e / srce exhibited significant differences between the healthy cats and those with hcm . however , multivariate regression analysis showed no significant relationships between ste variables and patient s condition , gender and age ( table 6table 6.multivariate regression analysis to evaluate relathionships between ste variables and patient s condition , gender and ageparameterp valuesrpatient s condition0.1370.507gender0.1090.577age0.3350.740srrspatient s condition0.0180.930gender0.1260.526age0.0380.846srrepatient s condition0.2910.172gender0.0070.970age0.2340.239scpatient s condition0.1090.597gender0.1240.527age0.1480.448srcspatient s srce : peak circumferential strain rate during early diastole . ) a fusion of e and a in srr was observed in 9 cats and in src in 11 , respectively , out of the 17 cats with hcm ( 52.9 and 64.7% each ) . relatively dominant hypertrophy in intraventricular wall and lv free wall was seen in 10 and 3 cats , respectively , out of the cats with hcm ( 59 and 18% each ) . comparison of segmental strain and strain rate variables between lv wall segments with hypertrophy ( thickness of lv wall during end - diastole is 6 mm or more ) and those without hypertrophy ( the same is less than 6 mm ) in the cats with hcm revealed significant differences in sr and srre ( table 7table 7.comparison of segmental radial strain and strain rate variables between left ventricular wall segments with and without hypertrophy in cats with hcmparameterunitnon - hypertrophic segmentshypertrophic segmentsp valuesegmental lv wall thickness in diastolemean sdmm4.86 0.59(53)6.86 0.90(49)<0.001rangemm3.65.96.09.3sr%47.27 17.15(53)37.72 17.68(49)0.007srrs / sec3.82 1.13(53)3.51 1.06(49)0.15srre / sec3.34 1.36(41)2.68 1.27(44)0.024sc%23.26 8.54(53)23.03 6.89(49)0.88srcs / sec3.69 1.22(53)3.45 1.18(49)0.32srce / sec3.41 segmental radial variables ( sr and srrs ) and corresponding lv segmental wall thickness also showed significant relations , though their correlations were weak ( sr , r= 0.23 p=0.018 ; srrs , r= 0.20 p=0.047 ; and srre , no significant relation was observed in segmental circumferential variables ( sc , r= 0.038 p=0.707 ; srcs , r= 0.081 p=0.418 ; and srce , in the present study , we assessed the feasibility of analysis for radial and circumferential strain variables using ste in cats . because of the relatively high heart rates of cats , it was anticipated that a somewhat inferior resolution in 2d images may have compromised the ste analysis . therefore , it might prove less accurate to assess ste parameters using time intervals in cats , compared with those in dogs and humans . in the present study , ea fusion was frequently exhibited in the circumferential strain rate rather than in the radial one . the inter- and intraobserver variabilities of strain analysis in cats appeared to be satisfactory in the present study . sedation with a combination of acepromazine and buprenorphine did not significantly influence strain and strain rate variables using ste in the present study . we evaluated whether or not it is possible to find significant difference of ste variables in cats with overt heart disease that may be detectable by morphological assessment using echocardiogram , in comparison with healthy cats . ste analysis in the present study could detect significant decrease of global radial and circumferential strain rate during early diastole in the cats with hcm , compared with healthy cats . in the present study , since almost cats with hcm did not have congestive heart failure and even left atrial enlargement , reduced global srre and srce may suggest the presence of impaired lv diastolic function due to lv hypertrophy and fibrosis in relatively early stage . however , since multivariate regression analysis did not show significant relationships between the ste variables and patient s condition , global ste variables in radial and circumferential directions could not detect impaired lv myocardial function . actually , in some studies in human patients with hcm , circumferential strain and strain variables have increased abnormally compared with controls in contrast to the studies described above and the present study [ 10 , 15 ] . values of circumferential strain also have been suggested to be different among layers of lv ( epicardial , mid - myocardial and endocardial layers ) in normal human subjects . further assessments for circumferential strain and strain variables of the layers separately may be needed in cats . on the other hand , it has been suggested that circumferential strain variables increase or preserved appear to maintain to the normal lv ejection fraction as a compensation for longitudinal and radial strains [ 26 , 42 ] . further assessment of each change of strain variables in the 3 directions among different regions of lv in hcm of cats is needed . in the present study , systolic indices of global ste variables including sr , sc , srrs and srcs did not decrease significantly in cats with hcm compared with controls . however , a significant decrease in longitudinal strain using tdi in cats with hcm has been previously reported . in the present study , a significant difference was seen in segmental radial strain and strain rate variables between hypertrophic and non - hypertrophic segments . furthermore , segmental radial strain and strain rate variables had significant correlation with segmental lv thickness . assessing lv functions segmentally using ste , local ventricular dysfunctions could be detected even in the radial direction in the present study . in addition , segmental values of strain and strain rate variables in radial direction are more invariant than those in circumferential direction among the lv 6 segments of normal subjects in the present study . second , few cats with hcm were given medications , such as atenolol , at their examinations , and the fact that such medications may influence lv systolic and diastolic functions shall be taken into consideration [ 30 , 41 ] . age - specific change in lv function longitudinal strain and strain rate have been reported to decrease significantly in an older population compared with a young one in a human study , though no significant difference was seen in the radial and circumferential strain and strain rate in systole and early diastole . further studies to assess gender- and age - specific differences in strain and strain rate variables using ste are warranted even in cats . in conclusion , radial and circumferential strain analysis using ste was considered clinically feasible for assessing cardiac functions in cats . in lv segments of cats with hcm , reduced segmental radial strain and strain rate variables have possibility to have relations with lv segmental hypertrophy . in short axis image of lv in cats with hcm , radial strain and strain rate variables have possibility to detect myocardial dysfunctions locally and early .

five hundred twenty - six patients with a clinical diagnosis of hsp were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . blood samples and clinical data were collected after written consent was obtained from all study participants . standardized clinical assessments were performed and included demographic information , family history , developmental history , and hsp core symptoms ( age at onset , lower extremity weakness and spasticity , hyperreflexia , extensor plantar responses , and bladder dysfunction ) . other clinical symptoms such as progressive cognitive deficits , ataxia , extrapyramidal movements , swallowing difficulties , and ocular motor abnormalities were also assessed . for a subset of patients ( n = 65 ) , disability was evaluated using the spatax - eurospa disability score , ranging from 0 ( no disability ) to 7 ( severe disability / confined to bed ) . we also used the spastic paraplegia rating scale ( sprs ) , a previously validated disease - specific outcome measure for a subset of patients ( n = 48 ) . the sprs is a composite measure with the following subdomains : speed of gait , climbing stairs , quality of gait , arising from chair , quality of spasticity , weakness and contractures , and bladder dysfunction . all subdomains were rated on a scale between 0 and 4 with a maximum total score of 52 . ancillary investigations such as brain mri , electrophysiologic tests , and metabolic studies were also evaluated . patients with cerebellar signs were screened for mutations in fxn , sacs , and the common spinocerebellar ataxias ( scas 18 ) prior to being included in the study . this study was approved by the research ethics boards of all institutions involved in the study ( figure 1 ) . patients with a clinical diagnosis of hereditary spastic paraplegia ( hsp ) were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . genomic dna was extracted from the peripheral blood according to standard procedures . for a subset of the cohort from the province of ontario ( 37 patients ) , all exons and flanking intron sequences of a panel of 51 genes known to cause hsp were sequenced both in forward and reverse directions at the hospital for sick children using next - generation sequencing techniques . sanger validation was performed according to standard protocols using bigdye terminator v1.1 ( life technologies , carlsbad , ca ) , and sequencing products were separated on an applied biosystems model 3730 capillary sequencer ( life technologies ) and analyzed using seqpilot software ( jsi medical systems , kippenheim , germany ) . whole - exome sequencing was performed on 108 samples from 51 families without a genetic diagnosis , using the agilent sureselect human all exon v4 kit for capture and targeted enrichment of the exome . the captured samples were sequenced in the illumina hiseq 2000/2500 system ( genome qubec innovation centre , montreal , qc , canada ) . the reads were then aligned against the human genome ( grch37 assembly ) using burrows - wheeler aligner . the exome data were then screened for all known hsp - causing genes and mutations . for the purpose of statistical analysis mean ( sd ) , median , and interquartile range were calculated for continuous variables . we performed an ordered logistic regression to examine the association between ambulatory status and age at symptom onset . linear regressions were used to analyze the association between disability and age at onset , and logistic regression was used to evaluate the association between disability and abnormal brain mri . we investigated the association between genetic diagnoses and outcomes of interest including age at onset , disease duration , abnormal bladder function , swallowing difficulties , hsp - related symptoms , learning disabilities , speech delay , progressive cognitive deficits , motor delay , ambulatory status , age achieved walking , and disability scores . for each outcome of interest bonferroni correction technique was then used to adjust for multiple testing ( adjusted p value threshold is 0.01 ) . logistic regressions were performed for binary outcomes . in cases of data point separation issues , we conducted a multiple linear regression analysis to examine the association between disability score ( assessed by the sprs ) and variables such as genes of interest , age at evaluation , disease duration , abnormal brain mri , and ataxia . five hundred twenty - six patients with a clinical diagnosis of hsp were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . blood samples and clinical data were collected after written consent was obtained from all study participants . standardized clinical assessments were performed and included demographic information , family history , developmental history , and hsp core symptoms ( age at onset , lower extremity weakness and spasticity , hyperreflexia , extensor plantar responses , and bladder dysfunction ) . other clinical symptoms such as progressive cognitive deficits , ataxia , extrapyramidal movements , swallowing difficulties , and ocular motor abnormalities were also assessed . for a subset of patients ( n = 65 ) , disability was evaluated using the spatax - eurospa disability score , ranging from 0 ( no disability ) to 7 ( severe disability / confined to bed ) . we also used the spastic paraplegia rating scale ( sprs ) , a previously validated disease - specific outcome measure for a subset of patients ( n = 48 ) . the sprs is a composite measure with the following subdomains : speed of gait , climbing stairs , quality of gait , arising from chair , quality of spasticity , weakness and contractures , and bladder dysfunction . all subdomains were rated on a scale between 0 and 4 with a maximum total score of 52 . ancillary investigations such as brain mri , electrophysiologic tests , and metabolic studies were also evaluated . patients with cerebellar signs were screened for mutations in fxn , sacs , and the common spinocerebellar ataxias ( scas 18 ) prior to being included in the study . this study was approved by the research ethics boards of all institutions involved in the study ( figure 1 ) . patients with a clinical diagnosis of hereditary spastic paraplegia ( hsp ) were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . genomic dna was extracted from the peripheral blood according to standard procedures . for a subset of the cohort from the province of ontario ( 37 patients ) , all exons and flanking intron sequences of a panel of 51 genes known to cause hsp were sequenced both in forward and reverse directions at the hospital for sick children using next - generation sequencing techniques . sanger validation was performed according to standard protocols using bigdye terminator v1.1 ( life technologies , carlsbad , ca ) , and sequencing products were separated on an applied biosystems model 3730 capillary sequencer ( life technologies ) and analyzed using seqpilot software ( jsi medical systems , kippenheim , germany ) . whole - exome sequencing was performed on 108 samples from 51 families without a genetic diagnosis , using the agilent sureselect human all exon v4 kit for capture and targeted enrichment of the exome . the captured samples were sequenced in the illumina hiseq 2000/2500 system ( genome qubec innovation centre , montreal , qc , canada ) . the reads were then aligned against the human genome ( grch37 assembly ) using burrows - wheeler aligner . the exome data were then screened for all known hsp - causing genes and mutations . for the purpose of statistical analysis , we analyzed only the 150 patients with a genetically confirmed diagnosis . mean ( sd ) , median , and interquartile range were calculated for continuous variables . we performed an ordered logistic regression to examine the association between ambulatory status and age at symptom onset . linear regressions were used to analyze the association between disability and age at onset , and logistic regression was used to evaluate the association between disability and abnormal brain mri . we investigated the association between genetic diagnoses and outcomes of interest including age at onset , disease duration , abnormal bladder function , swallowing difficulties , hsp - related symptoms , learning disabilities , speech delay , progressive cognitive deficits , motor delay , ambulatory status , age achieved walking , and disability scores . for each outcome of interest , we performed 5 univariate analyses with each gene as the covariate separately . bonferroni correction technique was then used to adjust for multiple testing ( adjusted p value threshold is 0.01 ) . logistic regressions were performed for binary outcomes . in cases of data point separation issues , we conducted a multiple linear regression analysis to examine the association between disability score ( assessed by the sprs ) and variables such as genes of interest , age at evaluation , disease duration , abnormal brain mri , and ataxia . of the 526 patients who met the inclusion criteria , 150 ( 28.5% ) patients from 58 families were confirmed to have a genetic diagnosis . the most frequent were spast ( spg4 , 72 patients , 48% ) , atl1 ( spg3a , 24 patients , 16% ) , spg11 ( 12 patients , 8% ) , spg7 ( 10 patients , 7% ) , and kiaa0196 ( spg8 , 8 patients , 5% ) . mutations were also found in plp1 ( spg2 , 5 patients , 3% ) , slc2a1 ( 5 patients , 3% ) , cyp7b1 ( spg5a , 4 patients , 3% ) , vamp1 ( 3 patients , 2% ) , als2 ( 2 patients , 1% ) , kif5a ( spg10 , 1 patient , 0.6% ) , slc16a2 ( spg22 , 1 patient , 0.6% ) , zfyve26 ( spg15 , 1 patient , 0.6% ) , fa2h ( spg35 , 1 patient , 0.6% ) , and pnpla6 ( spg39 , 1 patient , 0.6% ) ( figure 2 and table e-1 ) . a total of 26 novel mutations were found in our cohort ; they were absent in publically available databases ( single nucleotide polymorphism database , 1000 genomes project , and exome aggregation consortium ) and were predicted to be pathogenic when evaluated using standard prediction programs ( sift , polyphen , and mutation taster ) . spg4 and spg3a were the most frequent autosomal dominant hereditary spastic paraplegia ( hsp ) , while spg11 followed by spg7 were the most frequent autosomal recessive hsp . ethnic group was self - reported , and 65% of the cohort was from north america , 29% from europe , 2% from the middle east , and 4% from asia . parental consanguinity was reported for 12/141 patients ( 8.5% ) . a positive family history ( first - degree family members with symptoms of hsp ) the mean age at evaluation of our cohort was 39.2 years ( sd 22.1 , range 182 years ) , and the mean age at onset of symptoms was 18.9 years ( sd 18.6 , range 067 years ) . the majority of patients acquired independent walking by the age of 2 years ( range less than 1 to 13 years ) . the most common clinical complaints , in addition to the core features of spasticity , weakness , and urinary disturbance , were dysarthria ( 33% ) , sensory abnormalities ( 35% ) , motor delay ( 24% ) , peripheral neuropathy ( 23% ) , speech delay ( 18% ) , and ataxia ( 17% ) . less common symptoms included ocular motor disturbance ( 11% ) , intentional tremor ( 11% ) , and seizures ( 3% ) . the most common abnormalities reported were thin corpus callosum ( 6 patients , 10% ) , followed by cerebellar atrophy ( 5 patients , 8% ) , and white matter abnormalities ( 4 patients , 7% ) . at the time of evaluation for this study , 36/56 patients ( 64% ) required an aid for ambulation with the mean age at onset of mobility aid use of 33.6 years ( sd 23.3 , range 268 years ) . twelve patients required the use of a cane ( 21% ) and 3 used a walker ( 5% ) . approximately one - third of our cohort was independent with respect to ambulation ( 20/56 patients , 36% ) ; however , 21 patients ( 38% ) were using a wheelchair . the mean sprs for the subset of our cohort for which this was available ( 48 patients ) was 19 ( sd 11 , range 239 ) , and the majority of patients ( 48/65 , 74% ) had spatax - eurospa disability scores within the mild to moderate range . on univariate analysis , there was no statistically significant association between age at symptom onset and ambulatory status , nor was there a statistically significant association between age at symptom onset and sprs score when adjusted for age at evaluation . there was a clear association between abnormal brain mri and disability as measured by the sprs , as patients with abnormal brain mri had a mean higher sprs score of 10.49 compared to those with normal brain imaging ( p = 0.0037 ) . linear regression analysis with bonferroni correction revealed that patients with spg4 and spg7 were more likely to develop symptoms later compared to other hsp subtypes , with a mean age at symptom onset of 24.6 ( standard error [ se ] 3.2 ) years for spg4 ( p = 0.0017 ) and 33 ( se 6.0 ) years for spg7 ( p = 0.013 ) . patients with spg3a were more likely to present at a younger age , with a mean age at symptom onset of 10.1 ( se 4.4 ) years ( p = 0.022 ) ( figure 3 ) . spg4 appeared to be associated with greater risk of bladder dysfunction ( odds ratio ( or ) 2.24 , 95% confidence interval ( ci ) 1.034.86 , p = 0.04 ) , and patients with spg3a less likely to develop bladder dysfunction ( or 0.30 , 95% ci 0.090.99 , p = 0.04 ) , but these observations were not statistically significant following bonferroni correction . patients with spg4 and spg7 were more likely to develop symptoms later compared to other hereditary spastic paraplegia subtypes , while patients with spg3a were more likely to present at a younger age . the horizontal line inside the rectangle shows the median , and the whiskers above and below the box represent the minimum and maximum values of the ages at symptom onset . similarly , spg11 appeared to be associated with increased pain due to hsp - related symptoms compared to other genetic diagnoses ( or 5.44 , 95% ci 1.0827.37 , p = 0.039 ) , but this was not statistically significant following bonferroni correction . spg2 appeared to be associated with an increased risk of speech delay ( or 18.33 , 95% ci 1.71196.19 , p = 0.016 ) and motor delay ( or 11.8 , 95% ci 1.14122.1 , p = 0.04 ) , and spg4 less likely to have motor delay ( or 0.10 , 95% ci 0.010.78 , p = 0.028 ) , but these observations were no longer statistically significant after bonferroni correction . spg11 was strongly associated with an increased risk of learning disability ( or 30.59 , 95% ci 3.58261.19 , p = 0.0018 ) and progressive cognitive deficits ( or 87.75 , 95% ci 14.04548.24 , p < 0.0001 ) , compared to other genetic subtypes of hsp . patients with spg4 were significantly less likely to have learning disability compared to those with mutations in other genes ( or 0.06 , 95% ci 0.010.51 , p = 0.0096 ) . multiple linear regression analysis revealed that spg3a patients had better functional outcomes with a mean lower sprs score of 11.2 ( se 5.3 ) compared to those with mutations in other genes ( p = 0.04 ) , after adjusting for genotype , disease duration , and brain mri status . abnormal brain mri was associated with a mean higher total sprs score of 10.4 ( se 4.0 ) and worse functional outcomes compared with patients with normal brain mri ( p = 0.014 ) . of the 526 patients who met the inclusion criteria , 150 ( 28.5% ) patients from 58 families were confirmed to have a genetic diagnosis . the most frequent were spast ( spg4 , 72 patients , 48% ) , atl1 ( spg3a , 24 patients , 16% ) , spg11 ( 12 patients , 8% ) , spg7 ( 10 patients , 7% ) , and kiaa0196 ( spg8 , 8 patients , 5% ) . mutations were also found in plp1 ( spg2 , 5 patients , 3% ) , slc2a1 ( 5 patients , 3% ) , cyp7b1 ( spg5a , 4 patients , 3% ) , vamp1 ( 3 patients , 2% ) , als2 ( 2 patients , 1% ) , kif5a ( spg10 , 1 patient , 0.6% ) , slc16a2 ( spg22 , 1 patient , 0.6% ) , zfyve26 ( spg15 , 1 patient , 0.6% ) , fa2h ( spg35 , 1 patient , 0.6% ) , and pnpla6 ( spg39 , 1 patient , 0.6% ) ( figure 2 and table e-1 ) . a total of 26 novel mutations were found in our cohort ; they were absent in publically available databases ( single nucleotide polymorphism database , 1000 genomes project , and exome aggregation consortium ) and were predicted to be pathogenic when evaluated using standard prediction programs ( sift , polyphen , and mutation taster ) . spg4 and spg3a were the most frequent autosomal dominant hereditary spastic paraplegia ( hsp ) , while spg11 followed by spg7 were the most frequent autosomal recessive hsp . ethnic group was self - reported , and 65% of the cohort was from north america , 29% from europe , 2% from the middle east , and 4% from asia . parental consanguinity was reported for 12/141 patients ( 8.5% ) . a positive family history ( first - degree family members with symptoms of hsp ) the mean age at evaluation of our cohort was 39.2 years ( sd 22.1 , range 182 years ) , and the mean age at onset of symptoms was 18.9 years ( sd 18.6 , range 067 years ) . the majority of patients acquired independent walking by the age of 2 years ( range less than 1 to 13 years ) . the most common clinical complaints , in addition to the core features of spasticity , weakness , and urinary disturbance , were dysarthria ( 33% ) , sensory abnormalities ( 35% ) , motor delay ( 24% ) , peripheral neuropathy ( 23% ) , speech delay ( 18% ) , and ataxia ( 17% ) . less common symptoms included ocular motor disturbance ( 11% ) , intentional tremor ( 11% ) , and seizures ( 3% ) . the most common abnormalities reported were thin corpus callosum ( 6 patients , 10% ) , followed by cerebellar atrophy ( 5 patients , 8% ) , and white matter abnormalities ( 4 patients , 7% ) . at the time of evaluation for this study , 36/56 patients ( 64% ) required an aid for ambulation with the mean age at onset of mobility aid use of 33.6 years ( sd 23.3 , range 268 years ) . twelve patients required the use of a cane ( 21% ) and 3 used a walker ( 5% ) . approximately one - third of our cohort was independent with respect to ambulation ( 20/56 patients , 36% ) ; however , 21 patients ( 38% ) were using a wheelchair . the mean sprs for the subset of our cohort for which this was available ( 48 patients ) was 19 ( sd 11 , range 239 ) , and the majority of patients ( 48/65 , 74% ) had spatax - eurospa disability scores within the mild to moderate range . on univariate analysis , there was no statistically significant association between age at symptom onset and ambulatory status , nor was there a statistically significant association between age at symptom onset and sprs score when adjusted for age at evaluation . there was a clear association between abnormal brain mri and disability as measured by the sprs , as patients with abnormal brain mri had a mean higher sprs score of 10.49 compared to those with normal brain imaging ( p = 0.0037 ) . linear regression analysis with bonferroni correction revealed that patients with spg4 and spg7 were more likely to develop symptoms later compared to other hsp subtypes , with a mean age at symptom onset of 24.6 ( standard error [ se ] 3.2 ) years for spg4 ( p = 0.0017 ) and 33 ( se 6.0 ) years for spg7 ( p = 0.013 ) . patients with spg3a were more likely to present at a younger age , with a mean age at symptom onset of 10.1 ( se 4.4 ) years ( p = 0.022 ) ( figure 3 ) . spg4 appeared to be associated with greater risk of bladder dysfunction ( odds ratio ( or ) 2.24 , 95% confidence interval ( ci ) 1.034.86 , p = 0.04 ) , and patients with spg3a less likely to develop bladder dysfunction ( or 0.30 , 95% ci 0.090.99 , p = 0.04 ) , but these observations were not statistically significant following bonferroni correction . patients with spg4 and spg7 were more likely to develop symptoms later compared to other hereditary spastic paraplegia subtypes , while patients with spg3a were more likely to present at a younger age . the horizontal line inside the rectangle shows the median , and the whiskers above and below the box represent the minimum and maximum values of the ages at symptom onset . similarly , spg11 appeared to be associated with increased pain due to hsp - related symptoms compared to other genetic diagnoses ( or 5.44 , 95% ci 1.0827.37 , p = 0.039 ) , but this was not statistically significant following bonferroni correction . spg2 appeared to be associated with an increased risk of speech delay ( or 18.33 , 95% ci 1.71196.19 , p = 0.016 ) and motor delay ( or 11.8 , 95% ci 1.14122.1 , p = 0.04 ) , and spg4 less likely to have motor delay ( or 0.10 , 95% ci 0.010.78 , p = 0.028 ) , but these observations were no longer statistically significant after bonferroni correction . spg11 was strongly associated with an increased risk of learning disability ( or 30.59 , 95% ci 3.58261.19 , p = 0.0018 ) and progressive cognitive deficits ( or 87.75 , 95% ci 14.04548.24 , p < 0.0001 ) , compared to other genetic subtypes of hsp . patients with spg4 were significantly less likely to have learning disability compared to those with mutations in other genes ( or 0.06 , 95% ci 0.010.51 , p = 0.0096 ) . multiple linear regression analysis revealed that spg3a patients had better functional outcomes with a mean lower sprs score of 11.2 ( se 5.3 ) compared to those with mutations in other genes ( p = 0.04 ) , after adjusting for genotype , disease duration , and brain mri status . abnormal brain mri was associated with a mean higher total sprs score of 10.4 ( se 4.0 ) and worse functional outcomes compared with patients with normal brain mri ( p = 0.014 ) . hsps are clinically and genetically heterogeneous , and only a few population - based studies , mainly in relatively homogeneous populations , have been reported . we conducted a cross - sectional study of a large , ethnically diverse cohort of patients with hsp in canada using standardized clinical assessments and specifically assessed functional outcomes using validated rating scales . as in previously published population - based studies , spg4 and spg3a were the most frequent ad - hsp . spg4 accounts for up to 45% of ad - hsp and up to 20% of sporadic cases , while spg3a accounts for nearly 10% of ad - hsp . spg8 was present in 5% of our cohort , which is more common compared to previous reports . spg11 followed by spg7 were the most frequent ar - hsp , which is also in keeping with previously published studies . the primary analysis of the exome data was focused on known hsp genes ; however , mutations were also found in genes not typically considered hsp genes in patients who met the clinical criteria for hsp ( 1 patient with a mutation in slc16a2 , 3 patients with mutations in vamp1 , and 4 patients from a single family with mutations in slc2a1 ) . this has been observed by other groups and further highlights the clinical and genetic heterogeneity of hsp . in contrast to comparable neurodegenerative disorders such as the scas , very little is known about functional outcomes in patients with hsp , and the majority of studies are focused exclusively on spg4 and spg3a . this is likely due to very recent advances in genetic diagnoses of hsp compared to the common scas and highlights the need for good natural history data to identify parameters which influence clinical outcomes of hsp . there is one study which examined the correlation between disease duration and spasticity in patients with hsp , but unfortunately , the genetic diagnoses were not reported . another study identified the degree of spasticity as being the most important contributing factor for wheelchair dependence . higher sprs scores have been shown to be inversely correlated with quality of life in a previous study . as the majority of patients in our cohort who met the clinical criteria for hsp did not have a genetic diagnosis , this illustrates the need for studies of clinical parameters which influence prognosis and outcomes for these patients . to our knowledge , only 1 recent study has focused on functional outcomes in a large genetically confirmed hsp population , the majority of which were adults with spg4 of german ancestry . in contrast to the findings from this study , we did not observe a statistically significant association between disability scores and age at symptom onset or disease duration . this may reflect the underlying differences between our patient populations ( ethnically diverse population from canada vs primarily german ethnicity ) and the large proportion of pediatric patients in our study . in our cohort , patients with spg4 and spg3a had less severe disability scores compared with other hsp genetic subtypes , and patients with spg11 and spg2 had more severe scores , although the samples sizes were too small for this observation to reach statistical significance . bladder dysfunction is a recurrent and disabling symptom , which was present in a large proportion of our cohort , with the exception of patients with spg3a , which may contribute to the better functional outcomes observed in this subgroup of patients . the clear correlation between abnormal brain mri and increased disability scores in our cohort confirms that brain imaging is an essential tool for clinical diagnosis and also for prognostication . the diagnostic utility of brain imaging to identify specific features of genetic subtypes of hsp such as spg2 , spg7 , spg11 , or spg35 is well established . as an example , patients with spg11 have thinning or agenesis ( partial or complete ) of the corpus callosum . they also demonstrate prominent cognitive impairment and learning disabilities , and some patients with spg11 mutations present with a very rare phenotype ( kjellin syndrome ) where mental retardation is a predominant feature . the clinical spectrum associated with spg7 mutations is variable , but specific brain mri findings such as cerebellar atrophy is common , which is consistent with ataxia being the first clinical symptom in many of these patients . prospective studies of brain imaging in patients with hsp will be necessary to define the cns phenotypes of the genetic hsp subtypes . the use of advanced techniques such as fractional anisotropy reduction , spinal morphometry , or diffusor tensor imaging brain will allow identification of imaging biomarkers which could be used to monitor the progression of the disease , and eventually used as outcome measures for future clinical trials . one major strength of our study was the inclusion of both adult and pediatric patients , which enabled accurate assessment of age at symptom onset , and other clinical parameters essential for delineating the natural history of these disorders . our study was limited by the relatively small sample sizes of the genetic subtypes of hsp , with the exception of spg3a , spg4 , spg11 , and spg7 . the clinical variability between patients with the same genetic subtype , and even within the same family , renders accurate genotype - phenotype correlations difficult . large prospective studies of well - phenotyped patients are required to define the natural history of the various hsp subtypes and to design appropriate clinical trials of therapies for these complex disorders . hsp is usually not a life - limiting disorder but can be associated with serious disability , and accurate genetic diagnosis , genetic counseling , and management of symptoms are essential to optimizing care for patients with hsp and their families . the canadian hsp population is characterized by considerable clinical and genetic heterogeneity , yet we observed meaningful genotype - phenotype correlations such as spg4 and spg7 presenting at later onset compared to spg3a . spg11 is strongly associated with abnormal brain mri but also with prominent learning disabilities , progressive cognitive deficits , and poor functional outcomes . patients with spg3a and spg4 have less disability and are less likely to develop cognitive deficits . brain mri is the most useful tool to predict functional outcomes and disabilities and should always be integrated in the clinical evaluation of any patient with suspected hsp . accurate molecular characterization of well - phenotyped cohorts and international collaboration to increase the power of future studies will be critical to establish the natural history of these rare degenerative disorders to enable future clinical trials . nicolas dupr was responsible for the study concept and design ; acquisition of data ; analysis and interpretation ; and critical revision of the manuscript for important intellectual content . ziv gan - or performed the analysis of the whole - exome sequencing data and critically reviewed the manuscript for important intellectual content . shiyi chen performed the statistical analyses of the clinical data and critically reviewed the manuscript for important intellectual content . anil venkitachalam , jean - denis brisson , jodi warman - chardon , sohnee ahmed , setareh ashtiani , heather macdonald , and noreen mohsin contributed clinical data and critically reviewed the manuscript for important intellectual content . karim mourabit - amari coordinated clinical molecular diagnostic testing ; contributed clinical laboratory data ; and critically reviewed the manuscript for important intellectual content . pierre provencher and kym boycott contributed clinical data and critically reviewed the manuscript for important intellectual content . dimitri j stavropoulos performed the clinical molecular testing ; sanger validation of variants and critically reviewed the manuscript for important intellectual content . patrick a dion performed the analysis of the whole - exome data and critically reviewed the manuscript for important intellectual content . peter n. ray performed the clinical molecular testing ; sanger validation of variants and critically reviewed the manuscript for important intellectual content . oksana suchowersky was responsible for the study concept and design ; acquisition of data ; and critical revision of the manuscript for important intellectual content . guy a. rouleau was responsible for the study concept and design ; acquisition of data ; analysis and interpretation ; and critical revision of the manuscript for important intellectual content . grace yoon was responsible for the study concept and design ; acquisition of data ; analysis and interpretation ; critical revision of the manuscript for important intellectual content ; and overall study supervision . this study was funded by cihr emerging team grant , in collaboration with the canadian organization for rare disorders ( cord ) , grant number rn127580 - 260005 . the funding organization had no role in design and conduct of the study ; collection , management , analysis , and interpretation of the data ; and preparation , review , or approval of the manuscript . the corresponding author had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis . dr . dupr has received travel funding / speaker honoraria from actelion pharmaceuticals and has served on the editorial board of cerebellum & ataxia . gan - or has received travel funding and speaker honoraria from sanofi - genzyme and has received research support from the canadian institute of health research , mcgill university , and the michael j. fox foundation . macdonald has received research support from cihr , in collaboration with the canadian organization for rare disorders ( cord ) . boycott has served on the editorial boards of human molecular genetics , genetics , and molecular genetics and genomic medicine . suchowersky has received travel funding / speaker honoraria from the american academy of neurology ; has served on the editorial boards of the canadian journal of neurological sciences and nature reviews neurology ; has received publishing royalties from uptodate for chapters on diagnosis of huntington disease , treatment of huntington disease , and diagnosis of chorea ( and for yearly ongoing updates on these 3 chapters ) ; has served on the advisory boards of abbott ( abbvie ) and ipsen ; has received research support from cihr , nih , chdi , the toupin foundation , and abbvie pharmaceuticals ; has acted as a consultant for abbvie ; and is supported by the toupin research chair in neurology , university of alberta . rouleau has received grants from cihr , the als association , genome qubec , frsq , the als society of canada , and the michael j. fox foundation . yoon has received funding from cihr and the canadian organization for rare disorders ( cord ) .

objective : to describe the clinical , genetic , and epidemiologic features of hereditary spastic paraplegia ( hsp ) in canada and to determine which clinical , radiologic , and genetic factors determine functional outcomes for patients with hsp.methods:we conducted a multicenter observational study of patients who met clinical criteria for the diagnosis of hsp in the provinces of alberta , ontario , and quebec from 2012 to 2015 . characteristics of the participants were analyzed using descriptive statistics . the main outcome measure for a subset of the cohort ( n = 48 ) was the spastic paraplegia rating scale . we also used the spatax - eurospa disability stage ( disability score ) to assess disability ( n = 65).results : a total of 526 patients were identified with hsp across the country , and 150 patients had a confirmed genetic diagnosis . mutations were identified in 15 different genes ; the most common were spast ( spg4 , 48% ) , atl1 ( spg3a , 16% ) , spg11 ( 8% ) , spg7 ( 7% ) , and kiaa0196 ( spg8 , 5% ) . the diagnosis of spg4 was associated with older age at symptom onset ( p = 0.0017 ) . spg4 and spg3a were less associated with learning disabilities compared to other subtypes of hsp , and spg11 was strongly associated with progressive cognitive deficits ( odds ratio 87.75 , 95% confidence interval 14.04548.24 , p < 0.0001 ) . spg3a was associated with better functional outcomes compared to other hsp subtypes ( p = 0.04 ) on multivariate analysis . the strongest predictor of significant disability was abnormal brain mri ( p = 0.014).conclusions : the most important predictors of disability in our hsp cohort were spg11 mutations and abnormal brain mri . accurate molecular characterization of well - phenotyped cohorts and international collaboration are essential to establish the natural history of these rare neurodegenerative disorders .

METHODS Clinical assessment. Standard protocol approvals, registrations, and patient consents. Genetic analysis/mutation screening. Statistical analyses. RESULTS Genetic testing. Clinical features. Functional outcomes. Genotype-phenotype correlations. DISCUSSION CONCLUSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

five hundred twenty - six patients with a clinical diagnosis of hsp were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . for a subset of patients ( n = 65 ) , disability was evaluated using the spatax - eurospa disability score , ranging from 0 ( no disability ) to 7 ( severe disability / confined to bed ) . we also used the spastic paraplegia rating scale ( sprs ) , a previously validated disease - specific outcome measure for a subset of patients ( n = 48 ) . patients with a clinical diagnosis of hereditary spastic paraplegia ( hsp ) were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . for a subset of the cohort from the province of ontario ( 37 patients ) , all exons and flanking intron sequences of a panel of 51 genes known to cause hsp were sequenced both in forward and reverse directions at the hospital for sick children using next - generation sequencing techniques . linear regressions were used to analyze the association between disability and age at onset , and logistic regression was used to evaluate the association between disability and abnormal brain mri . we investigated the association between genetic diagnoses and outcomes of interest including age at onset , disease duration , abnormal bladder function , swallowing difficulties , hsp - related symptoms , learning disabilities , speech delay , progressive cognitive deficits , motor delay , ambulatory status , age achieved walking , and disability scores . in cases of data point separation issues , we conducted a multiple linear regression analysis to examine the association between disability score ( assessed by the sprs ) and variables such as genes of interest , age at evaluation , disease duration , abnormal brain mri , and ataxia . five hundred twenty - six patients with a clinical diagnosis of hsp were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . for a subset of patients ( n = 65 ) , disability was evaluated using the spatax - eurospa disability score , ranging from 0 ( no disability ) to 7 ( severe disability / confined to bed ) . we also used the spastic paraplegia rating scale ( sprs ) , a previously validated disease - specific outcome measure for a subset of patients ( n = 48 ) . patients with a clinical diagnosis of hereditary spastic paraplegia ( hsp ) were recruited from 6 major medical centers across canada ( toronto , quebec , montreal , edmonton , calgary , and ottawa ) from february 1 , 2012 , to january 31 , 2015 . for a subset of the cohort from the province of ontario ( 37 patients ) , all exons and flanking intron sequences of a panel of 51 genes known to cause hsp were sequenced both in forward and reverse directions at the hospital for sick children using next - generation sequencing techniques . linear regressions were used to analyze the association between disability and age at onset , and logistic regression was used to evaluate the association between disability and abnormal brain mri . we investigated the association between genetic diagnoses and outcomes of interest including age at onset , disease duration , abnormal bladder function , swallowing difficulties , hsp - related symptoms , learning disabilities , speech delay , progressive cognitive deficits , motor delay , ambulatory status , age achieved walking , and disability scores . in cases of data point separation issues , we conducted a multiple linear regression analysis to examine the association between disability score ( assessed by the sprs ) and variables such as genes of interest , age at evaluation , disease duration , abnormal brain mri , and ataxia . of the 526 patients who met the inclusion criteria , 150 ( 28.5% ) patients from 58 families were confirmed to have a genetic diagnosis . the most frequent were spast ( spg4 , 72 patients , 48% ) , atl1 ( spg3a , 24 patients , 16% ) , spg11 ( 12 patients , 8% ) , spg7 ( 10 patients , 7% ) , and kiaa0196 ( spg8 , 8 patients , 5% ) . a total of 26 novel mutations were found in our cohort ; they were absent in publically available databases ( single nucleotide polymorphism database , 1000 genomes project , and exome aggregation consortium ) and were predicted to be pathogenic when evaluated using standard prediction programs ( sift , polyphen , and mutation taster ) . spg4 and spg3a were the most frequent autosomal dominant hereditary spastic paraplegia ( hsp ) , while spg11 followed by spg7 were the most frequent autosomal recessive hsp . a positive family history ( first - degree family members with symptoms of hsp ) the mean age at evaluation of our cohort was 39.2 years ( sd 22.1 , range 182 years ) , and the mean age at onset of symptoms was 18.9 years ( sd 18.6 , range 067 years ) . the most common clinical complaints , in addition to the core features of spasticity , weakness , and urinary disturbance , were dysarthria ( 33% ) , sensory abnormalities ( 35% ) , motor delay ( 24% ) , peripheral neuropathy ( 23% ) , speech delay ( 18% ) , and ataxia ( 17% ) . the most common abnormalities reported were thin corpus callosum ( 6 patients , 10% ) , followed by cerebellar atrophy ( 5 patients , 8% ) , and white matter abnormalities ( 4 patients , 7% ) . the mean sprs for the subset of our cohort for which this was available ( 48 patients ) was 19 ( sd 11 , range 239 ) , and the majority of patients ( 48/65 , 74% ) had spatax - eurospa disability scores within the mild to moderate range . there was a clear association between abnormal brain mri and disability as measured by the sprs , as patients with abnormal brain mri had a mean higher sprs score of 10.49 compared to those with normal brain imaging ( p = 0.0037 ) . linear regression analysis with bonferroni correction revealed that patients with spg4 and spg7 were more likely to develop symptoms later compared to other hsp subtypes , with a mean age at symptom onset of 24.6 ( standard error [ se ] 3.2 ) years for spg4 ( p = 0.0017 ) and 33 ( se 6.0 ) years for spg7 ( p = 0.013 ) . patients with spg3a were more likely to present at a younger age , with a mean age at symptom onset of 10.1 ( se 4.4 ) years ( p = 0.022 ) ( figure 3 ) . spg4 appeared to be associated with greater risk of bladder dysfunction ( odds ratio ( or ) 2.24 , 95% confidence interval ( ci ) 1.034.86 , p = 0.04 ) , and patients with spg3a less likely to develop bladder dysfunction ( or 0.30 , 95% ci 0.090.99 , p = 0.04 ) , but these observations were not statistically significant following bonferroni correction . patients with spg4 and spg7 were more likely to develop symptoms later compared to other hereditary spastic paraplegia subtypes , while patients with spg3a were more likely to present at a younger age . the horizontal line inside the rectangle shows the median , and the whiskers above and below the box represent the minimum and maximum values of the ages at symptom onset . similarly , spg11 appeared to be associated with increased pain due to hsp - related symptoms compared to other genetic diagnoses ( or 5.44 , 95% ci 1.0827.37 , p = 0.039 ) , but this was not statistically significant following bonferroni correction . spg2 appeared to be associated with an increased risk of speech delay ( or 18.33 , 95% ci 1.71196.19 , p = 0.016 ) and motor delay ( or 11.8 , 95% ci 1.14122.1 , p = 0.04 ) , and spg4 less likely to have motor delay ( or 0.10 , 95% ci 0.010.78 , p = 0.028 ) , but these observations were no longer statistically significant after bonferroni correction . spg11 was strongly associated with an increased risk of learning disability ( or 30.59 , 95% ci 3.58261.19 , p = 0.0018 ) and progressive cognitive deficits ( or 87.75 , 95% ci 14.04548.24 , p < 0.0001 ) , compared to other genetic subtypes of hsp . patients with spg4 were significantly less likely to have learning disability compared to those with mutations in other genes ( or 0.06 , 95% ci 0.010.51 , p = 0.0096 ) . multiple linear regression analysis revealed that spg3a patients had better functional outcomes with a mean lower sprs score of 11.2 ( se 5.3 ) compared to those with mutations in other genes ( p = 0.04 ) , after adjusting for genotype , disease duration , and brain mri status . abnormal brain mri was associated with a mean higher total sprs score of 10.4 ( se 4.0 ) and worse functional outcomes compared with patients with normal brain mri ( p = 0.014 ) . of the 526 patients who met the inclusion criteria , 150 ( 28.5% ) patients from 58 families were confirmed to have a genetic diagnosis . the most frequent were spast ( spg4 , 72 patients , 48% ) , atl1 ( spg3a , 24 patients , 16% ) , spg11 ( 12 patients , 8% ) , spg7 ( 10 patients , 7% ) , and kiaa0196 ( spg8 , 8 patients , 5% ) . a total of 26 novel mutations were found in our cohort ; they were absent in publically available databases ( single nucleotide polymorphism database , 1000 genomes project , and exome aggregation consortium ) and were predicted to be pathogenic when evaluated using standard prediction programs ( sift , polyphen , and mutation taster ) . spg4 and spg3a were the most frequent autosomal dominant hereditary spastic paraplegia ( hsp ) , while spg11 followed by spg7 were the most frequent autosomal recessive hsp . a positive family history ( first - degree family members with symptoms of hsp ) the mean age at evaluation of our cohort was 39.2 years ( sd 22.1 , range 182 years ) , and the mean age at onset of symptoms was 18.9 years ( sd 18.6 , range 067 years ) . the most common clinical complaints , in addition to the core features of spasticity , weakness , and urinary disturbance , were dysarthria ( 33% ) , sensory abnormalities ( 35% ) , motor delay ( 24% ) , peripheral neuropathy ( 23% ) , speech delay ( 18% ) , and ataxia ( 17% ) . the most common abnormalities reported were thin corpus callosum ( 6 patients , 10% ) , followed by cerebellar atrophy ( 5 patients , 8% ) , and white matter abnormalities ( 4 patients , 7% ) . the mean sprs for the subset of our cohort for which this was available ( 48 patients ) was 19 ( sd 11 , range 239 ) , and the majority of patients ( 48/65 , 74% ) had spatax - eurospa disability scores within the mild to moderate range . there was a clear association between abnormal brain mri and disability as measured by the sprs , as patients with abnormal brain mri had a mean higher sprs score of 10.49 compared to those with normal brain imaging ( p = 0.0037 ) . linear regression analysis with bonferroni correction revealed that patients with spg4 and spg7 were more likely to develop symptoms later compared to other hsp subtypes , with a mean age at symptom onset of 24.6 ( standard error [ se ] 3.2 ) years for spg4 ( p = 0.0017 ) and 33 ( se 6.0 ) years for spg7 ( p = 0.013 ) . patients with spg3a were more likely to present at a younger age , with a mean age at symptom onset of 10.1 ( se 4.4 ) years ( p = 0.022 ) ( figure 3 ) . spg4 appeared to be associated with greater risk of bladder dysfunction ( odds ratio ( or ) 2.24 , 95% confidence interval ( ci ) 1.034.86 , p = 0.04 ) , and patients with spg3a less likely to develop bladder dysfunction ( or 0.30 , 95% ci 0.090.99 , p = 0.04 ) , but these observations were not statistically significant following bonferroni correction . patients with spg4 and spg7 were more likely to develop symptoms later compared to other hereditary spastic paraplegia subtypes , while patients with spg3a were more likely to present at a younger age . similarly , spg11 appeared to be associated with increased pain due to hsp - related symptoms compared to other genetic diagnoses ( or 5.44 , 95% ci 1.0827.37 , p = 0.039 ) , but this was not statistically significant following bonferroni correction . spg2 appeared to be associated with an increased risk of speech delay ( or 18.33 , 95% ci 1.71196.19 , p = 0.016 ) and motor delay ( or 11.8 , 95% ci 1.14122.1 , p = 0.04 ) , and spg4 less likely to have motor delay ( or 0.10 , 95% ci 0.010.78 , p = 0.028 ) , but these observations were no longer statistically significant after bonferroni correction . spg11 was strongly associated with an increased risk of learning disability ( or 30.59 , 95% ci 3.58261.19 , p = 0.0018 ) and progressive cognitive deficits ( or 87.75 , 95% ci 14.04548.24 , p < 0.0001 ) , compared to other genetic subtypes of hsp . patients with spg4 were significantly less likely to have learning disability compared to those with mutations in other genes ( or 0.06 , 95% ci 0.010.51 , p = 0.0096 ) . multiple linear regression analysis revealed that spg3a patients had better functional outcomes with a mean lower sprs score of 11.2 ( se 5.3 ) compared to those with mutations in other genes ( p = 0.04 ) , after adjusting for genotype , disease duration , and brain mri status . abnormal brain mri was associated with a mean higher total sprs score of 10.4 ( se 4.0 ) and worse functional outcomes compared with patients with normal brain mri ( p = 0.014 ) . we conducted a cross - sectional study of a large , ethnically diverse cohort of patients with hsp in canada using standardized clinical assessments and specifically assessed functional outcomes using validated rating scales . as in previously published population - based studies , spg4 and spg3a were the most frequent ad - hsp . the primary analysis of the exome data was focused on known hsp genes ; however , mutations were also found in genes not typically considered hsp genes in patients who met the clinical criteria for hsp ( 1 patient with a mutation in slc16a2 , 3 patients with mutations in vamp1 , and 4 patients from a single family with mutations in slc2a1 ) . in contrast to comparable neurodegenerative disorders such as the scas , very little is known about functional outcomes in patients with hsp , and the majority of studies are focused exclusively on spg4 and spg3a . as the majority of patients in our cohort who met the clinical criteria for hsp did not have a genetic diagnosis , this illustrates the need for studies of clinical parameters which influence prognosis and outcomes for these patients . in our cohort , patients with spg4 and spg3a had less severe disability scores compared with other hsp genetic subtypes , and patients with spg11 and spg2 had more severe scores , although the samples sizes were too small for this observation to reach statistical significance . bladder dysfunction is a recurrent and disabling symptom , which was present in a large proportion of our cohort , with the exception of patients with spg3a , which may contribute to the better functional outcomes observed in this subgroup of patients . the diagnostic utility of brain imaging to identify specific features of genetic subtypes of hsp such as spg2 , spg7 , spg11 , or spg35 is well established . they also demonstrate prominent cognitive impairment and learning disabilities , and some patients with spg11 mutations present with a very rare phenotype ( kjellin syndrome ) where mental retardation is a predominant feature . the clinical spectrum associated with spg7 mutations is variable , but specific brain mri findings such as cerebellar atrophy is common , which is consistent with ataxia being the first clinical symptom in many of these patients . one major strength of our study was the inclusion of both adult and pediatric patients , which enabled accurate assessment of age at symptom onset , and other clinical parameters essential for delineating the natural history of these disorders . our study was limited by the relatively small sample sizes of the genetic subtypes of hsp , with the exception of spg3a , spg4 , spg11 , and spg7 . large prospective studies of well - phenotyped patients are required to define the natural history of the various hsp subtypes and to design appropriate clinical trials of therapies for these complex disorders . hsp is usually not a life - limiting disorder but can be associated with serious disability , and accurate genetic diagnosis , genetic counseling , and management of symptoms are essential to optimizing care for patients with hsp and their families . spg11 is strongly associated with abnormal brain mri but also with prominent learning disabilities , progressive cognitive deficits , and poor functional outcomes . brain mri is the most useful tool to predict functional outcomes and disabilities and should always be integrated in the clinical evaluation of any patient with suspected hsp . accurate molecular characterization of well - phenotyped cohorts and international collaboration to increase the power of future studies will be critical to establish the natural history of these rare degenerative disorders to enable future clinical trials . suchowersky has received travel funding / speaker honoraria from the american academy of neurology ; has served on the editorial boards of the canadian journal of neurological sciences and nature reviews neurology ; has received publishing royalties from uptodate for chapters on diagnosis of huntington disease , treatment of huntington disease , and diagnosis of chorea ( and for yearly ongoing updates on these 3 chapters ) ; has served on the advisory boards of abbott ( abbvie ) and ipsen ; has received research support from cihr , nih , chdi , the toupin foundation , and abbvie pharmaceuticals ; has acted as a consultant for abbvie ; and is supported by the toupin research chair in neurology , university of alberta .

information for this review article was derived from the extensive one health community records of the ohc and the ohi autonomous pro bono team , one health web searches conducted in the fall of 2016 , and the professional contacts and experiences of the authors . both the ohc ( since 2009 ) and ohi ( since 2006 ) have worked in global one health endeavors for more than 10 years and are currently integrated into a wide network of one health stakeholders . furthermore , a call for information was disseminated to the ohc global one health community listserv . participants were asked to populate an online google document with information on one health training and research activities ( entries were verified and one health activities were summarized ) . some of the most noteworthy programs are highlighted in this article , and additional programs and courses are listed in table 1 . the authors learn new one health activities almost daily . full text descriptions of all one health activities in north america would markedly exceed this journal 's word count limits . academic one health training , research , and outreach activities in north america , stroud , usa , 2016 a version of this table that includes url links active at the time of publication is available as an online supplementary file . ohc , one health commission ; ohi , one health initiative ; mph , masters of public health ; mhs , master of health sciences , lmu - cvm , lincoln memorial university college of veterinary medicine . the authors acknowledge that there are likely additional one health training , research , and outreach efforts in north america that were inadvertently missed in this review . readers are encouraged to share additional information on their one health activities in north america with the corresponding author to update this listing and expand future summaries . as discussed earlier , the more traditional setting for one health training and research is in academia , but non - academic players also recognize the growing interest in one health and have responded enthusiastically . non - academic one health leadership activities , including those initiated by state and national governmental agencies , are listed in table 2 . location of known academic one health training , research , and outreach programs in north america as of august 2016 . blue stars denote universities that offer formal one health , academic credit - earning programs , including undergraduate majors / minors as well as certificate , masters , or phd programs . gray stars denote universities with no academic credit - earning program but with other significant one health training , research , or outreach activities . non - academic one health training , research , and outreach activities in north america , stroud , usa , 2016 a version of this table that includes url links current at time of publication is available as a supplementary file . ohitf , one health initiative task force ; ohc , one health commission ; cdc , centers for disease control and prevention ; usaid , us agency for international development ; usda , us department of agriculture ; aphis , animal & plant health inspection service ; umn , university of minnesota . thirteen institutions in north america offer major / minor , certificate , masters , or phd programs grounded in the one health concept . the ross university college of veterinary medicine offers a master of science ( msc ) one health degree ; the virginia - maryland college of veterinary medicine offers a masters of public health ( mph ) grounded in an interdisciplinary one health approach ; and tufts university offers a masters in conservation medicine ( mcm ) that uses a one health approach . cornell university will offer an mph degree with a focus on epidemiology , food systems , infectious disease , and sustainability starting in fall 2017 . the university of florida offers a master of health sciences ( mhs ) in one health as well as what is believed to be the first one health phd program created and developed originally by one of the coauthors of this article ( g.c.g . ) . the university of washington offers its mph students a training program in occupational health at the human - animal interface ( ohhai ) that promotes a one health approach . the university of tennessee operates a joint dvm - mph program that integrates one health concepts into its curriculum . similarly , two university of illinois campuses , chicago and urbana - champaign , have made a partnership to operate a joint doctor of veterinary medicine ( dvm)-mph . at the undergraduate level , the chicago campus also offers a bachelor of public health degree with a one health theme , and the university of california , davis offers a global disease biology major . fontbonne university offers an undergraduate one health certificate , and in canada , the university of saskatchewan offers a graduate certificate in one health . while relatively few academic institutions operate designated degree or certificate programs , many have one health focused degrees , programs , groups , or initiatives . due to space reason , in this article we limit this discussion to only the largest programs ( listed in alphabetical order ) with additional institutions known to provide significant one health training listed in table 1 . duke university is home to the duke one health training program and duke one health research team . during the last 9 years , this one health training program ( initially offered at iowa state university and then at the university of florida ) has trained three cohorts of one health trainees . the program involves 3.5 weeks of intense graduate training in one health ( four courses ) and often engages international professional and graduate students from 10 or more countries ( 26 ) . duke faculty , along with faculty from ncsu , the unc , and other institutions in the raleigh - durham - chapel hill research triangle park area , participate in the nc ohc . since 2010 , this group has overseen a one health intellectual exchange group discussion series at the north carolina biotechnology center and an interinstitutional course called one health : philosophy to practical integration , cross listed at duke university , ncsu , and unc and offered to area graduate and undergraduate students . duke university also partners with duke - national university of singapore ( nus ) medical university singapore and duke kunshan university , china to exchange graduate students and professionals conducting one health training and research ( 27 ) . duke university has a national institute of health ( nih)-funded d43 training program for teams of us and mongolian professionals to conduct 2 years of one health zoonotic disease research . finally , duke university is conducting nih - funded r01 of zoonotic influenza transmission with multiple research institution partners in china ( 28 ) . ohio state university 's ( osu ) recently retired dean of the college of veterinary medicine ( cvm ) , dr . lonnie j. king [ dvm , ms , mpa ] has been an extraordinary one health leader . after chairing the 20072008 ohitf , dr . king was instrumental in taking the one health conversation into us national arenas . among his various efforts , one was organizing one health workshops in the national academy of sciences ( 29 ) . under his leadership , the osu cvm expanded programs that take an integrated approach to addressing animal , environmental , and human health , and it offers a mph with a veterinary public health specialization ( 30 ) . individuals and colleges outside the cvm have also expanded their oh research , training , and outreach activities . ohio state faculty and staff have created itunes u courses related to their one health projects ( 31 ) . an osu student organization , buckeyes without borders , was formed in 2009 to bring together graduate students from a wide range of disciplines , including audiology , dentistry , medicine , nursing , occupational therapy , optometry , pharmacy , physical therapy , public health , social work , speech language pathology , and veterinary medicine ( 32 ) . the ohio state global ohi was launched in 2009 , and it partnered with ohio state 's seven health sciences colleges with 19 institutes in ethiopia and the rest in the united states . training students ( from ethiopia and the united states ) in one health summer institutes is one of the osu health sciences - ethiopia partnership 's priority areas to build capacity and strengthen collaboration ( 33 ) . the initiative has brought in additional partners and begun to extend its reach into eastern africa , brazil , and southeast asia ( 34 ) . osu 's efforts have involved not only students but also the broader one health community . in 2015 , osu was a co - organizer of the third international congress on pathogens at the human animal interface ( icophai ) ( 35 ) . the osu extension division organizes one health conferences where public health officials , veterinarians , and extension workers can learn about and discuss issues relevant to public , veterinary medical , and environmental health ( 36 ) . the texas a&m university ( tamu ) ohi was started in 2011 by the deans and faculty of the college of veterinary medicine & biomedical sciences and the college of medicine ( 37 ) . the initiative provides a one health learning community for undergraduate students aimed at introducing the one health concept to students early in their educational careers . for faculty , a tamu one health grand challenge funding program was initiated in 2014 ; four major oh research themes were identified , and a plan was implemented to bring faculty from across tamu into collaborative , inter-/transdisciplinary research partnerships . information on some of these partnerships , along with early outcomes , can be found on the tamu website ( 38 ) . the tamu one health on - campus summer research program provides an opportunity for two professional and/or graduate students to participate in a 13-week hands - on research program at the university . one health student travel grants are awarded to selected students to present one health research at scientific conferences . the tamu oh program oversees a one health seminar series ( 39 ) and has implemented outreach programs in nicaragua and china . a final opportunity for one health outreach at tamu is the student one health association ( soha ) , which is open to all undergraduate , graduate , and professional students ( 40 ) . the university of california , davis ( uc davis ) one health institute was created in 2009 as a result of a strong commitment to the one health approach ( 41 ) . the institute leads the predict project which is a part of the usaid ept program . in partnership with usaid , wcs , ecohealth alliance , metabiota , and the smithsonian institution , predict researchers contribute to global surveillance efforts to detect pathogens of pandemic potential and prevent spillover between wildlife and people ( 42 ) . uc davis wildlife health center operates the ohi lab , which serves as a primary research facility for predict ( 43 ) . the one health institute leads the health for animals and livelihood improvement ( hali ) project , a collaborative , capacity - building program that brings together researchers from the united states and tanzania to study the effects of zoonotic diseases and water management practices on individuals living within tanzania 's ruaha ecosystem ( 44 ) . uc davis is also home to the calvin schwabe project , which has been named in honor of the former uc davis school of veterinary medicine professor ( calvin schwabe ) who coined the term the calvin schwabe project offers a wide range of oh opportunities to veterinary students , encouraging them to approach their future careers with an appreciation of the interconnectedness of animal , environmental , and human health ( 45 ) . outside the cvm , the uc davis global health department program offers one health graduate and undergraduate seminars , as well as one health in action , an intensive , 4-week field course , teaching students to apply the one health approach to complex problems ( 46 ) . uc davis has extended its one health activities to include students within the larger university of california system . the uc global health institute ( ucghi ) was established as a partnership between the uc davis and uc riverside , but its programs and opportunities are available to students at any of the 10 uc campuses ( 47 ) . one of the three centers of expertise initially established within ucghi was a one health center , specializing in research into problems that occur at the human - water - animal - food interface ( 47 , 48 ) . the center offers oh student research fellowships to both graduate and professional students interested in integrating a one health approach into their global health research efforts ( 49 ) . the university of florida 's emerging pathogens institute is home to its one health center of excellence for research and training ( which was first organized by one of the coauthors of this article ( g.c.g ) . the center oversees the quarterly online one health newsletter , which publishes one health papers as well as event(s ) details , to subscribers from a wide range of disciplines ( 50 ) . the university of florida s department of environmental and global health launched two degree programs that immerse trainees in the one health concept : an mhs , one health concentration , and a phd of public health , one health concentration , as mentioned previously ( 5153 ) . the summer training program now offered by duke university was first operated as a certificate program at the university of florida under the leadership of dr . the university of pennsylvania 's school of veterinary medicine ( penn vet ) has had a long established and notable one health program , and one health education continues to be a high priority under the current dean , joan c. hendricks [ vmd , phd ] ( 54 ) . the relationship between animal health and human health is the core of its teaching tradition so that students can learn first - hand how veterinary medicine and research impacts human lives as well as the lives of animals . in 2015 , in partnership with the pennsylvania farm bureau , a commonwealth one health scholarship was initiated , providing a full tuition subsidy for 4 years of veterinary school at penn vet . the first two recipients demonstrated a strong commitment to food animal medicine . for faculty , deans of the four health schools at the university of pennsylvania the perelman school of medicine , the school of nursing science , the school of dental medicine , and the school of veterinary medicine annually present a one health award for significant , collaborative research conducted by faculty , highlighting those engaged in professional education bridging two or more of the schools with outreach and innovation in training and service in clinics or to the community . the university of saskatchewan sponsors a 3-day one health leadership experience each fall , where as many as 200 professionals from multiple disciplines meet to discuss employing one health in modern complex problems settings . the university of saskatchewan also hosts a one health research development grants competition that has funded nine projects in 2 years ( 56 ) . students for one health ( soh ) groups have formed all over the world to further one health education and awareness , often forging paths at their universities and educating academic faculty . they implement local discussions on one health topics , and some go into local communities to hold one health they are extremely comfortable in interacting with students studying in disciplines other than their own . it is very important to give students the chance to form relationships across disciplines very early in their training as these relationships will follow them throughout their careers , increasing their future comfort for working across professions . realizing that these students are the next generation of one health leaders , the ohc supports any student group working for one health by providing an soh web page , who 's who in soh page , an soh listserv ( 57 ) , and an online meeting platform so that they can connect with each other . soh groups that we are aware of are included at the bottom of table 1 . soh groups that we are not yet aware of are encouraged to share information about their groups and their one health activities . as mentioned earlier , one health education is not confined to a traditional academic setting . as interest in , and the need for , taking a multidisciplinary or transdisciplinary approach to addressing complex problems has grown , visionary researchers and health professionals have sought to integrate one health approaches into their work . many non - academic organizations discussed below ( and additional ones listed in table 2 ) , including for - profit corporations , have embraced one health and are actively leading one health training and research . the association of american veterinary medical colleges ( aavmc ) maintains and supports a standing one health committee and hosted in 2015 a competition of one health case studies , illustrating applications of one health approaches . these studies , along with facilitator materials , are available online and free to use for teaching in many settings ( 58 ) . they are a part of aavmc 's one health interprofessional education initiative , an effort to integrate one health training into veterinary degree programs around the united states . the aavmc demonstrated its commitment to educating about the concept by making one health the focus of its 2014 annual conference , entitled the canadian veterinary medical association ( cvma ) has demonstrated its commitment to promoting one health by making the theme of its 2016 animal health week the georgia aquarium leads a one ocean , one health initiative , making a commitment to recognize the interconnectedness of human , animal , and environmental well - being and not study animals in isolation ( 61 ) . one of its research and conservation goals is to build the one ocean one health research institute in marineland , florida , which could establish itself as a pioneer of the one health concept in ocean science ( 62 ) . the kansas city area life sciences institute has led multiple one health conferences , including a 2016 one health innovations symposium that was hosted in partnership with the schools of veterinary medicine at university of missouri and kansas state university . this 2-day symposium focused on the translational and comparative aspects of human and animal medicine ( 63 ) . the one health academy , founded in 2006 in washington , dc , oversees monthly gatherings of health professionals and government officials interested in dialogue , social engagement , learning , and collaboration . by offering networking opportunities , educational presentations and opportunities for discussion , networking and mentoring , these events allow federal , non - profit , clinical , and private industry leaders to interact directly and form relationships that often lead to collaborations ( 64 ) . the one health commission , created in 2009 as a result of recommendations by the ohitf , is a globally focused 501(c)(3 ) non - profit organization that seeks to connect one health advocates , to create networks and teams that can work together across disciplines , and to educate about one health and one health issues ( 63 ) . based in the research triangle park region of north carolina , usa , the commission is governed by a board of directors comprising representatives from organizations that fund its work . it serves as an action arm for one health , a gateway for active education and exchange of knowledge , sharing of resources , and collaborative projects that reach beyond disciplinary boundaries . its volunteer one health action teams identify knowledge and education gaps and take actions to address them . to encourage the next generation of one health leaderst , the ohc supports soh groups and includes elected student representatives on its board of directors ( 65 ) . the one health initiative autonomous pro bono team ( see non - academic one health training , research , and outreach section ) . the zoobiquity initiative and conferences were started by barbara natterson - horowitz , md , a professor of medicine in the ucla division of cardiology at the david geffen school of medicine , who noticed the many parallels between human and animal health conditions and set out to raise awareness among her colleagues . in 2011 , dr . natterson - horowitz founded the ongoing annual zoobiquity conference series to bring together human and animal medicine leaders for direct discussions about how to diagnose and treat the conditions that afflict animals and humans alike . in 2012 , dr . natterson - horowitz and coauthor kathryn bowers published their book zoobiquity in which they highlighted the many conditions that plague both humans and animals and advocated for a cross - species approach to health care . the zoobiquity research initiative also started a program that brings together ucla medical students and uc davis veterinary students for discussions of mutual health care interest ( 66 ) . this valuable project highlights and promotes many collaborative , comparative medicine / translational medicine research achievements over previous decades . as evidenced from table 1 and the aforementioned discussions , a number of training programs in north america provide opportunities for one health research . students and investigators seeking research funding and other opportunities may look to the ohc website which maintains a list of funding and grant opportunities . the commission also requests that the one health community send future opportunities to be posted and shared on this web page ( 67 ) . funding for one health research has often come from the us government . as of september 2016 , a search of the grants.gov website with the keyword the usaid has also devoted financial and human resources to oh research in training ; its ept programs 1 and 2 have awarded grants to investigators to build a one health workforce in developing countries that are most threatened by emerging diseases with animal , environmental , and human health implications . one health research also receives non - governmental support in the united states : the gates foundation and several universities have sponsored significant one health research . the canadian government has demonstrated its support for one health . one health researchers have been successful in applying for grants offered specifically for collaborative / interdisciplinary work . the collaborative health research projects ( chrps ) , operated jointly by the natural sciences and engineering research council of canada ( nserc ) and canadian institutes of health research ( cihr ) , have provided research funding to programs such as the one health certificate program at the university of saskatchewan . government agencies of canada are themselves embracing the one health concept in their programs and research projects : the public health agency of canada claims to be pursuing activities that are consistent with a one health approach , and its centre for food - borne , environmental and zoonotic infectious diseases ( cfezid ) hosted the one world one health expert consultation in winnipeg , manitoba in 2009 ( 69 , 70 ) . among the agency 's one health - consistent activities are the canadian integrated program for antimicrobial resistance surveillance ( cipars ) , which takes a one health approach to combat amr that spans human , animal , agricultural , food , and environmental sectors , and foodnet , canada , a comprehensive sentinel site surveillance system ( 71 ) . thirteen institutions in north america offer major / minor , certificate , masters , or phd programs grounded in the one health concept . the ross university college of veterinary medicine offers a master of science ( msc ) one health degree ; the virginia - maryland college of veterinary medicine offers a masters of public health ( mph ) grounded in an interdisciplinary one health approach ; and tufts university offers a masters in conservation medicine ( mcm ) that uses a one health approach . cornell university will offer an mph degree with a focus on epidemiology , food systems , infectious disease , and sustainability starting in fall 2017 . the university of florida offers a master of health sciences ( mhs ) in one health as well as what is believed to be the first one health phd program created and developed originally by one of the coauthors of this article ( g.c.g . ) . the university of washington offers its mph students a training program in occupational health at the human - animal interface ( ohhai ) that promotes a one health approach . the university of tennessee operates a joint dvm - mph program that integrates one health concepts into its curriculum . similarly , two university of illinois campuses , chicago and urbana - champaign , have made a partnership to operate a joint doctor of veterinary medicine ( dvm)-mph . at the undergraduate level , the chicago campus also offers a bachelor of public health degree with a one health theme , and the university of california , davis offers a global disease biology major . fontbonne university offers an undergraduate one health certificate , and in canada , the university of saskatchewan offers a graduate certificate in one health . while relatively few academic institutions operate designated degree or certificate programs , many have one health focused degrees , programs , groups , or initiatives . due to space reason , in this article we limit this discussion to only the largest programs ( listed in alphabetical order ) with additional institutions known to provide significant one health training listed in table 1 . duke university is home to the duke one health training program and duke one health research team . during the last 9 years , this one health training program ( initially offered at iowa state university and then at the university of florida ) has trained three cohorts of one health trainees . the program involves 3.5 weeks of intense graduate training in one health ( four courses ) and often engages international professional and graduate students from 10 or more countries ( 26 ) . duke faculty , along with faculty from ncsu , the unc , and other institutions in the raleigh - durham - chapel hill research triangle park area , participate in the nc ohc . since 2010 , this group has overseen a one health intellectual exchange group discussion series at the north carolina biotechnology center and an interinstitutional course called one health : philosophy to practical integration , cross listed at duke university , ncsu , and unc and offered to area graduate and undergraduate students . duke university also partners with duke - national university of singapore ( nus ) medical university singapore and duke kunshan university , china to exchange graduate students and professionals conducting one health training and research ( 27 ) . duke university has a national institute of health ( nih)-funded d43 training program for teams of us and mongolian professionals to conduct 2 years of one health zoonotic disease research . finally , duke university is conducting nih - funded r01 of zoonotic influenza transmission with multiple research institution partners in china ( 28 ) . ohio state university 's ( osu ) recently retired dean of the college of veterinary medicine ( cvm ) , dr . lonnie j. king [ dvm , ms , mpa ] has been an extraordinary one health leader . after chairing the 20072008 ohitf , dr . king was instrumental in taking the one health conversation into us national arenas . among his various efforts , one was organizing one health workshops in the national academy of sciences ( 29 ) . under his leadership , the osu cvm expanded programs that take an integrated approach to addressing animal , environmental , and human health , and it offers a mph with a veterinary public health specialization ( 30 ) . individuals and colleges outside the cvm have also expanded their oh research , training , and outreach activities . ohio state faculty and staff have created itunes u courses related to their one health projects ( 31 ) . an osu student organization , buckeyes without borders , was formed in 2009 to bring together graduate students from a wide range of disciplines , including audiology , dentistry , medicine , nursing , occupational therapy , optometry , pharmacy , physical therapy , public health , social work , speech language pathology , and veterinary medicine ( 32 ) . the ohio state global ohi was launched in 2009 , and it partnered with ohio state 's seven health sciences colleges with 19 institutes in ethiopia and the rest in the united states . training students ( from ethiopia and the united states ) in one health summer institutes is one of the osu health sciences - ethiopia partnership 's priority areas to build capacity and strengthen collaboration ( 33 ) . the initiative has brought in additional partners and begun to extend its reach into eastern africa , brazil , and southeast asia ( 34 ) . osu 's efforts have involved not only students but also the broader one health community . in 2015 , osu was a co - organizer of the third international congress on pathogens at the human animal interface ( icophai ) ( 35 ) . the osu extension division organizes one health conferences where public health officials , veterinarians , and extension workers can learn about and discuss issues relevant to public , veterinary medical , and environmental health ( 36 ) . the texas a&m university ( tamu ) ohi was started in 2011 by the deans and faculty of the college of veterinary medicine & biomedical sciences and the college of medicine ( 37 ) . the initiative provides a one health learning community for undergraduate students aimed at introducing the one health concept to students early in their educational careers . for faculty , a tamu one health grand challenge funding program was initiated in 2014 ; four major oh research themes were identified , and a plan was implemented to bring faculty from across tamu into collaborative , inter-/transdisciplinary research partnerships . information on some of these partnerships , along with early outcomes , can be found on the tamu website ( 38 ) . the tamu one health on - campus summer research program provides an opportunity for two professional and/or graduate students to participate in a 13-week hands - on research program at the university . one health student travel grants are awarded to selected students to present one health research at scientific conferences . the tamu oh program oversees a one health seminar series ( 39 ) and has implemented outreach programs in nicaragua and china . a final opportunity for one health outreach at tamu is the student one health association ( soha ) , which is open to all undergraduate , graduate , and professional students ( 40 ) . the university of california , davis ( uc davis ) one health institute was created in 2009 as a result of a strong commitment to the one health approach ( 41 ) . the institute leads the predict project which is a part of the usaid ept program . in partnership with usaid , wcs , ecohealth alliance , metabiota , and the smithsonian institution , predict researchers contribute to global surveillance efforts to detect pathogens of pandemic potential and prevent spillover between wildlife and people ( 42 ) . uc davis wildlife health center operates the ohi lab , which serves as a primary research facility for predict ( 43 ) . the one health institute leads the health for animals and livelihood improvement ( hali ) project , a collaborative , capacity - building program that brings together researchers from the united states and tanzania to study the effects of zoonotic diseases and water management practices on individuals living within tanzania 's ruaha ecosystem ( 44 ) . uc davis is also home to the calvin schwabe project , which has been named in honor of the former uc davis school of veterinary medicine professor ( calvin schwabe ) who coined the term one medicine ( 40 ) . the calvin schwabe project offers a wide range of oh opportunities to veterinary students , encouraging them to approach their future careers with an appreciation of the interconnectedness of animal , environmental , and human health ( 45 ) . outside the cvm , the uc davis global health department program offers one health graduate and undergraduate seminars , as well as one health in action , an intensive , 4-week field course , teaching students to apply the one health approach to complex problems ( 46 ) . uc davis has extended its one health activities to include students within the larger university of california system . the uc global health institute ( ucghi ) was established as a partnership between the uc davis and uc riverside , but its programs and opportunities are available to students at any of the 10 uc campuses ( 47 ) . centers of expertise initially established within ucghi was a one health center , specializing in research into problems that occur at the human - water - animal - food interface ( 47 , 48 ) . the center offers oh student research fellowships to both graduate and professional students interested in integrating a one health approach into their global health research efforts ( 49 ) . the university of florida 's emerging pathogens institute is home to its one health center of excellence for research and training ( which was first organized by one of the coauthors of this article ( g.c.g ) . the center oversees the quarterly online one health newsletter , which publishes one health papers as well as event(s ) details , to subscribers from a wide range of disciplines ( 50 ) . the university of florida s department of environmental and global health launched two degree programs that immerse trainees in the one health concept : an mhs , one health concentration , and a phd of public health , one health concentration , as mentioned previously ( 5153 ) . the summer training program now offered by duke university was first operated as a certificate program at the university of florida under the leadership of dr . the university of pennsylvania 's school of veterinary medicine ( penn vet ) has had a long established and notable one health program , and one health education continues to be a high priority under the current dean , joan c. hendricks [ vmd , phd ] ( 54 ) . the relationship between animal health and human health is the core of its teaching tradition so that students can learn first - hand how veterinary medicine and research impacts human lives as well as the lives of animals . in 2015 , in partnership with the pennsylvania farm bureau , a commonwealth one health scholarship was initiated , providing a full tuition subsidy for 4 years of veterinary school at penn vet . for faculty , deans of the four health schools at the university of pennsylvania the perelman school of medicine , the school of nursing science , the school of dental medicine , and the school of veterinary medicine annually present a one health award for significant , collaborative research conducted by faculty , highlighting those engaged in professional education bridging two or more of the schools with outreach and innovation in training and service in clinics or to the community . the university of saskatchewan sponsors a 3-day one health leadership experience each fall , where as many as 200 professionals from multiple disciplines meet to discuss employing one health in modern complex problems settings . the university of saskatchewan also hosts a one health research development grants competition that has funded nine projects in 2 years ( 56 ) . students for one health ( soh ) groups have formed all over the world to further one health education and awareness , often forging paths at their universities and educating academic faculty . they implement local discussions on one health topics , and some go into local communities to hold one health they are extremely comfortable in interacting with students studying in disciplines other than their own . it is very important to give students the chance to form relationships across disciplines very early in their training as these relationships will follow them throughout their careers , increasing their future comfort for working across professions . realizing that these students are the next generation of one health leaders , the ohc supports any student group working for one health by providing an soh web page , who 's who in soh page , an soh listserv ( 57 ) , and an online meeting platform so that they can connect with each other . soh groups that we are aware of are included at the bottom of table 1 . soh groups that we are not yet aware of are encouraged to share information about their groups and their one health activities . as mentioned earlier , as interest in , and the need for , taking a multidisciplinary or transdisciplinary approach to addressing complex problems has grown , visionary researchers and health professionals have sought to integrate one health approaches into their work . many non - academic organizations discussed below ( and additional ones listed in table 2 ) , including for - profit corporations , have embraced one health and are actively leading one health training and research . the association of american veterinary medical colleges ( aavmc ) maintains and supports a standing one health committee and hosted in 2015 a competition of one health case studies , illustrating applications of one health approaches . these studies , along with facilitator materials , are available online and free to use for teaching in many settings ( 58 ) . they are a part of aavmc 's one health interprofessional education initiative , an effort to integrate one health training into veterinary degree programs around the united states . the aavmc demonstrated its commitment to educating about the concept by making one health the focus of its 2014 annual conference , entitled one health in veterinary medical education ( 59 ) . national us and canada veterinary medical associations actively support the one health concept . the canadian veterinary medical association ( cvma ) has demonstrated its commitment to promoting one health by making the theme of its 2016 animal health week the georgia aquarium leads a one ocean , one health initiative , making a commitment to recognize the interconnectedness of human , animal , and environmental well - being and not study animals in isolation ( 61 ) . one of its research and conservation goals is to build the one ocean one health research institute in marineland , florida , which could establish itself as a pioneer of the one health concept in ocean science ( 62 ) . the kansas city area life sciences institute has led multiple one health conferences , including a 2016 one health innovations symposium that was hosted in partnership with the schools of veterinary medicine at university of missouri and kansas state university . this 2-day symposium focused on the translational and comparative aspects of human and animal medicine ( 63 ) . the one health academy , founded in 2006 in washington , dc , oversees monthly gatherings of health professionals and government officials interested in dialogue , social engagement , learning , and collaboration . by offering networking opportunities , educational presentations and opportunities for discussion , networking and mentoring , these events allow federal , non - profit , clinical , and private industry leaders to interact directly and form relationships that often lead to collaborations ( 64 ) . the one health commission , created in 2009 as a result of recommendations by the ohitf , is a globally focused 501(c)(3 ) non - profit organization that seeks to connect one health advocates , to create networks and teams that can work together across disciplines , and to educate about one health and one health issues ( 63 ) . based in the research triangle park region of north carolina , usa , the commission is governed by a board of directors comprising representatives from organizations that fund its work . it serves as an action arm for one health , a gateway for active education and exchange of knowledge , sharing of resources , and collaborative projects that reach beyond disciplinary boundaries . its volunteer one health action teams identify knowledge and education gaps and take actions to address them . to encourage the next generation of one health leaderst , the ohc supports soh groups and includes elected student representatives on its board of directors ( 65 ) . the one health initiative autonomous pro bono team ( see non - academic one health training , research , and outreach section ) . the zoobiquity initiative and conferences were started by barbara natterson - horowitz , md , a professor of medicine in the ucla division of cardiology at the david geffen school of medicine , who noticed the many parallels between human and animal health conditions and set out to raise awareness among her colleagues . in 2011 , natterson - horowitz founded the ongoing annual zoobiquity conference series to bring together human and animal medicine leaders for direct discussions about how to diagnose and treat the conditions that afflict animals and humans alike . in 2012 , dr . natterson - horowitz and coauthor kathryn bowers published their book zoobiquity in which they highlighted the many conditions that plague both humans and animals and advocated for a cross - species approach to health care . the zoobiquity research initiative also started a program that brings together ucla medical students and uc davis veterinary students for discussions of mutual health care interest ( 66 ) . this valuable project highlights and promotes many collaborative , comparative medicine / translational medicine research achievements over previous decades . thirteen institutions in north america offer major / minor , certificate , masters , or phd programs grounded in the one health concept . the ross university college of veterinary medicine offers a master of science ( msc ) one health degree ; the virginia - maryland college of veterinary medicine offers a masters of public health ( mph ) grounded in an interdisciplinary one health approach ; and tufts university offers a masters in conservation medicine ( mcm ) that uses a one health approach . cornell university will offer an mph degree with a focus on epidemiology , food systems , infectious disease , and sustainability starting in fall 2017 . the university of florida offers a master of health sciences ( mhs ) in one health as well as what is believed to be the first one health phd program created and developed originally by one of the coauthors of this article ( g.c.g . ) . the university of washington offers its mph students a training program in occupational health at the human - animal interface ( ohhai ) that promotes a one health approach . the university of tennessee operates a joint dvm - mph program that integrates one health concepts into its curriculum . similarly , two university of illinois campuses , chicago and urbana - champaign , have made a partnership to operate a joint doctor of veterinary medicine ( dvm)-mph . at the undergraduate level , the chicago campus also offers a bachelor of public health degree with a one health theme , and the university of california , davis offers a global disease biology major . fontbonne university offers an undergraduate one health certificate , and in canada , the university of saskatchewan offers a graduate certificate in one health . while relatively few academic institutions operate designated degree or certificate programs , many have one health focused degrees , programs , groups , or initiatives . due to space reason , in this article we limit this discussion to only the largest programs ( listed in alphabetical order ) with additional institutions known to provide significant one health training listed in table 1 . duke university is home to the duke one health training program and duke one health research team . during the last 9 years , this one health training program ( initially offered at iowa state university and then at the university of florida ) has trained three cohorts of one health trainees . the program involves 3.5 weeks of intense graduate training in one health ( four courses ) and often engages international professional and graduate students from 10 or more countries ( 26 ) . duke faculty , along with faculty from ncsu , the unc , and other institutions in the raleigh - durham - chapel hill research triangle park area , participate in the nc ohc . since 2010 , this group has overseen a one health intellectual exchange group discussion series at the north carolina biotechnology center and an interinstitutional course called one health : philosophy to practical integration , cross listed at duke university , ncsu , and unc and offered to area graduate and undergraduate students . duke university also partners with duke - national university of singapore ( nus ) medical university singapore and duke kunshan university , china to exchange graduate students and professionals conducting one health training and research ( 27 ) . duke university has a national institute of health ( nih)-funded d43 training program for teams of us and mongolian professionals to conduct 2 years of one health zoonotic disease research . finally , duke university is conducting nih - funded r01 of zoonotic influenza transmission with multiple research institution partners in china ( 28 ) . ohio state university 's ( osu ) recently retired dean of the college of veterinary medicine ( cvm ) , dr . lonnie j. king [ dvm , ms , mpa ] has been an extraordinary one health leader . after chairing the 20072008 ohitf , dr . king was instrumental in taking the one health conversation into us national arenas . among his various efforts , one was organizing one health workshops in the national academy of sciences ( 29 ) . under his leadership , the osu cvm expanded programs that take an integrated approach to addressing animal , environmental , and human health , and it offers a mph with a veterinary public health specialization ( 30 ) . individuals and colleges outside the cvm have also expanded their oh research , training , and outreach activities . ohio state faculty and staff have created itunes u courses related to their one health projects ( 31 ) . an osu student organization , buckeyes without borders , was formed in 2009 to bring together graduate students from a wide range of disciplines , including audiology , dentistry , medicine , nursing , occupational therapy , optometry , pharmacy , physical therapy , public health , social work , speech language pathology , and veterinary medicine ( 32 ) . the ohio state global ohi was launched in 2009 , and it partnered with ohio state 's seven health sciences colleges with 19 institutes in ethiopia and the rest in the united states . training students ( from ethiopia and the united states ) in one health summer institutes is one of the osu health sciences - ethiopia partnership 's priority areas to build capacity and strengthen collaboration ( 33 ) . the initiative has brought in additional partners and begun to extend its reach into eastern africa , brazil , and southeast asia ( 34 ) . osu 's efforts have involved not only students but also the broader one health community . in 2015 , osu was a co - organizer of the third international congress on pathogens at the human animal interface ( icophai ) ( 35 ) . the osu extension division organizes one health conferences where public health officials , veterinarians , and extension workers can learn about and discuss issues relevant to public , veterinary medical , and environmental health ( 36 ) . the texas a&m university ( tamu ) ohi was started in 2011 by the deans and faculty of the college of veterinary medicine & biomedical sciences and the college of medicine ( 37 ) . the initiative provides a one health learning community for undergraduate students aimed at introducing the one health concept to students early in their educational careers . for faculty , a tamu one health grand challenge funding program was initiated in 2014 ; four major oh research themes were identified , and a plan was implemented to bring faculty from across tamu into collaborative , inter-/transdisciplinary research partnerships . information on some of these partnerships , along with early outcomes , can be found on the tamu website ( 38 ) . the tamu one health on - campus summer research program provides an opportunity for two professional and/or graduate students to participate in a 13-week hands - on research program at the university . one health student travel grants are awarded to selected students to present one health research at scientific conferences . the tamu oh program oversees a one health seminar series ( 39 ) and has implemented outreach programs in nicaragua and china . a final opportunity for one health outreach at tamu is the student one health association ( soha ) , which is open to all undergraduate , graduate , and professional students ( 40 ) . the university of california , davis ( uc davis ) one health institute was created in 2009 as a result of a strong commitment to the one health approach ( 41 ) . the institute leads the predict project which is a part of the usaid ept program . in partnership with usaid , wcs , ecohealth alliance , metabiota , and the smithsonian institution , predict researchers contribute to global surveillance efforts to detect pathogens of pandemic potential and prevent spillover between wildlife and people ( 42 ) . uc davis wildlife health center operates the ohi lab , which serves as a primary research facility for predict ( 43 ) . the one health institute leads the health for animals and livelihood improvement ( hali ) project , a collaborative , capacity - building program that brings together researchers from the united states and tanzania to study the effects of zoonotic diseases and water management practices on individuals living within tanzania 's ruaha ecosystem ( 44 ) . uc davis is also home to the calvin schwabe project , which has been named in honor of the former uc davis school of veterinary medicine professor ( calvin schwabe ) who coined the term one medicine ( 40 ) . the calvin schwabe project offers a wide range of oh opportunities to veterinary students , encouraging them to approach their future careers with an appreciation of the interconnectedness of animal , environmental , and human health ( 45 ) . outside the cvm , the uc davis global health department program offers one health graduate and undergraduate seminars , as well as one health in action , an intensive , 4-week field course , teaching students to apply the one health approach to complex problems ( 46 ) . uc davis has extended its one health activities to include students within the larger university of california system . the uc global health institute ( ucghi ) was established as a partnership between the uc davis and uc riverside , but its programs and opportunities are available to students at any of the 10 uc campuses ( 47 ) . one of the three centers of expertise initially established within ucghi was a one health center , specializing in research into problems that occur at the human - water - animal - food interface ( 47 , 48 ) . the center offers oh student research fellowships to both graduate and professional students interested in integrating a one health approach into their global health research efforts ( 49 ) . the university of florida 's emerging pathogens institute is home to its one health center of excellence for research and training ( which was first organized by one of the coauthors of this article ( g.c.g ) . the center oversees the quarterly online one health newsletter , which publishes one health papers as well as event(s ) details , to subscribers from a wide range of disciplines ( 50 ) . the university of florida s department of environmental and global health launched two degree programs that immerse trainees in the one health concept : an mhs , one health concentration , and a phd of public health , one health concentration , as mentioned previously ( 5153 ) . the summer training program now offered by duke university was first operated as a certificate program at the university of florida under the leadership of dr . the university of pennsylvania 's school of veterinary medicine ( penn vet ) has had a long established and notable one health program , and one health education continues to be a high priority under the current dean , joan c. hendricks [ vmd , phd ] ( 54 ) . the relationship between animal health and human health is the core of its teaching tradition so that students can learn first - hand how veterinary medicine and research impacts human lives as well as the lives of animals . in 2015 , in partnership with the pennsylvania farm bureau , a commonwealth one health scholarship was initiated , providing a full tuition subsidy for 4 years of veterinary school at penn vet . for faculty , deans of the four health schools at the university of pennsylvania the perelman school of medicine , the school of nursing science , the school of dental medicine , and the school of veterinary medicine annually present a one health award for significant , collaborative research conducted by faculty , highlighting those engaged in professional education bridging two or more of the schools with outreach and innovation in training and service in clinics or to the community . the university of saskatchewan sponsors a 3-day one health leadership experience each fall , where as many as 200 professionals from multiple disciplines meet to discuss employing one health in modern complex problems settings . the university of saskatchewan also hosts a one health research development grants competition that has funded nine projects in 2 years ( 56 ) . students for one health ( soh ) groups have formed all over the world to further one health education and awareness , often forging paths at their universities and educating academic faculty . they implement local discussions on one health topics , and some go into local communities to hold one health they are extremely comfortable in interacting with students studying in disciplines other than their own . it is very important to give students the chance to form relationships across disciplines very early in their training as these relationships will follow them throughout their careers , increasing their future comfort for working across professions . realizing that these students are the next generation of one health leaders , the ohc supports any student group working for one health by providing an soh web page , who 's who in soh page , an soh listserv ( 57 ) , and an online meeting platform so that they can connect with each other . soh groups that we are aware of are included at the bottom of table 1 . soh groups that we are not yet aware of are encouraged to share information about their groups and their one health activities . as mentioned earlier , one health education is not confined to a traditional academic setting . as interest in , and the need for , taking a multidisciplinary or transdisciplinary approach to addressing complex problems has grown , visionary researchers and health professionals have sought to integrate one health approaches into their work . many non - academic organizations discussed below ( and additional ones listed in table 2 ) , including for - profit corporations , have embraced one health and are actively leading one health training and research . the association of american veterinary medical colleges ( aavmc ) maintains and supports a standing one health committee and hosted in 2015 a competition of one health case studies , illustrating applications of one health approaches . these studies , along with facilitator materials , are available online and free to use for teaching in many settings ( 58 ) . they are a part of aavmc 's one health interprofessional education initiative , an effort to integrate one health training into veterinary degree programs around the united states . the aavmc demonstrated its commitment to educating about the concept by making one health the focus of its 2014 annual conference , entitled the canadian veterinary medical association ( cvma ) has demonstrated its commitment to promoting one health by making the theme of its 2016 animal health week the georgia aquarium leads a one ocean , one health initiative , making a commitment to recognize the interconnectedness of human , animal , and environmental well - being and not study animals in isolation ( 61 ) . one of its research and conservation goals is to build the one ocean one health research institute in marineland , florida , which could establish itself as a pioneer of the one health concept in ocean science ( 62 ) . the kansas city area life sciences institute has led multiple one health conferences , including a 2016 one health innovations symposium that was hosted in partnership with the schools of veterinary medicine at university of missouri and kansas state university . this 2-day symposium focused on the translational and comparative aspects of human and animal medicine ( 63 ) . the one health academy , founded in 2006 in washington , dc , oversees monthly gatherings of health professionals and government officials interested in dialogue , social engagement , learning , and collaboration . by offering networking opportunities , educational presentations and opportunities for discussion , networking and mentoring , these events allow federal , non - profit , clinical , and private industry leaders to interact directly and form relationships that often lead to collaborations ( 64 ) . the one health commission , created in 2009 as a result of recommendations by the ohitf , is a globally focused 501(c)(3 ) non - profit organization that seeks to connect one health advocates , to create networks and teams that can work together across disciplines , and to educate about one health and one health issues ( 63 ) . based in the research triangle park region of north carolina , usa , the commission is governed by a board of directors comprising representatives from organizations that fund its work . it serves as an action arm for one health , a gateway for active education and exchange of knowledge , sharing of resources , and collaborative projects that reach beyond disciplinary boundaries . its volunteer one health action teams identify knowledge and education gaps and take actions to address them . to encourage the next generation of one health leaderst , the ohc supports soh groups and includes elected student representatives on its board of directors ( 65 ) . the one health initiative autonomous pro bono team ( see non - academic one health training , research , and outreach section ) . the zoobiquity initiative and conferences were started by barbara natterson - horowitz , md , a professor of medicine in the ucla division of cardiology at the david geffen school of medicine , who noticed the many parallels between human and animal health conditions and set out to raise awareness among her colleagues . in 2011 , natterson - horowitz founded the ongoing annual zoobiquity conference series to bring together human and animal medicine leaders for direct discussions about how to diagnose and treat the conditions that afflict animals and humans alike . in 2012 , dr . natterson - horowitz and coauthor kathryn bowers published their book zoobiquity in which they highlighted the many conditions that plague both humans and animals and advocated for a cross - species approach to health care . the zoobiquity research initiative also started a program that brings together ucla medical students and uc davis veterinary students for discussions of mutual health care interest ( 66 ) . this valuable project highlights and promotes many collaborative , comparative medicine / translational medicine research achievements over previous decades . as evidenced from table 1 and the aforementioned discussions , a number of training programs in north america provide opportunities for one health research . students and investigators seeking research funding and other opportunities may look to the ohc website which maintains a list of funding and grant opportunities . the commission also requests that the one health community send future opportunities to be posted and shared on this web page ( 67 ) . funding for one health research has often come from the us government . as of september 2016 , a search of the grants.gov website with the keyword the usaid has also devoted financial and human resources to oh research in training ; its ept programs 1 and 2 have awarded grants to investigators to build a one health workforce in developing countries that are most threatened by emerging diseases with animal , environmental , and human health implications . one health research also receives non - governmental support in the united states : the gates foundation and several universities have sponsored significant one health research . the canadian government has demonstrated its support for one health . one health researchers have been successful in applying for grants offered specifically for collaborative / interdisciplinary work . the collaborative health research projects ( chrps ) , operated jointly by the natural sciences and engineering research council of canada ( nserc ) and canadian institutes of health research ( cihr ) , have provided research funding to programs such as the one health certificate program at the university of saskatchewan . government agencies of canada are themselves embracing the one health concept in their programs and research projects : the public health agency of canada claims to be pursuing activities that are consistent with a one health approach , and its centre for food - borne , environmental and zoonotic infectious diseases ( cfezid ) hosted the one world one health expert consultation in winnipeg , manitoba in 2009 ( 69 , 70 ) . among the agency 's one health - consistent activities are the canadian integrated program for antimicrobial resistance surveillance ( cipars ) , which takes a one health approach to combat amr that spans human , animal , agricultural , food , and environmental sectors , and foodnet , canada , a comprehensive sentinel site surveillance system ( 71 ) . in north america , it is clear that one health has gained considerable recognition over the past decade and is being rapidly recognized as an efficacious and expeditious approach to address today 's most complex problems . many one health stakeholders see it as a means to accomplish the united nations sustainable development goals and a viable path to planetary health ( 72 ) . numerous academic , government , and private partners are providing one health training and research opportunities . yet , in our opinion , it is unclear whether the one health movement will evolve into a mainstream discipline or remain an those arguing for the one health sciences to become a discipline encourage that path because it would provide opportunities for young professionals to pursue careers in one health . those arguing against depth of training that specialties provide and urge that young professionals choose their discipline , hone their skills , and do their work in a one health way . it may be that our future path lies somewhere between the two scenarios , with some one health specialists trained to be experts in the areas that fall at the intersections of animal , environmental , and human health . meanwhile , there is an ever growing global understanding and appreciation of the value of the one health paradigm shift that will make this bridging across disciplines the default way of doing business at all levels of academia , research , government , policy , and law . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

backgroundthe one health ( oh ) concept , formerly referred to as one medicine in the later part of the 20th century , has gained exceptional popularity in the early 21st century , and numerous academic and non - academic institutions have developed one health programs.objectivesto summarize one health training , research , and outreach activities originating in north america.methodswe used data from extensive electronic records maintained by the one health commission ( ohc ) ( www.onehealthcommission.org/ ) and the one health initiative ( www.onehealthinitiative.com/ ) and from web - based searches , combined with the corporate knowledge of the authors and their professional contacts . finally , a call was released to members of the ohc 's global one health community listserv , asking that they populate a google document with information on one health training , research , and outreach activities in north american academic and non - academic institutions.resultsa current snapshot of north american one health training , research , and outreach activities as of august 2016 has evolved.conclusionsit is clear that the one health concept has gained considerable recognition during the first decade of the 21st century , with numerous current training and research activities carried out among north american academic , non - academic , government , corporate , and non - profit entities .

Methods Results and discussion Academic One Health training opportunities Certificates, Masters, and PhD programs Academic One Health training, research, and outreach programs Non-academic One Health training, research, and outreach One Health research activities Conclusions Supplementary Material Conflict of interest and funding

information for this review article was derived from the extensive one health community records of the ohc and the ohi autonomous pro bono team , one health web searches conducted in the fall of 2016 , and the professional contacts and experiences of the authors . both the ohc ( since 2009 ) and ohi ( since 2006 ) have worked in global one health endeavors for more than 10 years and are currently integrated into a wide network of one health stakeholders . furthermore , a call for information was disseminated to the ohc global one health community listserv . participants were asked to populate an online google document with information on one health training and research activities ( entries were verified and one health activities were summarized ) . full text descriptions of all one health activities in north america would markedly exceed this journal 's word count limits . academic one health training , research , and outreach activities in north america , stroud , usa , 2016 a version of this table that includes url links active at the time of publication is available as an online supplementary file . ohc , one health commission ; ohi , one health initiative ; mph , masters of public health ; mhs , master of health sciences , lmu - cvm , lincoln memorial university college of veterinary medicine . the authors acknowledge that there are likely additional one health training , research , and outreach efforts in north america that were inadvertently missed in this review . readers are encouraged to share additional information on their one health activities in north america with the corresponding author to update this listing and expand future summaries . as discussed earlier , the more traditional setting for one health training and research is in academia , but non - academic players also recognize the growing interest in one health and have responded enthusiastically . non - academic one health leadership activities , including those initiated by state and national governmental agencies , are listed in table 2 . location of known academic one health training , research , and outreach programs in north america as of august 2016 . gray stars denote universities with no academic credit - earning program but with other significant one health training , research , or outreach activities . non - academic one health training , research , and outreach activities in north america , stroud , usa , 2016 a version of this table that includes url links current at time of publication is available as a supplementary file . thirteen institutions in north america offer major / minor , certificate , masters , or phd programs grounded in the one health concept . the university of florida offers a master of health sciences ( mhs ) in one health as well as what is believed to be the first one health phd program created and developed originally by one of the coauthors of this article ( g.c.g . ) duke university also partners with duke - national university of singapore ( nus ) medical university singapore and duke kunshan university , china to exchange graduate students and professionals conducting one health training and research ( 27 ) . individuals and colleges outside the cvm have also expanded their oh research , training , and outreach activities . training students ( from ethiopia and the united states ) in one health summer institutes is one of the osu health sciences - ethiopia partnership 's priority areas to build capacity and strengthen collaboration ( 33 ) . the texas a&m university ( tamu ) ohi was started in 2011 by the deans and faculty of the college of veterinary medicine & biomedical sciences and the college of medicine ( 37 ) . uc davis is also home to the calvin schwabe project , which has been named in honor of the former uc davis school of veterinary medicine professor ( calvin schwabe ) who coined the term the calvin schwabe project offers a wide range of oh opportunities to veterinary students , encouraging them to approach their future careers with an appreciation of the interconnectedness of animal , environmental , and human health ( 45 ) . the university of florida s department of environmental and global health launched two degree programs that immerse trainees in the one health concept : an mhs , one health concentration , and a phd of public health , one health concentration , as mentioned previously ( 5153 ) . in 2015 , in partnership with the pennsylvania farm bureau , a commonwealth one health scholarship was initiated , providing a full tuition subsidy for 4 years of veterinary school at penn vet . for faculty , deans of the four health schools at the university of pennsylvania the perelman school of medicine , the school of nursing science , the school of dental medicine , and the school of veterinary medicine annually present a one health award for significant , collaborative research conducted by faculty , highlighting those engaged in professional education bridging two or more of the schools with outreach and innovation in training and service in clinics or to the community . they implement local discussions on one health topics , and some go into local communities to hold one health they are extremely comfortable in interacting with students studying in disciplines other than their own . realizing that these students are the next generation of one health leaders , the ohc supports any student group working for one health by providing an soh web page , who 's who in soh page , an soh listserv ( 57 ) , and an online meeting platform so that they can connect with each other . many non - academic organizations discussed below ( and additional ones listed in table 2 ) , including for - profit corporations , have embraced one health and are actively leading one health training and research . they are a part of aavmc 's one health interprofessional education initiative , an effort to integrate one health training into veterinary degree programs around the united states . one of its research and conservation goals is to build the one ocean one health research institute in marineland , florida , which could establish itself as a pioneer of the one health concept in ocean science ( 62 ) . by offering networking opportunities , educational presentations and opportunities for discussion , networking and mentoring , these events allow federal , non - profit , clinical , and private industry leaders to interact directly and form relationships that often lead to collaborations ( 64 ) . the one health commission , created in 2009 as a result of recommendations by the ohitf , is a globally focused 501(c)(3 ) non - profit organization that seeks to connect one health advocates , to create networks and teams that can work together across disciplines , and to educate about one health and one health issues ( 63 ) . it serves as an action arm for one health , a gateway for active education and exchange of knowledge , sharing of resources , and collaborative projects that reach beyond disciplinary boundaries . the one health initiative autonomous pro bono team ( see non - academic one health training , research , and outreach section ) . as evidenced from table 1 and the aforementioned discussions , a number of training programs in north america provide opportunities for one health research . the commission also requests that the one health community send future opportunities to be posted and shared on this web page ( 67 ) . as of september 2016 , a search of the grants.gov website with the keyword the usaid has also devoted financial and human resources to oh research in training ; its ept programs 1 and 2 have awarded grants to investigators to build a one health workforce in developing countries that are most threatened by emerging diseases with animal , environmental , and human health implications . one health research also receives non - governmental support in the united states : the gates foundation and several universities have sponsored significant one health research . the collaborative health research projects ( chrps ) , operated jointly by the natural sciences and engineering research council of canada ( nserc ) and canadian institutes of health research ( cihr ) , have provided research funding to programs such as the one health certificate program at the university of saskatchewan . government agencies of canada are themselves embracing the one health concept in their programs and research projects : the public health agency of canada claims to be pursuing activities that are consistent with a one health approach , and its centre for food - borne , environmental and zoonotic infectious diseases ( cfezid ) hosted the one world one health expert consultation in winnipeg , manitoba in 2009 ( 69 , 70 ) . thirteen institutions in north america offer major / minor , certificate , masters , or phd programs grounded in the one health concept . at the undergraduate level , the chicago campus also offers a bachelor of public health degree with a one health theme , and the university of california , davis offers a global disease biology major . duke university also partners with duke - national university of singapore ( nus ) medical university singapore and duke kunshan university , china to exchange graduate students and professionals conducting one health training and research ( 27 ) . individuals and colleges outside the cvm have also expanded their oh research , training , and outreach activities . training students ( from ethiopia and the united states ) in one health summer institutes is one of the osu health sciences - ethiopia partnership 's priority areas to build capacity and strengthen collaboration ( 33 ) . the osu extension division organizes one health conferences where public health officials , veterinarians , and extension workers can learn about and discuss issues relevant to public , veterinary medical , and environmental health ( 36 ) . for faculty , a tamu one health grand challenge funding program was initiated in 2014 ; four major oh research themes were identified , and a plan was implemented to bring faculty from across tamu into collaborative , inter-/transdisciplinary research partnerships . the calvin schwabe project offers a wide range of oh opportunities to veterinary students , encouraging them to approach their future careers with an appreciation of the interconnectedness of animal , environmental , and human health ( 45 ) . outside the cvm , the uc davis global health department program offers one health graduate and undergraduate seminars , as well as one health in action , an intensive , 4-week field course , teaching students to apply the one health approach to complex problems ( 46 ) . the university of florida s department of environmental and global health launched two degree programs that immerse trainees in the one health concept : an mhs , one health concentration , and a phd of public health , one health concentration , as mentioned previously ( 5153 ) . in 2015 , in partnership with the pennsylvania farm bureau , a commonwealth one health scholarship was initiated , providing a full tuition subsidy for 4 years of veterinary school at penn vet . for faculty , deans of the four health schools at the university of pennsylvania the perelman school of medicine , the school of nursing science , the school of dental medicine , and the school of veterinary medicine annually present a one health award for significant , collaborative research conducted by faculty , highlighting those engaged in professional education bridging two or more of the schools with outreach and innovation in training and service in clinics or to the community . they implement local discussions on one health topics , and some go into local communities to hold one health they are extremely comfortable in interacting with students studying in disciplines other than their own . realizing that these students are the next generation of one health leaders , the ohc supports any student group working for one health by providing an soh web page , who 's who in soh page , an soh listserv ( 57 ) , and an online meeting platform so that they can connect with each other . as mentioned earlier , as interest in , and the need for , taking a multidisciplinary or transdisciplinary approach to addressing complex problems has grown , visionary researchers and health professionals have sought to integrate one health approaches into their work . many non - academic organizations discussed below ( and additional ones listed in table 2 ) , including for - profit corporations , have embraced one health and are actively leading one health training and research . one of its research and conservation goals is to build the one ocean one health research institute in marineland , florida , which could establish itself as a pioneer of the one health concept in ocean science ( 62 ) . by offering networking opportunities , educational presentations and opportunities for discussion , networking and mentoring , these events allow federal , non - profit , clinical , and private industry leaders to interact directly and form relationships that often lead to collaborations ( 64 ) . the one health commission , created in 2009 as a result of recommendations by the ohitf , is a globally focused 501(c)(3 ) non - profit organization that seeks to connect one health advocates , to create networks and teams that can work together across disciplines , and to educate about one health and one health issues ( 63 ) . the one health initiative autonomous pro bono team ( see non - academic one health training , research , and outreach section ) . thirteen institutions in north america offer major / minor , certificate , masters , or phd programs grounded in the one health concept . at the undergraduate level , the chicago campus also offers a bachelor of public health degree with a one health theme , and the university of california , davis offers a global disease biology major . during the last 9 years , this one health training program ( initially offered at iowa state university and then at the university of florida ) has trained three cohorts of one health trainees . duke university also partners with duke - national university of singapore ( nus ) medical university singapore and duke kunshan university , china to exchange graduate students and professionals conducting one health training and research ( 27 ) . individuals and colleges outside the cvm have also expanded their oh research , training , and outreach activities . the ohio state global ohi was launched in 2009 , and it partnered with ohio state 's seven health sciences colleges with 19 institutes in ethiopia and the rest in the united states . the osu extension division organizes one health conferences where public health officials , veterinarians , and extension workers can learn about and discuss issues relevant to public , veterinary medical , and environmental health ( 36 ) . the texas a&m university ( tamu ) ohi was started in 2011 by the deans and faculty of the college of veterinary medicine & biomedical sciences and the college of medicine ( 37 ) . for faculty , a tamu one health grand challenge funding program was initiated in 2014 ; four major oh research themes were identified , and a plan was implemented to bring faculty from across tamu into collaborative , inter-/transdisciplinary research partnerships . the one health institute leads the health for animals and livelihood improvement ( hali ) project , a collaborative , capacity - building program that brings together researchers from the united states and tanzania to study the effects of zoonotic diseases and water management practices on individuals living within tanzania 's ruaha ecosystem ( 44 ) . the calvin schwabe project offers a wide range of oh opportunities to veterinary students , encouraging them to approach their future careers with an appreciation of the interconnectedness of animal , environmental , and human health ( 45 ) . the university of florida s department of environmental and global health launched two degree programs that immerse trainees in the one health concept : an mhs , one health concentration , and a phd of public health , one health concentration , as mentioned previously ( 5153 ) . for faculty , deans of the four health schools at the university of pennsylvania the perelman school of medicine , the school of nursing science , the school of dental medicine , and the school of veterinary medicine annually present a one health award for significant , collaborative research conducted by faculty , highlighting those engaged in professional education bridging two or more of the schools with outreach and innovation in training and service in clinics or to the community . realizing that these students are the next generation of one health leaders , the ohc supports any student group working for one health by providing an soh web page , who 's who in soh page , an soh listserv ( 57 ) , and an online meeting platform so that they can connect with each other . many non - academic organizations discussed below ( and additional ones listed in table 2 ) , including for - profit corporations , have embraced one health and are actively leading one health training and research . they are a part of aavmc 's one health interprofessional education initiative , an effort to integrate one health training into veterinary degree programs around the united states . the aavmc demonstrated its commitment to educating about the concept by making one health the focus of its 2014 annual conference , entitled the canadian veterinary medical association ( cvma ) has demonstrated its commitment to promoting one health by making the theme of its 2016 animal health week the georgia aquarium leads a one ocean , one health initiative , making a commitment to recognize the interconnectedness of human , animal , and environmental well - being and not study animals in isolation ( 61 ) . one of its research and conservation goals is to build the one ocean one health research institute in marineland , florida , which could establish itself as a pioneer of the one health concept in ocean science ( 62 ) . by offering networking opportunities , educational presentations and opportunities for discussion , networking and mentoring , these events allow federal , non - profit , clinical , and private industry leaders to interact directly and form relationships that often lead to collaborations ( 64 ) . the one health commission , created in 2009 as a result of recommendations by the ohitf , is a globally focused 501(c)(3 ) non - profit organization that seeks to connect one health advocates , to create networks and teams that can work together across disciplines , and to educate about one health and one health issues ( 63 ) . it serves as an action arm for one health , a gateway for active education and exchange of knowledge , sharing of resources , and collaborative projects that reach beyond disciplinary boundaries . the one health initiative autonomous pro bono team ( see non - academic one health training , research , and outreach section ) . as evidenced from table 1 and the aforementioned discussions , a number of training programs in north america provide opportunities for one health research . the commission also requests that the one health community send future opportunities to be posted and shared on this web page ( 67 ) . as of september 2016 , a search of the grants.gov website with the keyword the usaid has also devoted financial and human resources to oh research in training ; its ept programs 1 and 2 have awarded grants to investigators to build a one health workforce in developing countries that are most threatened by emerging diseases with animal , environmental , and human health implications . the collaborative health research projects ( chrps ) , operated jointly by the natural sciences and engineering research council of canada ( nserc ) and canadian institutes of health research ( cihr ) , have provided research funding to programs such as the one health certificate program at the university of saskatchewan . government agencies of canada are themselves embracing the one health concept in their programs and research projects : the public health agency of canada claims to be pursuing activities that are consistent with a one health approach , and its centre for food - borne , environmental and zoonotic infectious diseases ( cfezid ) hosted the one world one health expert consultation in winnipeg , manitoba in 2009 ( 69 , 70 ) . among the agency 's one health - consistent activities are the canadian integrated program for antimicrobial resistance surveillance ( cipars ) , which takes a one health approach to combat amr that spans human , animal , agricultural , food , and environmental sectors , and foodnet , canada , a comprehensive sentinel site surveillance system ( 71 ) . in north america , it is clear that one health has gained considerable recognition over the past decade and is being rapidly recognized as an efficacious and expeditious approach to address today 's most complex problems . numerous academic , government , and private partners are providing one health training and research opportunities . it may be that our future path lies somewhere between the two scenarios , with some one health specialists trained to be experts in the areas that fall at the intersections of animal , environmental , and human health . meanwhile , there is an ever growing global understanding and appreciation of the value of the one health paradigm shift that will make this bridging across disciplines the default way of doing business at all levels of academia , research , government , policy , and law .

we used data from the outcomes registry for better informed treatment of atrial fibrillation ( oribt - af ) , a prospective us registry of af patients in the community , managed by primary care physicians , cardiologists , and/or electrophysiologists . a nationally representative sample of sites was recruited , with diversity by geography and practice type . eligible patients were 18 years of age or older , with electrocardiographically documented af that was not the result of a reversible cause . sites enrolled consecutive patients that met the inclusion criteria without exclusions , and patients were expected to have clinical follow - up every 6 months for at least 2 years . the patients medical record served as the primary source of data , supplemented by the treating physician s input and external medical records . all data were entered in a web - based case report form , and site management and study coordination were performed by the duke clinical research institute . data collection included sections on demographics , medical history , af history ( including symptoms ) , medical and interventional therapies , vital signs , laboratory and echocardiographic measures , and incident procedures and adverse events . specifically , the patients resting heart rate in the clinic , their european heart rhythm association ( ehra ) symptom score ( i to iv ) , and medicines ( including anticoagulation , antiarrhythmic drugs , and rate control agents ) were recorded at baseline and at each follow - up . additional information on the orbit - af rationale and design has been reported previously.10 in order to assess the impact of resting heart rate in af , the primary analysis cohort for the present study included only patients in orbit - af with permanent af at baseline . patients without heart rate recorded at baseline were also excluded . in analyses of outcomes , patients without any follow - up visits were excluded . the population was stratified by baseline resting heart rate : < 60 ; 60 to 79 ; 80 to 109 ; and 110 bpm . baseline characteristics , medical history , af history , and medical therapies were compared among these groups . using these data , the correlation between heart rate and ehra score was assessed using all available visits for patients in the study population . we then assessed the relationship between resting heart rate and subsequent clinical outcomes , after adjustment for known confounders . additional outcomes included : cause - specific death ; cause - specific hospitalization ; the composite of stroke or systemic embolism ( sse ; adjudicated through primary source documentation review at the coordinating center ) or major bleeding as defined by the international society of thrombosis and haemostasis11 ; the composite of myocardial infarction ( mi ) , coronary revascularization , or new - onset heart failure ; and a composite of all adverse events ( sse , major bleeding , new heart failure , mi , coronary revascularization , hospitalization , or death ) . tests for the interaction effect between baseline antiarrhythmic drug therapy and heart rate on outcomes were performed . the relationships between baseline beta - blocker ( bb ) use , baseline calcium - channel blocker use ( nondihydropyridine , [ nd - ccb ] ) , and clinical outcomes were also assessed . sensitivity analyses measuring the association between heart rate and clinical outcomes in patients with all af types were also performed . univariate data across groups stratified by baseline heart rate are presented as percentages for categorical variables and medians ( interquartile range ; iqr ) or means ( sd ) for continuous variables . variables were compared using the chi - square test for categorical variables and continuous variables were compared using the kruskal - wallis test . in order to describe the unadjusted association between heart rate and ehra scores using data from every visit ( including baseline and all follow - up assessments ) , we compared heart rates across symptom status as defined by ehra scores ( i : no symptoms , ii : mild , iii : severe , iv : disabling ) using box plots . this included all visits for each patient , where both heart rate and ehra score were recorded . we tested for a difference in heart rate across ehra score using linear generalized estimating equations with heart rate as the response and ehra a 4-level categorical variable , and a compound symmetry correlation structure to account for repeated measurements in each patient . next , we evaluated the adjusted association between baseline heart rate and baseline ehra score , we present a risk estimate ( ie , odds ratio [ or ] ) and corresponding 95% confidence interval ( ci ) and p value from ordinal logistic regression . the regression model for ehra score at baseline was developed based on risk factors from the candidate baseline characteristics ( tables s1 and s2 ) using backward selection , with an alpha for exclusion of 0.05 . all continuous variables ( including heart rate ) were tested for linearity , and nonlinear relationships were accounted for using linear splines . to describe the association between resting heart rate and clinical outcomes ( listed above ) , we determined risk estimates ( ie , hazard ratio [ hr ] ) and corresponding 95% cis and p values using cox regression where longitudinally updated heart rate , as a continuous variable , is included as a time - dependent covariate , along with adjustment for baseline risk factors . empirical standard errors were also used to account for correlation between patients at the same site . adjustment risk factors were based on previously developed outcomes models in this population,12,13 which include all statistically significant covariates based on backward selection and =0.05 , selected from a large candidate list ( tables s1 and s2 ) . the time - dependent heart rate covariate was tested for linearity , and nonlinear relationships were illustrated a priori using restricted cubic splines . this provided a flexible relationship that , in all cases , could be approximated by piece - wise linear splines , which were used to estimate hrs within appropriate ranges of heart rate ( defined by the observed inflection point ) . to assess the interaction between heart rate and the use of antiarrhythmic therapy for each outcome , we included one additional interaction term in the model . to determine the effect of rate control therapy on outcomes , propensity scores for nd - ccb versus bb use were generated using inclusive final covariates for 3 endpoints ( all - cause death , cardiovascular death , noncardiovascular death ) . the outcome model was weighted using the inverse propensity score ( ipw ) for getting nd - ccb to minimize confounding and to incorporate nd - ccb ( binary ) therapy . the effectiveness of the ipw was evaluated using cramer s phi ( v ) and r. we calculated risk statistics ( ie , hr , corresponding 95% ci , and p value ) for nd - ccb versus bb by cox regression with robust covariance . missing data among the baseline covariates used for multivariable adjustment ( not heart rate ) were handled with single imputation , using mcmc and regression methods in sas . missing data in these variables was < 4% for all covariates , except left ventricular ejection fraction ( lvef ; 10% ) , left atrial diameter ( 14% ) , serum creatinine ( 7% ) , and hematocrit ( 10% ) . intermittent missing values in longitudinal heart rate were handled by a last value carried forward approach . several sensitivity analyses of the relationship between heart rate and clinical outcomes were performed . to test the durability of our findings , the above analysis was repeated in the overall orbit - af population , including all types of af . in response to peer review , we used linear regression modeling of baseline heart rate as an outcome to calculate the r for all baseline patient characteristics ( assess factors associated with increased heart rate ) . next , we calculated the variance of heart rate across different patients at the same point in time , and also for the same patient at different time points , using a mixed model for longitudinal heart rate . the orbit - af registry was approved by the duke university institutional review board ( irb ) , and all sites received irb approval pursuant to local regulations . all patients provided written informed consent , and analyses of the aggregate , deidentified data were performed by the duke clinical research institute using sas software ( version 9.3 ; sas institute inc . , cary , nc ) . univariate data across groups stratified by baseline heart rate are presented as percentages for categorical variables and medians ( interquartile range ; iqr ) or means ( sd ) for continuous variables . variables were compared using the chi - square test for categorical variables and continuous variables were compared using the kruskal - wallis test . in order to describe the unadjusted association between heart rate and ehra scores using data from every visit ( including baseline and all follow - up assessments ) , we compared heart rates across symptom status as defined by ehra scores ( i : no symptoms , ii : mild , iii : severe , iv : disabling ) using box plots . this included all visits for each patient , where both heart rate and ehra score were recorded . we tested for a difference in heart rate across ehra score using linear generalized estimating equations with heart rate as the response and ehra a 4-level categorical variable , and a compound symmetry correlation structure to account for repeated measurements in each patient . next , we evaluated the adjusted association between baseline heart rate and baseline ehra score , we present a risk estimate ( ie , odds ratio [ or ] ) and corresponding 95% confidence interval ( ci ) and p value from ordinal logistic regression . the regression model for ehra score at baseline was developed based on risk factors from the candidate baseline characteristics ( tables s1 and s2 ) using backward selection , with an alpha for exclusion of 0.05 . all continuous variables ( including heart rate ) were tested for linearity , and nonlinear relationships were accounted for using linear splines . to describe the association between resting heart rate and clinical outcomes ( listed above ) , we determined risk estimates ( ie , hazard ratio [ hr ] ) and corresponding 95% cis and p values using cox regression where longitudinally updated heart rate , as a continuous variable , is included as a time - dependent covariate , along with adjustment for baseline risk factors . empirical standard errors were also used to account for correlation between patients at the same site . adjustment risk factors were based on previously developed outcomes models in this population,12,13 which include all statistically significant covariates based on backward selection and =0.05 , selected from a large candidate list ( tables s1 and s2 ) . the time - dependent heart rate covariate was tested for linearity , and nonlinear relationships were illustrated a priori using restricted cubic splines . this provided a flexible relationship that , in all cases , could be approximated by piece - wise linear splines , which were used to estimate hrs within appropriate ranges of heart rate ( defined by the observed inflection point ) . to assess the interaction between heart rate and the use of antiarrhythmic therapy for each outcome , we included one additional interaction term in the model . to determine the effect of rate control therapy on outcomes , propensity scores for nd - ccb versus bb use were generated using inclusive final covariates for 3 endpoints ( all - cause death , cardiovascular death , noncardiovascular death ) . the outcome model was weighted using the inverse propensity score ( ipw ) for getting nd - ccb to minimize confounding and to incorporate nd - ccb ( binary ) therapy . the effectiveness of the ipw was evaluated using cramer s phi ( v ) and r. we calculated risk statistics ( ie , hr , corresponding 95% ci , and p value ) for nd - ccb versus bb by cox regression with robust covariance . missing data among the baseline covariates used for multivariable adjustment ( not heart rate ) were handled with single imputation , using mcmc and regression methods in sas . missing data in these variables was < 4% for all covariates , except left ventricular ejection fraction ( lvef ; 10% ) , left atrial diameter ( 14% ) , serum creatinine ( 7% ) , and hematocrit ( 10% ) . intermittent missing values in longitudinal heart rate were handled by a last value carried forward approach . several sensitivity analyses of the relationship between heart rate and clinical outcomes were performed . to test the durability of our findings , the above analysis was repeated in the overall orbit - af population , including all types of af . in response to peer review , we used linear regression modeling of baseline heart rate as an outcome to calculate the r for all baseline patient characteristics ( assess factors associated with increased heart rate ) . next , we calculated the variance of heart rate across different patients at the same point in time , and also for the same patient at different time points , using a mixed model for longitudinal heart rate . the orbit - af registry was approved by the duke university institutional review board ( irb ) , and all sites received irb approval pursuant to local regulations . all patients provided written informed consent , and analyses of the aggregate , deidentified data were performed by the duke clinical research institute using sas software ( version 9.3 ; sas institute inc . , cary , nc ) . the overall orbit - af population included 10 132 patients from 176 us practices . we excluded 50 patients for missing baseline heart rate and 7270 patients for having nonpermanent af . patients with permanent af who were excluded for other reasons were largely similar to patients included in the analysis . this resulted in a primary study cohort of 2812 patients with permanent af , enrolled from june 2010 through august 2011 . the median follow - up was 24 months ( 25th and 75th percentile : 18 , 30 months ) and included a total of 12 299 heart rate measurements for all patients throughout the study period . at baseline , 7.4% ( n=207 ) had a heart rate < 60 bpm ; 62% ( n=1755 ) 60 to 79 bpm ; 29% ( n=817 ) 80 to 109 bpm ; and 1.2% ( n=33 ) 110 bpm . patients with heart rate < 60 bpm were less likely to be female ( 32% ; p=0.048 ) , to be african american ( 3.4% ; p=0.001 ) , and had the lowest prevalence of sinus node dysfunction ( 10% ; p=0.0048 ) . there were no significant differences among the heart rate groups with respect to age ( p=0.1 ) , previous cerebrovascular disease ( p=0.1 ) , heart failure status ( p=0.2 ) , cha2ds2-vasc scores ( p=0.8 ) , renal function ( p=0.5),14 or left ventricular function ( p=0.4 ) . demographics , past medical history , and laboratory studies by baseline resting heart rate values are presented as percentage or median ( interquartile range ) , unless noted otherwise . p values were calculated across groups using the chi - square test for categorical variables and the kruskal - wallis test for continuous variables . bpm indicates beats per minute ; cad , coronary artery disease ; chf , congestive heart failure ; ef , ejection fraction ; mi , myocardial infarction ; nyha , new york heart association heart failure class ; tia , transient ischemic attack . as calculated by the cockcroft - gault formula.14 previous and current therapies for af , across these groups , patients with lower resting heart rate ( < 60 bpm ) were least likely to have received an antiarrhythmic drug previously ( 23% ; p=0.01 ) and were more likely to be treated with nd - ccb ( 21% ; p=0.001 ) and anticoagulation ( 90% ; p=0.009 ) at baseline . baseline use of antiarrhythmic drug ( p=0.8 ) and digoxin ( p=0.5 ) was balanced across groups . atrial fibrillation history and management by baseline resting heart rate values are presented as percentage . p values were calculated across groups using the chi - square test for categorical variables and the kruskal - wallis test for continuous variables . ace - i indicates angiotensin - converting enzyme inhibitor ; af , atrial fibrillation ; arb , angiotensin ii receptor blocker ; av , atrioventricular ; bpm , beats per minute . unadjusted assessment of heart rate and ehra class included 12 299 visits for all patients documenting resting heart rate and ehra score : 2701 patients at baseline ; 2402 at 6 months ; 2153 at 12 months ; 1957 at 18 months ; 1792 at 24 months ; 960 at 30 months ; 310 at 36 months ; and 24 recorded at early study termination . there were 6797 measurements correlating with no symptoms ( ehra class i ) , 4414 with mild symptoms ( class ii ) , 1000 with severe symptoms ( class iii ) , and 88 with disabling symptoms ( class iv ) . there was a significant association between increasing heart rate and worse concomitant ehra symptom class across visits ( figure1 ; p<0.0001 ) . in adjusted analyses , the relationship between baseline - only resting heart rate and concomitant , baseline ehra symptom class was found to be linear , and increasing heart rate at baseline was significantly associated with more - severe baseline ehra symptom class ( adjusted or , 1.04 per 5 bpm increase ; 95% ci , 1.01 to 1.08 ; p=0.007 ; see table s3 ) . distribution of 12 299 observations of resting heart rate versus concomitant ehra symptom score in 2812 patients with permanent af . owing to multiple follow - up visits , individual patients may contribute multiple observations of heart rate and ehra score . the p value is derived by testing for the overall significance of ehra score levels from the correlated errors model , which yielded a coefficient of 1.11 for mild ehra ( vs. no symptoms ) , 2.06 for severe ehra ( vs no symptoms ) , and 2.36 for disabling ehra ( vs. no symptoms ) . af indicates atrial fibrillation ; bpm , beats per minute ; ehra , european heart rhythm association ; iqr , interquartile range . overall event rates , as well as unadjusted and adjusted hazards for clinical events , are shown in table3 . unadjusted outcomes demonstrated a j - shaped relationship between resting time - dependent heart rate and all - cause mortality ( unadjusted hr per 5-bpm decrease in heart rate 65 bpm , 1.10 ; 95% ci , 0.96 to 1.25 ; unadjusted hr per 5-bpm increase in heart rate > 65 bpm , 1.07 ; 95% ci , 1.03 to 1.12 ) . in multivariable analysis using heart rate as a continuous , time - dependent covariate , the relationship between heart rate and cause - specific mortality remained nonlinear , with an inflection point at 65 bpm . the adjusted hrs of these splines , with 95% cis , are shown in figure2a through 2c . decreasing heart rate 65 bpm was associated with increasing all - cause mortality ( adjusted hr , 1.15 per 5-bpm increase ; 95% ci , 1.01 to 1.32 ; p=0.04 ) , and increasing heart rate > 65 bpm was associated with worse all - cause mortality ( adjusted hr , 1.10 per 5-bpm increase ; 95% ci , 1.05 to 1.15 ; p<0.0001 ) . unadjusted and adjusted association between increasing heart rate and clinical outcomes denominators may differ owing to competing risks . bpm indicates beats per minute ; ci , confidence interval ; hr , hazard ratio ; mi , myocardial infarction ; sse , stroke or systemic embolism . relationship between time - dependent resting heart rate and clinical outcome among 2812 patients with permanent af . adjusted hazard ratios ( with 95% cis ) of increasing heart rate ( using the mean heart rate of 73 bpm as the referent ) for ( a ) all - cause mortality , ( b ) cardiovascular death , and ( c ) noncardiovascular death . af indicates atrial fibrillation ; bpm , beats per minute ; ci , confidence interval . linear splines were also derived for the composite endpoint of all adverse events ( sse , major bleeding , new heart failure , mi , revascularization , all - cause hospitalization , and all - cause death ) . heart rates below and above 65 bpm were associated with worse outcomes ( adjusted hr , 1.10 per 5-bpm decrease 65 bpm ; 95% ci , 1.02 to 1.19 ; adjusted hr , 1.03 per 5-bpm increase > 65 bpm ; 95% ci , 1.00 to 1.06 ) . overall 227 patients ( 8% ) were receiving antiarrhythmic therapy at baseline , most commonly amiodarone ( n=105 ; 3.7% ) . interaction testing was performed between heart rate and baseline antiarrhythmic therapy for each of the clinical outcomes ( sse , major bleeding , new heart failure , mi , revascularization , cause - specific hospitalization , and cause - specific death ) . a significant interaction ( pinteraction<0.05 ) there was a significant association between increased heart rate and noncardiovascular death among patients not on an antiarrhythmic drug at baseline ( n=2496 ; 92% ; adjusted hr , 1.08 per 5-bpm increase ; 95% ci , 1.02 to 1.15 ; p=0.01 ) , but not for patients receiving an antiarrhythmic drug at baseline ( n=215 ; 8% ; adjusted hr , 0.75 per 5-bpm increase ; 95% ci , 0.52 to 1.07 ; p=0.1 , pinteraction=0.02 ) . propensity score modeling to assess different associations between nd - ccb and bb therapy and outcomes included 117 patients treated with nd - ccb and 1638 patients with bb ( n=1755 ) . cox regression models did not demonstrate significant differences in outcomes between groups : adjusted hr for nd - ccb ( versus bb ) for all - cause death , 0.99 ( 95% ci , 0.46 to 2.14 ; p=1.0 ) ; cardiovascular death , 0.97 ( 95% ci , 0.23 to 3.11 ; p=0.8 ) ; and noncardiovascular death , 1.29 ( 95% ci , 0.49 to 3.37 ; p=0.6 ) . in sensitivity analyses of patients with any type of af ( n=9648 ) , including nonpermanent forms of af , the associations between heart rate and clinical outcomes were consistent ( tables s3 and s4 ) . additional , exploratory sensitivity analyses were performed assessing the factors associated with baseline heart rate , heart rate change over time , and the association between baseline - only heart rate and subsequent clinical outcomes . other patient characteristics were minimally associated with baseline heart rate ( r=0.03 ) , and heart rate varied nearly twice as much within individuals over time as it did between patients ( variance 95 within individuals vs. 54 between individuals , in a mixed model for longitudinal heart rate ) . without updating heart rate as a time - dependent covariate , there were not significant associations between heart rate and clinical outcomes , unadjusted or adjusted ( tables s5 and s6 ) . unadjusted assessment of heart rate and ehra class included 12 299 visits for all patients documenting resting heart rate and ehra score : 2701 patients at baseline ; 2402 at 6 months ; 2153 at 12 months ; 1957 at 18 months ; 1792 at 24 months ; 960 at 30 months ; 310 at 36 months ; and 24 recorded at early study termination . there were 6797 measurements correlating with no symptoms ( ehra class i ) , 4414 with mild symptoms ( class ii ) , 1000 with severe symptoms ( class iii ) , and 88 with disabling symptoms ( class iv ) . there was a significant association between increasing heart rate and worse concomitant ehra symptom class across visits ( figure1 ; p<0.0001 ) . in adjusted analyses , the relationship between baseline - only resting heart rate and concomitant , baseline ehra symptom class was found to be linear , and increasing heart rate at baseline was significantly associated with more - severe baseline ehra symptom class ( adjusted or , 1.04 per 5 bpm increase ; 95% ci , 1.01 to 1.08 ; p=0.007 ; see table s3 ) . distribution of 12 299 observations of resting heart rate versus concomitant ehra symptom score in 2812 patients with permanent af . owing to multiple follow - up visits , individual patients may contribute multiple observations of heart rate and ehra score . the p value is derived by testing for the overall significance of ehra score levels from the correlated errors model , which yielded a coefficient of 1.11 for mild ehra ( vs. no symptoms ) , 2.06 for severe ehra ( vs no symptoms ) , and 2.36 for disabling ehra ( vs. no symptoms ) . af indicates atrial fibrillation ; bpm , beats per minute ; ehra , european heart rhythm association ; iqr , interquartile range . overall event rates , as well as unadjusted and adjusted hazards for clinical events , are shown in table3 . unadjusted outcomes demonstrated a j - shaped relationship between resting time - dependent heart rate and all - cause mortality ( unadjusted hr per 5-bpm decrease in heart rate 65 bpm , 1.10 ; 95% ci , 0.96 to 1.25 ; unadjusted hr per 5-bpm increase in heart rate > 65 bpm , 1.07 ; 95% ci , 1.03 to 1.12 ) . in multivariable analysis using heart rate as a continuous , time - dependent covariate , the relationship between heart rate and cause - specific mortality remained nonlinear , with an inflection point at 65 bpm . the adjusted hrs of these splines , with 95% cis , are shown in figure2a through 2c . decreasing heart rate 65 bpm was associated with increasing all - cause mortality ( adjusted hr , 1.15 per 5-bpm increase ; 95% ci , 1.01 to 1.32 ; p=0.04 ) , and increasing heart rate > 65 bpm was associated with worse all - cause mortality ( adjusted hr , 1.10 per 5-bpm increase ; 95% ci , 1.05 to 1.15 ; p<0.0001 ) . unadjusted and adjusted association between increasing heart rate and clinical outcomes denominators may differ owing to competing risks . bpm indicates beats per minute ; ci , confidence interval ; hr , hazard ratio ; mi , myocardial infarction ; sse , stroke or systemic embolism . relationship between time - dependent resting heart rate and clinical outcome among 2812 patients with permanent af . adjusted hazard ratios ( with 95% cis ) of increasing heart rate ( using the mean heart rate of 73 bpm as the referent ) for ( a ) all - cause mortality , ( b ) cardiovascular death , and ( c ) noncardiovascular death . af indicates atrial fibrillation ; bpm , beats per minute ; ci , confidence interval . linear splines were also derived for the composite endpoint of all adverse events ( sse , major bleeding , new heart failure , mi , revascularization , all - cause hospitalization , and all - cause death ) . heart rates below and above 65 bpm were associated with worse outcomes ( adjusted hr , 1.10 per 5-bpm decrease 65 bpm ; 95% ci , 1.02 to 1.19 ; adjusted hr , 1.03 per 5-bpm increase > 65 bpm ; 95% ci , 1.00 to 1.06 ) . overall 227 patients ( 8% ) were receiving antiarrhythmic therapy at baseline , most commonly amiodarone ( n=105 ; 3.7% ) . interaction testing was performed between heart rate and baseline antiarrhythmic therapy for each of the clinical outcomes ( sse , major bleeding , new heart failure , mi , revascularization , cause - specific hospitalization , and cause - specific death ) . a significant interaction ( pinteraction<0.05 ) there was a significant association between increased heart rate and noncardiovascular death among patients not on an antiarrhythmic drug at baseline ( n=2496 ; 92% ; adjusted hr , 1.08 per 5-bpm increase ; 95% ci , 1.02 to 1.15 ; p=0.01 ) , but not for patients receiving an antiarrhythmic drug at baseline ( n=215 ; 8% ; adjusted hr , 0.75 per 5-bpm increase ; 95% ci , 0.52 to 1.07 ; p=0.1 , pinteraction=0.02 ) . propensity score modeling to assess different associations between nd - ccb and bb therapy and outcomes included 117 patients treated with nd - ccb and 1638 patients with bb ( n=1755 ) . cox regression models did not demonstrate significant differences in outcomes between groups : adjusted hr for nd - ccb ( versus bb ) for all - cause death , 0.99 ( 95% ci , 0.46 to 2.14 ; p=1.0 ) ; cardiovascular death , 0.97 ( 95% ci , 0.23 to 3.11 ; p=0.8 ) ; and noncardiovascular death , 1.29 ( 95% ci , 0.49 to 3.37 ; p=0.6 ) . in sensitivity analyses of patients with any type of af ( n=9648 ) , including nonpermanent forms of af , the associations between heart rate and clinical outcomes were consistent ( tables s3 and s4 ) . additional , exploratory sensitivity analyses were performed assessing the factors associated with baseline heart rate , heart rate change over time , and the association between baseline - only heart rate and subsequent clinical outcomes . other patient characteristics were minimally associated with baseline heart rate ( r=0.03 ) , and heart rate varied nearly twice as much within individuals over time as it did between patients ( variance 95 within individuals vs. 54 between individuals , in a mixed model for longitudinal heart rate ) . without updating heart rate as a time - dependent covariate , there were not significant associations between heart rate and clinical outcomes , unadjusted or adjusted ( tables s5 and s6 ) . there is insufficient evidence to guide heart rate targets in patients with af ; however , these data provide several insights into current practice and outcomes associated with different heart rates in af . in this nation - wide community cohort of patients with permanent af , nearly all patients had resting heart rates < 110 bpm ( 99% ) and the majority ( 70% ) were < 80 bpm . however , we found that increasing heart rate above 65 bpm was associated with worse symptom class and lower survival rates , even after adjusting for baseline clinical factors . last , medical therapies appeared to have little impact on the relationship between heart rate and mortality . our results are not completely consistent with the us guidelines applicable during the study period ( 2011 ) , which designated strict heart rate control ( < 80 bpm ) as a class iii recommendation ( harm exceeds benefit)15 this recommendation was based mainly on a single trial,6 whereas previous studies had demonstrated adverse hemodynamic consequences of prolonged , uncontrolled ventricular rates.16 our data suggest that patients in community practice routinely ( 70% ) achieved more - stringent rate control ( below 80 ) and that the associated outcomes were more favorable so long as heart rate was 65 or greater . clinicians may have been reluctant to employ a modified guideline recommendation based upon a single study , given the previous accumulated clinical evidence . consistently , across endpoints and patient populations , increasing heart rate > 65 bpm was associated with worse outcomes , including all - cause and cause - specific mortality , as well as adverse cardiovascular events . these findings contrast with the results of previous studies comparing strict versus lenient heart rate control.6,17,18 the suggestion that strict rate control is unnecessary is predominantly based on the race ii trial , which randomized patients to each approach . yet , race ii was a noninferiority trial that was underpowered to detect a benefit of strict rate control : statistically , the trial could not exclude even a 4.6% absolute risk reduction in cardiovascular death , heart failure hospitalization , or stroke at 3 years with a strict rate control strategy ( in a binary comparison).6,19 additionally , follow - up heart rates in the 2 groups were closer than the targets suggested ( mean 85 bpm for the < 110 group vs. 76 bpm for the < 80 group ) . last , the relationship between heart rate and clinical outcomes , particularly mortality , could not be assessed . in contrast , the present analysis includes a significantly larger sample , and despite relatively low heart rates in this population , our data demonstrate a significant association between increased heart rate ( > 65 bpm , as a continuous covariate ) and adverse outcome . higher heart rates were associated with increased all - cause mortality in patients with or without antiarrhythmic therapy . though there was a significant interaction between antiarrhythmic use and heart rate for the endpoint of cardiovascular death , the sample was relatively small ( 8% ) and the cis were wide ( 95% ci , 0.52 to 1.07 ) . additionally , no difference in outcomes was observed in inverse propensity score models comparing nd - ccbs to bbs . whereas these are observational data and do not yield definitive conclusions , the findings support the hypothesis that choice of medical therapy may be a secondary consideration to the primary achievement of optimal resting heart rate . given the divergent treatment guidelines for rate control , our findings have several important clinical implications . though we can not definitively identify providers target heart rates in our study , the association between increasing heart rate and adverse outcomes suggests that strict rate control may be associated with superior outcomes . additional adequately powered , randomized , superiority studies to identify optimal rate control strategies are warranted , particularly given that there are more than 33 million individuals with af across the world . furthermore , there may be a floor effect , below which lowering heart rate is no longer beneficial ( and may be harmful ) . whereas physicians in practice appear to be comfortable with lower resting heart rates than previous guidelines dictated , identifying the optimal threshold , and the optimal therapies to achieve that threshold , will require further investigation . neither the extent of heart rate control nor the medical therapies were randomized , and thus residual and unmeasured confounding may exist . additionally , a small proportion of patients were lost to follow - up . though outcomes models included time - dependent covariates for heart rate , interim events or modifications to treatment were not included . there may have been other influences in outcome that were not captured by the multivariable models . additionally , few patients in this sample had heart rates 110 bpm ; therefore , conclusions regarding the increasing risk of very high heart rates are limited by power . however , the relative paucity of heart rates over 110 bpm may have led to underestimation of the impact of tachycardia on survival and other outcomes . in - depth analyses comparing and contrasting different rate control therapies were limited owing to smaller comparator subgroups . last , there also may be differences between patients enrolled in orbit - af and the broader af population . neither the extent of heart rate control nor the medical therapies were randomized , and thus residual and unmeasured confounding may exist . additionally , a small proportion of patients were lost to follow - up . though outcomes models included time - dependent covariates for heart rate , interim events or modifications to treatment were not included . there may have been other influences in outcome that were not captured by the multivariable models . additionally , few patients in this sample had heart rates 110 bpm ; therefore , conclusions regarding the increasing risk of very high heart rates are limited by power . however , the relative paucity of heart rates over 110 bpm may have led to underestimation of the impact of tachycardia on survival and other outcomes . in - depth analyses comparing and contrasting different rate control therapies were limited owing to smaller comparator subgroups . last , there also may be differences between patients enrolled in orbit - af and the broader af population . patients with permanent af in us community practice maintain relatively low resting heart rates , and increased heart rates are associated with worse symptom class . moreover , there is a j - shaped relationship between heart rate and mortality for patients with af , and this is a consistent finding across endpoints . these data support current american college of cardiology / american heart association guideline recommendations for strict rate control . the orbit - af registry is sponsored by janssen scientific affairs , llc ( raritan , nj ) . gersh reports modest dsmb / advisory board support from medtronic , baxter healthcare corporation , inspiremd , cardiovascular research foundation , ppd development , lp , boston scientific , and st . jude . reports : modest honoraria support form boehringer ingelheim and bayer ; modest consultant / advisory board to johnson & johnson , boehringer ingelheim , bristol - myers squibb , daiichi sankyo , pfizer , and ortho - mcneil - janssen . p.r.k . reports modest consultant / advisory board support from boehringer ingelheim , bristol - myers squibb , johnson & johnson , portola , merck , sanofi , and daiichi sankyo . mahaffey reports research grants from amgen , daiichi , johnson & johnson , medtronic , st . jude , and tenax ; modest consulting from the american college of cardiology , bayer , boehringer ingelheim , bristol - myers squibb , eli lilly , elsevier , epson , forest , medtronic , mt . sinai , myokardia , omithera , portola , purdue pharma , spring publishing , vindico , and webmd ; significant consulting from astrazeneca , cubist , glaxosmithkline , johnson & johnson , merck , and the medicines company , and modest equity interest in bioprint fitness . g.n . reports research grants from wyeth , reliant , medtronic , boston scientific , sanofi - aventis , and boehringer ingelheim and consultancies to wyeth , reliant , medtronic , boston scientific , sanofi - aventis , boehringer ingelheim , xention , pfizer , novartis , glaxosmithkline , and st . peterson reports : significant research grant support from eli lilly & company , janssen pharmaceuticals , inc , and the american heart association ; modest consultant / advisory board support from boehringer ingelheim , bristol - myers squibb , janssen pharmaceuticals , inc , pfizer , and genentech inc . piccini reports : significant research grant support from johnson & johnson / janssen pharmaceuticals ; significant other research support from boston scientific corporation , johnson & johnson pharmaceutical research & development ; modest consultant / advisory board support from medtronic , inc ; and significant consultant / advisory board support from johnson & johnson / janssen pharmaceuticals .

backgroundmost patients with atrial fibrillation ( af ) require rate control ; however , the optimal target heart rate remains under debate . we aimed to assess rate control and subsequent outcomes among patients with permanent af.methods and resultswe studied 2812 us outpatients with permanent af in the outcomes registry for better informed treatment of atrial fibrillation . resting heart rate was measured longitudinally and used as a time - dependent covariate in multivariable cox models of all - cause and cause - specific mortality during a median follow - up of 24 months . at baseline , 7.4% ( n=207 ) had resting heart rate < 60 beats per minute ( bpm ) , 62% ( n=1755 ) 60 to 79 bpm , 29% ( n=817 ) 80 to 109 bpm , and 1.2% ( n=33 ) 110 bpm . groups did not differ by age , previous cerebrovascular disease , heart failure status , cha2ds2-vasc scores , renal function , or left ventricular function . there were significant differences in race ( p=0.001 ) , sinus node dysfunction ( p=0.004 ) , and treatment with calcium - channel blockers ( p=0.006 ) and anticoagulation ( p=0.009 ) . in analyses of continuous heart rates , lower heart rate 65 bpm was associated with higher all - cause mortality ( adjusted hazard ratio [ hr ] , 1.15 per 5-bpm decrease ; 95% ci , 1.01 to 1.32 ; p=0.04 ) . similarly , increasing heart rate > 65 bpm was associated with higher all - cause mortality ( adjusted hr , 1.10 per 5-bpm increase ; 95% ci , 1.05 to 1.15 ; p<0.0001 ) . this relationship was consistent across endpoints and in a broader sensitivity analysis of permanent and nonpermanent af patients.conclusionsamong patients with permanent af , there is a j - shaped relationship between heart rate and mortality . these data support current guideline recommendations , and clinical trials are warranted to determine optimal rate control.clinical trial registrationurl : http://clinicaltrials.gov/. unique identifier : nct01165710 .

Clinical Trial Registration Methods Statistical Analyses Results Heart Rate and Symptoms Clinical Event Outcomes Medical Therapies Sensitivity Analyses Discussion Limitations Conclusions Sources of Funding Disclosures Supporting Information

we used data from the outcomes registry for better informed treatment of atrial fibrillation ( oribt - af ) , a prospective us registry of af patients in the community , managed by primary care physicians , cardiologists , and/or electrophysiologists . specifically , the patients resting heart rate in the clinic , their european heart rhythm association ( ehra ) symptom score ( i to iv ) , and medicines ( including anticoagulation , antiarrhythmic drugs , and rate control agents ) were recorded at baseline and at each follow - up . additional information on the orbit - af rationale and design has been reported previously.10 in order to assess the impact of resting heart rate in af , the primary analysis cohort for the present study included only patients in orbit - af with permanent af at baseline . the population was stratified by baseline resting heart rate : < 60 ; 60 to 79 ; 80 to 109 ; and 110 bpm . using these data , the correlation between heart rate and ehra score was assessed using all available visits for patients in the study population . we then assessed the relationship between resting heart rate and subsequent clinical outcomes , after adjustment for known confounders . additional outcomes included : cause - specific death ; cause - specific hospitalization ; the composite of stroke or systemic embolism ( sse ; adjudicated through primary source documentation review at the coordinating center ) or major bleeding as defined by the international society of thrombosis and haemostasis11 ; the composite of myocardial infarction ( mi ) , coronary revascularization , or new - onset heart failure ; and a composite of all adverse events ( sse , major bleeding , new heart failure , mi , coronary revascularization , hospitalization , or death ) . sensitivity analyses measuring the association between heart rate and clinical outcomes in patients with all af types were also performed . in order to describe the unadjusted association between heart rate and ehra scores using data from every visit ( including baseline and all follow - up assessments ) , we compared heart rates across symptom status as defined by ehra scores ( i : no symptoms , ii : mild , iii : severe , iv : disabling ) using box plots . to describe the association between resting heart rate and clinical outcomes ( listed above ) , we determined risk estimates ( ie , hazard ratio [ hr ] ) and corresponding 95% cis and p values using cox regression where longitudinally updated heart rate , as a continuous variable , is included as a time - dependent covariate , along with adjustment for baseline risk factors . to assess the interaction between heart rate and the use of antiarrhythmic therapy for each outcome , we included one additional interaction term in the model . the effectiveness of the ipw was evaluated using cramer s phi ( v ) and r. we calculated risk statistics ( ie , hr , corresponding 95% ci , and p value ) for nd - ccb versus bb by cox regression with robust covariance . several sensitivity analyses of the relationship between heart rate and clinical outcomes were performed . in order to describe the unadjusted association between heart rate and ehra scores using data from every visit ( including baseline and all follow - up assessments ) , we compared heart rates across symptom status as defined by ehra scores ( i : no symptoms , ii : mild , iii : severe , iv : disabling ) using box plots . to describe the association between resting heart rate and clinical outcomes ( listed above ) , we determined risk estimates ( ie , hazard ratio [ hr ] ) and corresponding 95% cis and p values using cox regression where longitudinally updated heart rate , as a continuous variable , is included as a time - dependent covariate , along with adjustment for baseline risk factors . to assess the interaction between heart rate and the use of antiarrhythmic therapy for each outcome , we included one additional interaction term in the model . the effectiveness of the ipw was evaluated using cramer s phi ( v ) and r. we calculated risk statistics ( ie , hr , corresponding 95% ci , and p value ) for nd - ccb versus bb by cox regression with robust covariance . several sensitivity analyses of the relationship between heart rate and clinical outcomes were performed . the median follow - up was 24 months ( 25th and 75th percentile : 18 , 30 months ) and included a total of 12 299 heart rate measurements for all patients throughout the study period . at baseline , 7.4% ( n=207 ) had a heart rate < 60 bpm ; 62% ( n=1755 ) 60 to 79 bpm ; 29% ( n=817 ) 80 to 109 bpm ; and 1.2% ( n=33 ) 110 bpm . patients with heart rate < 60 bpm were less likely to be female ( 32% ; p=0.048 ) , to be african american ( 3.4% ; p=0.001 ) , and had the lowest prevalence of sinus node dysfunction ( 10% ; p=0.0048 ) . there were no significant differences among the heart rate groups with respect to age ( p=0.1 ) , previous cerebrovascular disease ( p=0.1 ) , heart failure status ( p=0.2 ) , cha2ds2-vasc scores ( p=0.8 ) , renal function ( p=0.5),14 or left ventricular function ( p=0.4 ) . as calculated by the cockcroft - gault formula.14 previous and current therapies for af , across these groups , patients with lower resting heart rate ( < 60 bpm ) were least likely to have received an antiarrhythmic drug previously ( 23% ; p=0.01 ) and were more likely to be treated with nd - ccb ( 21% ; p=0.001 ) and anticoagulation ( 90% ; p=0.009 ) at baseline . ace - i indicates angiotensin - converting enzyme inhibitor ; af , atrial fibrillation ; arb , angiotensin ii receptor blocker ; av , atrioventricular ; bpm , beats per minute . there was a significant association between increasing heart rate and worse concomitant ehra symptom class across visits ( figure1 ; p<0.0001 ) . in adjusted analyses , the relationship between baseline - only resting heart rate and concomitant , baseline ehra symptom class was found to be linear , and increasing heart rate at baseline was significantly associated with more - severe baseline ehra symptom class ( adjusted or , 1.04 per 5 bpm increase ; 95% ci , 1.01 to 1.08 ; p=0.007 ; see table s3 ) . distribution of 12 299 observations of resting heart rate versus concomitant ehra symptom score in 2812 patients with permanent af . unadjusted outcomes demonstrated a j - shaped relationship between resting time - dependent heart rate and all - cause mortality ( unadjusted hr per 5-bpm decrease in heart rate 65 bpm , 1.10 ; 95% ci , 0.96 to 1.25 ; unadjusted hr per 5-bpm increase in heart rate > 65 bpm , 1.07 ; 95% ci , 1.03 to 1.12 ) . in multivariable analysis using heart rate as a continuous , time - dependent covariate , the relationship between heart rate and cause - specific mortality remained nonlinear , with an inflection point at 65 bpm . decreasing heart rate 65 bpm was associated with increasing all - cause mortality ( adjusted hr , 1.15 per 5-bpm increase ; 95% ci , 1.01 to 1.32 ; p=0.04 ) , and increasing heart rate > 65 bpm was associated with worse all - cause mortality ( adjusted hr , 1.10 per 5-bpm increase ; 95% ci , 1.05 to 1.15 ; p<0.0001 ) . relationship between time - dependent resting heart rate and clinical outcome among 2812 patients with permanent af . adjusted hazard ratios ( with 95% cis ) of increasing heart rate ( using the mean heart rate of 73 bpm as the referent ) for ( a ) all - cause mortality , ( b ) cardiovascular death , and ( c ) noncardiovascular death . af indicates atrial fibrillation ; bpm , beats per minute ; ci , confidence interval . linear splines were also derived for the composite endpoint of all adverse events ( sse , major bleeding , new heart failure , mi , revascularization , all - cause hospitalization , and all - cause death ) . heart rates below and above 65 bpm were associated with worse outcomes ( adjusted hr , 1.10 per 5-bpm decrease 65 bpm ; 95% ci , 1.02 to 1.19 ; adjusted hr , 1.03 per 5-bpm increase > 65 bpm ; 95% ci , 1.00 to 1.06 ) . interaction testing was performed between heart rate and baseline antiarrhythmic therapy for each of the clinical outcomes ( sse , major bleeding , new heart failure , mi , revascularization , cause - specific hospitalization , and cause - specific death ) . a significant interaction ( pinteraction<0.05 ) there was a significant association between increased heart rate and noncardiovascular death among patients not on an antiarrhythmic drug at baseline ( n=2496 ; 92% ; adjusted hr , 1.08 per 5-bpm increase ; 95% ci , 1.02 to 1.15 ; p=0.01 ) , but not for patients receiving an antiarrhythmic drug at baseline ( n=215 ; 8% ; adjusted hr , 0.75 per 5-bpm increase ; 95% ci , 0.52 to 1.07 ; p=0.1 , pinteraction=0.02 ) . cox regression models did not demonstrate significant differences in outcomes between groups : adjusted hr for nd - ccb ( versus bb ) for all - cause death , 0.99 ( 95% ci , 0.46 to 2.14 ; p=1.0 ) ; cardiovascular death , 0.97 ( 95% ci , 0.23 to 3.11 ; p=0.8 ) ; and noncardiovascular death , 1.29 ( 95% ci , 0.49 to 3.37 ; p=0.6 ) . in sensitivity analyses of patients with any type of af ( n=9648 ) , including nonpermanent forms of af , the associations between heart rate and clinical outcomes were consistent ( tables s3 and s4 ) . additional , exploratory sensitivity analyses were performed assessing the factors associated with baseline heart rate , heart rate change over time , and the association between baseline - only heart rate and subsequent clinical outcomes . other patient characteristics were minimally associated with baseline heart rate ( r=0.03 ) , and heart rate varied nearly twice as much within individuals over time as it did between patients ( variance 95 within individuals vs. 54 between individuals , in a mixed model for longitudinal heart rate ) . without updating heart rate as a time - dependent covariate , there were not significant associations between heart rate and clinical outcomes , unadjusted or adjusted ( tables s5 and s6 ) . unadjusted assessment of heart rate and ehra class included 12 299 visits for all patients documenting resting heart rate and ehra score : 2701 patients at baseline ; 2402 at 6 months ; 2153 at 12 months ; 1957 at 18 months ; 1792 at 24 months ; 960 at 30 months ; 310 at 36 months ; and 24 recorded at early study termination . there was a significant association between increasing heart rate and worse concomitant ehra symptom class across visits ( figure1 ; p<0.0001 ) . in adjusted analyses , the relationship between baseline - only resting heart rate and concomitant , baseline ehra symptom class was found to be linear , and increasing heart rate at baseline was significantly associated with more - severe baseline ehra symptom class ( adjusted or , 1.04 per 5 bpm increase ; 95% ci , 1.01 to 1.08 ; p=0.007 ; see table s3 ) . distribution of 12 299 observations of resting heart rate versus concomitant ehra symptom score in 2812 patients with permanent af . unadjusted outcomes demonstrated a j - shaped relationship between resting time - dependent heart rate and all - cause mortality ( unadjusted hr per 5-bpm decrease in heart rate 65 bpm , 1.10 ; 95% ci , 0.96 to 1.25 ; unadjusted hr per 5-bpm increase in heart rate > 65 bpm , 1.07 ; 95% ci , 1.03 to 1.12 ) . in multivariable analysis using heart rate as a continuous , time - dependent covariate , the relationship between heart rate and cause - specific mortality remained nonlinear , with an inflection point at 65 bpm . decreasing heart rate 65 bpm was associated with increasing all - cause mortality ( adjusted hr , 1.15 per 5-bpm increase ; 95% ci , 1.01 to 1.32 ; p=0.04 ) , and increasing heart rate > 65 bpm was associated with worse all - cause mortality ( adjusted hr , 1.10 per 5-bpm increase ; 95% ci , 1.05 to 1.15 ; p<0.0001 ) . bpm indicates beats per minute ; ci , confidence interval ; hr , hazard ratio ; mi , myocardial infarction ; sse , stroke or systemic embolism . relationship between time - dependent resting heart rate and clinical outcome among 2812 patients with permanent af . adjusted hazard ratios ( with 95% cis ) of increasing heart rate ( using the mean heart rate of 73 bpm as the referent ) for ( a ) all - cause mortality , ( b ) cardiovascular death , and ( c ) noncardiovascular death . af indicates atrial fibrillation ; bpm , beats per minute ; ci , confidence interval . heart rates below and above 65 bpm were associated with worse outcomes ( adjusted hr , 1.10 per 5-bpm decrease 65 bpm ; 95% ci , 1.02 to 1.19 ; adjusted hr , 1.03 per 5-bpm increase > 65 bpm ; 95% ci , 1.00 to 1.06 ) . interaction testing was performed between heart rate and baseline antiarrhythmic therapy for each of the clinical outcomes ( sse , major bleeding , new heart failure , mi , revascularization , cause - specific hospitalization , and cause - specific death ) . a significant interaction ( pinteraction<0.05 ) there was a significant association between increased heart rate and noncardiovascular death among patients not on an antiarrhythmic drug at baseline ( n=2496 ; 92% ; adjusted hr , 1.08 per 5-bpm increase ; 95% ci , 1.02 to 1.15 ; p=0.01 ) , but not for patients receiving an antiarrhythmic drug at baseline ( n=215 ; 8% ; adjusted hr , 0.75 per 5-bpm increase ; 95% ci , 0.52 to 1.07 ; p=0.1 , pinteraction=0.02 ) . cox regression models did not demonstrate significant differences in outcomes between groups : adjusted hr for nd - ccb ( versus bb ) for all - cause death , 0.99 ( 95% ci , 0.46 to 2.14 ; p=1.0 ) ; cardiovascular death , 0.97 ( 95% ci , 0.23 to 3.11 ; p=0.8 ) ; and noncardiovascular death , 1.29 ( 95% ci , 0.49 to 3.37 ; p=0.6 ) . in sensitivity analyses of patients with any type of af ( n=9648 ) , including nonpermanent forms of af , the associations between heart rate and clinical outcomes were consistent ( tables s3 and s4 ) . additional , exploratory sensitivity analyses were performed assessing the factors associated with baseline heart rate , heart rate change over time , and the association between baseline - only heart rate and subsequent clinical outcomes . without updating heart rate as a time - dependent covariate , there were not significant associations between heart rate and clinical outcomes , unadjusted or adjusted ( tables s5 and s6 ) . there is insufficient evidence to guide heart rate targets in patients with af ; however , these data provide several insights into current practice and outcomes associated with different heart rates in af . in this nation - wide community cohort of patients with permanent af , nearly all patients had resting heart rates < 110 bpm ( 99% ) and the majority ( 70% ) were < 80 bpm . however , we found that increasing heart rate above 65 bpm was associated with worse symptom class and lower survival rates , even after adjusting for baseline clinical factors . last , medical therapies appeared to have little impact on the relationship between heart rate and mortality . our results are not completely consistent with the us guidelines applicable during the study period ( 2011 ) , which designated strict heart rate control ( < 80 bpm ) as a class iii recommendation ( harm exceeds benefit)15 this recommendation was based mainly on a single trial,6 whereas previous studies had demonstrated adverse hemodynamic consequences of prolonged , uncontrolled ventricular rates.16 our data suggest that patients in community practice routinely ( 70% ) achieved more - stringent rate control ( below 80 ) and that the associated outcomes were more favorable so long as heart rate was 65 or greater . consistently , across endpoints and patient populations , increasing heart rate > 65 bpm was associated with worse outcomes , including all - cause and cause - specific mortality , as well as adverse cardiovascular events . yet , race ii was a noninferiority trial that was underpowered to detect a benefit of strict rate control : statistically , the trial could not exclude even a 4.6% absolute risk reduction in cardiovascular death , heart failure hospitalization , or stroke at 3 years with a strict rate control strategy ( in a binary comparison).6,19 additionally , follow - up heart rates in the 2 groups were closer than the targets suggested ( mean 85 bpm for the < 110 group vs. 76 bpm for the < 80 group ) . last , the relationship between heart rate and clinical outcomes , particularly mortality , could not be assessed . in contrast , the present analysis includes a significantly larger sample , and despite relatively low heart rates in this population , our data demonstrate a significant association between increased heart rate ( > 65 bpm , as a continuous covariate ) and adverse outcome . higher heart rates were associated with increased all - cause mortality in patients with or without antiarrhythmic therapy . though there was a significant interaction between antiarrhythmic use and heart rate for the endpoint of cardiovascular death , the sample was relatively small ( 8% ) and the cis were wide ( 95% ci , 0.52 to 1.07 ) . though we can not definitively identify providers target heart rates in our study , the association between increasing heart rate and adverse outcomes suggests that strict rate control may be associated with superior outcomes . patients with permanent af in us community practice maintain relatively low resting heart rates , and increased heart rates are associated with worse symptom class . moreover , there is a j - shaped relationship between heart rate and mortality for patients with af , and this is a consistent finding across endpoints . these data support current american college of cardiology / american heart association guideline recommendations for strict rate control .

the restorative properties of platelets , through the release of growth factors , have been implemented in various medical areas ( dentistry , dermatology , orthopaedic and plastic surgery , ophthalmology , etc . ) , because of their contribution to healing various tissues ( bone , skin , muscle , tendons , etc . ) . they may be injected , in situ , in the form of platelet concentrates , commonly known as prp ( platelet - rich plasma ) , obtained from autologous blood . this innovative treatment has piqued clinicians interest , particularly specialists in physical medicine and sports traumatology , more so because the production of prp is relatively easy . although intratendinous injection can slightly increase the blood concentration of growth factors ( particularly in vascular endothelial growth factor ( vegf ) ) , prp no longer appears on the list of doping products ( www.wada-ama.org ) , in contrast to other labile blood products . prp can neither improve muscle mass nor alter the transport of oxygen ( particularly because the presence of red blood cells alters prp quality ) . this literature review analyses articles on the use of prp in tendinous healing ( experimental and clinical studies ) published between 1 january 2012 and 31 december 2014 . the results of both pre - clinical and clinical studies were analyzed separately in this review and finally discussed together in order to trying to improve this therapeutic modality . articles were searched for using the medline and scopus databases , by entering , alone and/or combining , the following key words ( in french and in english ) : tenocytes , tendon , tendinopathy , prp , platelet - rich plasma . the papers with high level of proofs were preferentially selected . however , due to the small number of high level publications , most of the papers were accepted and discussed . forty seven publications relating to the use of prp were selected : 27 pre - clinical in vitro and in vivo animal studies and 20 clinical studies in humans . of these , five addressed lateral epicondylitis ; two addressed rotator cuff tendinopathies ; ten dealt with patellar tendinopathies ; and three looked at calcaneal tendinopathies . several lab studies ( in vitro and/or on animals ) have already demonstrated that prp accelerates the healing process and that each growth factor exercises a specific action during the tendon healing process . however , new studies allow us to better understand the effect of prp on tendon healing . mazzocca et al . , confirm that different prps stimulate cell proliferation ( muscle , bone and tendon ) . they did not , however , observe any significant difference between the different preparations used . in contrast , however , it appears that the most concentrated prps and those containing white blood cells are less efficient than less concentrated prps and strongly concentrated prps without white blood cells . moreover , this same team demonstrated in vitro that the anti - bacterial effect of prp against staphylococcus aureus , staphylococcus epidermis , propionibacterium acnes and methicillin - resistant staphylococcus aureus ( mrsa ) was not linked to the presence of white blood cells . in addition to the presence or otherwise of white blood cells in prp , galliera et al . observed that platelets actively controlled the recruitment of leukocytes through the intermediary of transforming growth factor ( tgf- ) . dragoo et al . , compared the inflammatory reaction caused by the infiltration of physiological liquid , blood , prps rich in white blood cells and prps low in white blood cells in rabbit tendons . five days after infiltration , the prps rich in white blood cells lead to a significant inflammatory cellular reaction , similar to that observed following blood infiltration . however , after 14 days , no difference was observed between the four groups . moreover , mccarrell et al . , assessed the effect of four prps ( prp with intermediate concentration of platelets 6.10 platelets/l ) and white blood cells ; prp with intermediate concentrations of platelets and reduced white blood cells ; prp with intermediate concentration of platelets and high concentration of white blood cells ( 12.10 platelets/l and white blood cells ) on the culture of horse tendons . they noted that the prp with an intermediate concentration of platelets and a high concentration of white blood cells led to higher expression of pro - inflammatory cytokines and catabolic mediators ( interleukin 1 ( il-1 ) , tumor necrosis factor ( tnf- ) ) as well as lower collagen synthesis . moreover , the prp with high concentrations of platelets and white blood cells led to a paradoxical effect on collagen synthesis , while also presenting an increase in inflammatory mediators . these observations were confirmed by boswell et al . , who also noted in equine tendon cultures that when platelet concentrations were too high ( despite low concentrations of white blood cells ) this reduced tendon metabolism and collagen synthesis . finally , giusti et al . , demonstrated in human tenocyte cultures that concentrations of between 5.10 and 1.10 platelets/l stimulated cell proliferation and migration as well as collagen production . on the other hand , higher platelet concentrations have an inhibitive effect both in terms of cell metabolism and collagen synthesis . in addition to the fact that prp stimulates cell proliferation and collagen synthesis , jo et al . , demonstrated that platelet activation through thrombin and calcium is more efficient that calcium activation alone . in vitro , however , prp does not enable the reversal of degenerative tendinopathies , characterised by the presence of lipid deposits , the accumulation of proteoglycans as well as the presence of calcification . however , still in vitro , prp does enable the differentiation of tenocyte stem cells and inhibits the differentiation in adipocytes , chondrocytes and osteocytes , which can hamper tendon healing . muto et al . , demonstrated that prp protects against the deleterious effects of triamcinolones on cells ( reduction in cell viability and stimulation of apoptosis ) from human rotator cuffs . , studied the effect of two different prps obtained from human blood , on isolated tenocytes in the long head of the biceps . they observed that corticoids and , particularly , local anaesthetics , alone or combined , reduced the positive effects of prp on tenocyte proliferation and viability . , who showed that local anaesthetics which may be used as painkillers during prp infiltration in clinical practice , could compromise the potential therapeutic potential of platelets . they reduce platelet aggregation but will not interfere with the release of their growth factors . finally , to reduce pain during infiltration , they recommend the use of smaller needles ( 30 g ) , because they do not alter platelet function . during the study of the effects of prp on biological activity of fibroblasts in human rotator cuffs , wang et al . , observed greater cell proliferation and collagen synthesis when prp was present in comparison with foetal veal serum . this is supported by other studies which show higher cell proliferation , better alignment of collagen fibres and better tendon bio - mechanical properties when prp is concentrated up to five times . kaux et al . , demonstrate , using rats achilles tendon lesion models that the early stages of healing ( five days ) are stimulated by prp : acute collagen synthesis and better traction resistance . within this group of prp , fernandez - sarmiento et al . , observed early healing progress , better collagen organisation , and a reduction in fibroblast and vascular density . these observations are confirmed by other series using lesion models of rats rotator cuffs . in an in vitro model on rabbits tendon cells and in vivo on rats achilles tendons , zhang et al . , proved that the anti - inflammatory effect of prp is essentially linked to hgf ( hepatocyte growth factor ) . hgf leads to a reduction in the expression of cyclooxygenases ( cox ) 1 and 2 , as well as prostaglandins ( pge2 ) . in terms of the growth factors involved in the healing process , solchaga et al . , noted that 10g platelet - derived growth factor ( pdgf - bb ) stimulated healing in rats achilles tendons , in contrast to 3g of pdgf - bb , triamcinolone , but also prp . , observed that vegf-111 , another vegf - a isoform , stimulated the early phases of the healing process in rats achilles tendons , by improving bio - mechanical properties . finally , in addition to platelet effectiveness on healing in line with a chemical tendinopathy model ( platelet and calcaneal tendons in rats ) , dallaudire et al . , did not show evidence of local toxicity of prp . in conclusion , the majority of pre - clinical studies show a beneficial effect of prp on the proliferation of tenocytes , collagen synthesis and the tendon healing process . in order to optimise efficiency , it would appear that platelet concentration should be lower than 10 platelets/l and that white blood cells should be absent . prps are used in the context of chronic tendinopathies in injectable form . for some years , it has been used increasingly regularly , more specifically in the area of physical medicine and sports traumatology . the objective is to heal tendinopathies which are particularly resistant to other conservative treatments ( eccentric physical therapy , shock waves , etc . ) and thus to avoid surgery . in a certain number of cases , the aim is also to reduce the duration of functional impotence and to encourage an early return to physical activity . however , currently the use of these prps remains a topic of debate and even controversy . table 1 presents the clinical studies discussed in this review . in a prospective three - month study , without a control group ( level 3 ) , silvestre et al . , report on the clinical evolution of 26 patients who had suffered from evolving lateral epicondylitis for more than six months and who had received prp infiltration . they noted that pain , assessed on simple verbal scale , was significantly improved , a well as the quick disabilities of the arm , shoulder and hand ( dash ) score . ultrasound observations showed the fissure had disappeared in 65% of patients , as well as a reduction in the pathological area and doppler hyperaemia in 27% of subjects . in their , compared the effect of prp or corticoid infiltration in 30 patients over a period of six weeks . they observed a positive change on the visual analogue scale ( vas ) and dash score within the two groups , but no significant difference between them . in their longitudinal six - month study ( level 4 ) of six patients who had received an injection of 3ml of prp under ultrasound , chaudhury et al . , observed a positive change in the tendon s ultrasound structure , with a trend towards increased vascularisation at the myotendinous junction . the randomised , controlled , double - blind study ( level 1 ) by krogh et al . , did not show any difference after prp , corticoid or physiological liquid infiltration in the context of epicondylitis over a period of three months . however , patients who received prp infiltration started to improve , while those who received corticoid infiltration ( resulting in fast improvement in pain in the very short term ) , indicated the progressive return of pain . recently , in their randomised and controlled study ( level 1 ) of 230 patients , mishra et al . , compared a prp group to an active control group receiving an injection of local anaesthetic . after 12 weeks , there was no difference between the two groups in terms of vas when extending the wrist against resistance and the patient - rated tennis elbow evaluation ( prtee ) self - assessment score . at the end of the 24 week follow - up period , significantly positive clinical changes were observed in the prp group of patients . to summarise , although studies with low levels of proof on the use of prp for lateral epicondylitis currently appear to be favourable , the small number of series with a high standard of proof are still contradictory and more randomised controlled studies are still needed . , ( level 1 ) compared the value of two prp infiltrations in 39 patients affected by tendinopathy of the rotator cuff , compared to two dry needle insertions guided by ultrasound , over a period of six months . after the first intervention however , after the second infiltration of prp , a clinical improvement ( in pain and mobility of the shoulder ) was observed in comparison with the control group . in their randomised , controlled study ( level 1 ) , kesikburun et al . , assessed the effect in 40 patients of an injection of either prp or physiological liquid . after 12 months , no difference could be observed between the two groups in terms of pain , quality of life , handicap and range of motion of the shoulder . to summarise , the very small number of studies with a high standard of proof on the use of prp for tendinopathies of the rotator cuff currently remain contradictory . up to now patellar tendinopathies the prospective , randomised and controlled ( level 1 ) study by almeida et al . , specified that prp improved healing at the puncture site ( patellar tendon ) required for ligamentoplasties ( kenneth - jones type ) of the anterior cruciate knee ligament . the prospective 18-month study ( level 3 ) by gosens et al . , compared 14 patients who had already received treatment ( cortocoids or ethoxysclerol and/or surgery ) with 22 patients who had never had any infiltrative or surgical treatment . after four weeks , the patients were able to gradually restart their sporting or recreational activities . an improvement in victorian institute of sport assessment for patellar tendinopathy ( visa - p ) scores , vas and pain during daily activities was seen in both groups , but more significantly so for the patients who had never had invasive treatment before prp infiltration . , report on the positive development and return to prior athletic form within six months of a 23-year old elite athlete ( level 4 ) who had received multidisciplinary treatment including prp infiltration for patellar tendinopathy . in their longitudinal study ( level 3 ) of 46 patients who had received three prp infiltrations two weeks apart , with a minimum monitoring period of up to 36 months ( average of 49 8 months ) , filardo et al . , demonstrated a positive evolution two months after infiltration , continuing at six months and until the end of the follow up period . patients with bilateral problems and whose symptoms had lasted the longest , however , evolved less well . in their randomised and controlled study ( level 1 ) , vetrano et al . , assessed the effect of prp over three treatments with focal shock waves in 46 patients suffering from patellar tendinopathy . however , after six and twelve months of follow up , the prp patients presented better progress than the group treated with shock waves . as part of their randomised and controlled study ( level 1 ) , dragoo et al . , compared the effect of prp infiltration to the introduction of a dry needle guided by ultrasound in 23 patients . although evolution at 12 weeks is better in the prp group , this beneficial effect dissipates over time and , at 26 weeks , no difference could be seen between the two groups . charousset et al . , followed 28 high level athletes ( level 4 ) who received three consecutive infiltrations of prp under ultrasound control . at the end of the two year follow - up period , the subjects reported improvements in their symptoms and function , enabling them to get back to their earlier athletic condition more quickly . moreover kaux et al . , followed 20 patients suffering from chronic patellar tendinopathy in a longitudinal cohort study ( level 3 ) following standardised prp infiltration , obtained by an apheresis machine combined with progressively intense sub - maximal eccentric physical exercise . evolution at six weeks and three months showed a significant reduction in pain and self - assessed scores , as well as in pain during maximal eccentric effort applied to the quadriceps , but no imaging improvements . two longitudinal studies ( level 3 and 4 ) relating to post - infiltration physical therapy have been published . the two programmes were based on eccentric physical therapy following a period of pain - relieving physical therapy and recovery of normal range of movement in the joint . however , the protocol used by kaux et al . , begins eccentric sub - maximal physical therapy earlier than that of van ark et al . these eccentric exercises are gradually combined with isometric work followed by concentric strengthening work on the quadriceps as well as proprioceptive exercises . at the end of the physical therapy treatment , which is supervised by a physiotherapist , report that three patients ( level 4 ) who had already received prp infiltration for unresponsive patellar tendinopathy , experienced an increase in painful symptoms leading to an inability to take part in sport , but also in increase in the thickness of the tendon and , in one patient , osteolysis of the patellar pole . fink et al . , also observed an osteolysis of the distal pole of the patella in one patient who had worsening pain and thickening of the patellar tendon after one infiltration of prp ( level 4 ) . kaux et al . , also published ( level 4 ) the case of a particularly exuberant inflammatory reaction in a type-1 diabetic patient following a prp infiltration for patellar tendinopathy . evolution was favourable at six months under pain - relieving and anti - inflammatory treatment . to summarise , studies on the use of prp for patellar tendinopathies currently appear to be favourable , but high standard of proof studies remain fairly contradictory . calcaneal tendinopathies over a period of three months , silvestre et al . , followed 32 patients ( level 3 ) with achilles tendonitis who had received ultrasound - guided prp infiltration . after one month , 22 patients were completely cured , and after two months , 28 were completely cured , with a favourable evolution in tendinous echostructure and a reduction in doppler hyperaemia at three months . only four patients did not see any clinical improvement and were operated upon . in their four - year longitudinal study ( level 3 ) of 27 patients suffering from chronic achilles tendinopathy , following three prp infiltrations two weeks apart , filardo et al . the patients who had suffered for the longest had more difficulties in returning to sporting activity . finally , murawski et al . , conducted a retrospective study ( level 4 ) on 32 patients over six months . at the end of the follow - up period , the seven others experienced no improvements in their pain and were operated upon . only four patients presented mri imaging improvements . to summarise , studies on the use of prp for calcaneal tendinopathies currently appear to be positive . however , no high standard of proof studies have taken place since the unfavourable study by de vos in 2010 which did not report any improvement after an infiltration of leukocyte rich prp compared to placebo ( saline injection ) . by releasing different platelet growth factors , prps are a new treatment for chronic tendinopathies : the ease of use , the relatively low cost , and the low invasive nature of the treatment are complementary arguments . up to now care should be taken , however , to ensure that any undesirable side effects are actually published . currently , only a bleeding disorder , a disorder of the skin covering ( infection , psoriasis ) next to the lesion could be reasonably considered as contra - indication of the use of prp in the treatment of tendinopathies . this treatment has been used for several years in different surgical specialities with favourable clinical results . despite the effectiveness of prp on tissue regeneration in vitro and in animals , the rare controlled , randomised and blind studies which exist appear to contradict one another . would this be different for certain voluminous tendons ( patellar ) compared to others ( epicondylian , rotator cuff , etc . ) ? however , as already observed in previous reviews on prp , the more recent studies can not conclude that prp is really efficient in the treatment of tendinopathy , even if most of the low level studies remain very encouraging . moreover , many of the authors of the articles reporting success of prp application ( essentially with leukocytes rich prp ) have conflicts of interest . prps are injected into the tendinous lesion , possibly guided by ultrasound ( under aseptic conditions ) to improve the accuracy of this treatment by a correct positioning of the needle . a percutaneous tenotomy using a needle ( guided by ultrasound ) could also be carried out during the infiltration . moreover , studies appear to be difficult to compare because there is no consensus on preparatory methods , qualitative characteristics of prp ( volume , platelet concentration , presence of leukocytes and erythrocytes , the infiltration technique , or the post - injection therapy protocol ) . indeed , different techniques collate different platelet volumes and concentrations : lab techniques which have been used in the past from an analytical perspective can be distinguished from recent commercial techniques . currently , the optimal concentration of platelets used in the treatment of tendinous lesions , has not yet been validated . however , the literature raises the following elements : a platelet concentration less than 10 platelets/l ( three to four times the blood platelet concentration ) would be optimal , while a concentration higher than 12.10 platelets would have a paradoxical effect of inhibiting collagen synthesis a total lack of white blood cells , which are likely to slow down healing due to the early occurrence of a higher local inflammatory reaction and the presence of pro - inflammatory factors ( cytokines and metalloproteinases ) likely to damage the extracellular matrix . the lack of red blood cells because these , once lysed , release various free radicals , similar to those of leukocytes , also likely to damage neighbouring tissues . anti - inflammatories are forbidden from day -10 to day + 21 , due to their inhibitory effect on prp action . it appears that studies where eccentric physical therapy takes place report better results than those with rest or classic rehabilitation techniques ; in fact , the prps trigger a healing process , subsequently developed through the vector of eccentric forces . the ioc coordinated the drafting of three articles on prp confirming the medical - sporting value of this treatment . it recommends that the injection is guided by ultrasound in order to ensure the correct position of the needle . a few minutes after infiltration , prps diffuse beyond the infiltrated area and the lesion site , singularly questioning the value of ultrasound - guided infiltrations . moreover , currently there is no consensus on the intratendinous and/or peritendinous site of the infiltration . experimentally , prp , through the release of different growth factors , stimulates tendinous healing . it should be examined whether prp improve tendinous healing in the same way in humans as in animals . up to now , even if prp seems to be safe and efficient to treat chronic tendinopathies non - responsive to classical conservative treatments , further high standard of proof series should enable this promising technique to be standardised and to optimise the clinical results .

objectives : the restorative properties of platelets , through the local release of growth factors , are used in various medical areas . this article reviews fundamental and clinical research relating to platelet - rich plasma applied to tendinous lesions.materials and method : articles in french and english , published between 1 january 2012 and 31 december 2014 . dealing with prp and tendons were searched for using the medline and scopus data bases.results : forty - seven articles were identified which addressed pre - clinical and clinical studies : 27 relating to in vitro and in vivo animal studies and 20 relating to human studies . of these , five addressed lateral epicondylitis , two addressed rotator cuff tendinopathies , ten dealt with patellar tendinopathies and three looked at achilles tendinopathies.conclusions : the majority of pre - clinical studies show that prp stimulates the tendon s healing process . however , clinical series remain more controversial and level 1 , controlled , randomised studies are still needed .

Introduction A. Pre-clinical studies B. Clinical studies: Discussion Conclusion Potential Conflicts of Interests

the restorative properties of platelets , through the release of growth factors , have been implemented in various medical areas ( dentistry , dermatology , orthopaedic and plastic surgery , ophthalmology , etc . ) , because of their contribution to healing various tissues ( bone , skin , muscle , tendons , etc . ) . they may be injected , in situ , in the form of platelet concentrates , commonly known as prp ( platelet - rich plasma ) , obtained from autologous blood . although intratendinous injection can slightly increase the blood concentration of growth factors ( particularly in vascular endothelial growth factor ( vegf ) ) , prp no longer appears on the list of doping products ( www.wada-ama.org ) , in contrast to other labile blood products . this literature review analyses articles on the use of prp in tendinous healing ( experimental and clinical studies ) published between 1 january 2012 and 31 december 2014 . the results of both pre - clinical and clinical studies were analyzed separately in this review and finally discussed together in order to trying to improve this therapeutic modality . articles were searched for using the medline and scopus databases , by entering , alone and/or combining , the following key words ( in french and in english ) : tenocytes , tendon , tendinopathy , prp , platelet - rich plasma . the papers with high level of proofs were preferentially selected . however , due to the small number of high level publications , most of the papers were accepted and discussed . forty seven publications relating to the use of prp were selected : 27 pre - clinical in vitro and in vivo animal studies and 20 clinical studies in humans . of these , five addressed lateral epicondylitis ; two addressed rotator cuff tendinopathies ; ten dealt with patellar tendinopathies ; and three looked at calcaneal tendinopathies . several lab studies ( in vitro and/or on animals ) have already demonstrated that prp accelerates the healing process and that each growth factor exercises a specific action during the tendon healing process . however , new studies allow us to better understand the effect of prp on tendon healing . mazzocca et al . , confirm that different prps stimulate cell proliferation ( muscle , bone and tendon ) . they did not , however , observe any significant difference between the different preparations used . in contrast , however , it appears that the most concentrated prps and those containing white blood cells are less efficient than less concentrated prps and strongly concentrated prps without white blood cells . moreover , this same team demonstrated in vitro that the anti - bacterial effect of prp against staphylococcus aureus , staphylococcus epidermis , propionibacterium acnes and methicillin - resistant staphylococcus aureus ( mrsa ) was not linked to the presence of white blood cells . observed that platelets actively controlled the recruitment of leukocytes through the intermediary of transforming growth factor ( tgf- ) . dragoo et al . , compared the inflammatory reaction caused by the infiltration of physiological liquid , blood , prps rich in white blood cells and prps low in white blood cells in rabbit tendons . five days after infiltration , the prps rich in white blood cells lead to a significant inflammatory cellular reaction , similar to that observed following blood infiltration . however , after 14 days , no difference was observed between the four groups . moreover , mccarrell et al . , assessed the effect of four prps ( prp with intermediate concentration of platelets 6.10 platelets/l ) and white blood cells ; prp with intermediate concentrations of platelets and reduced white blood cells ; prp with intermediate concentration of platelets and high concentration of white blood cells ( 12.10 platelets/l and white blood cells ) on the culture of horse tendons . they noted that the prp with an intermediate concentration of platelets and a high concentration of white blood cells led to higher expression of pro - inflammatory cytokines and catabolic mediators ( interleukin 1 ( il-1 ) , tumor necrosis factor ( tnf- ) ) as well as lower collagen synthesis . moreover , the prp with high concentrations of platelets and white blood cells led to a paradoxical effect on collagen synthesis , while also presenting an increase in inflammatory mediators . these observations were confirmed by boswell et al . , who also noted in equine tendon cultures that when platelet concentrations were too high ( despite low concentrations of white blood cells ) this reduced tendon metabolism and collagen synthesis . in addition to the fact that prp stimulates cell proliferation and collagen synthesis , jo et al . in vitro , however , prp does not enable the reversal of degenerative tendinopathies , characterised by the presence of lipid deposits , the accumulation of proteoglycans as well as the presence of calcification . however , still in vitro , prp does enable the differentiation of tenocyte stem cells and inhibits the differentiation in adipocytes , chondrocytes and osteocytes , which can hamper tendon healing . muto et al . , demonstrated that prp protects against the deleterious effects of triamcinolones on cells ( reduction in cell viability and stimulation of apoptosis ) from human rotator cuffs . , studied the effect of two different prps obtained from human blood , on isolated tenocytes in the long head of the biceps . they observed that corticoids and , particularly , local anaesthetics , alone or combined , reduced the positive effects of prp on tenocyte proliferation and viability . , who showed that local anaesthetics which may be used as painkillers during prp infiltration in clinical practice , could compromise the potential therapeutic potential of platelets . they reduce platelet aggregation but will not interfere with the release of their growth factors . finally , to reduce pain during infiltration , they recommend the use of smaller needles ( 30 g ) , because they do not alter platelet function . during the study of the effects of prp on biological activity of fibroblasts in human rotator cuffs , wang et al . , observed greater cell proliferation and collagen synthesis when prp was present in comparison with foetal veal serum . kaux et al . , demonstrate , using rats achilles tendon lesion models that the early stages of healing ( five days ) are stimulated by prp : acute collagen synthesis and better traction resistance . within this group of prp , fernandez - sarmiento et al . these observations are confirmed by other series using lesion models of rats rotator cuffs . in an in vitro model on rabbits tendon cells and in vivo on rats achilles tendons , zhang et al . hgf leads to a reduction in the expression of cyclooxygenases ( cox ) 1 and 2 , as well as prostaglandins ( pge2 ) . in terms of the growth factors involved in the healing process , solchaga et al . , noted that 10g platelet - derived growth factor ( pdgf - bb ) stimulated healing in rats achilles tendons , in contrast to 3g of pdgf - bb , triamcinolone , but also prp . , observed that vegf-111 , another vegf - a isoform , stimulated the early phases of the healing process in rats achilles tendons , by improving bio - mechanical properties . finally , in addition to platelet effectiveness on healing in line with a chemical tendinopathy model ( platelet and calcaneal tendons in rats ) , dallaudire et al . , did not show evidence of local toxicity of prp . in conclusion , the majority of pre - clinical studies show a beneficial effect of prp on the proliferation of tenocytes , collagen synthesis and the tendon healing process . prps are used in the context of chronic tendinopathies in injectable form . for some years , it has been used increasingly regularly , more specifically in the area of physical medicine and sports traumatology . the objective is to heal tendinopathies which are particularly resistant to other conservative treatments ( eccentric physical therapy , shock waves , etc . ) however , currently the use of these prps remains a topic of debate and even controversy . table 1 presents the clinical studies discussed in this review . in a prospective three - month study , without a control group ( level 3 ) , silvestre et al . , report on the clinical evolution of 26 patients who had suffered from evolving lateral epicondylitis for more than six months and who had received prp infiltration . ultrasound observations showed the fissure had disappeared in 65% of patients , as well as a reduction in the pathological area and doppler hyperaemia in 27% of subjects . in their , compared the effect of prp or corticoid infiltration in 30 patients over a period of six weeks . , observed a positive change in the tendon s ultrasound structure , with a trend towards increased vascularisation at the myotendinous junction . the randomised , controlled , double - blind study ( level 1 ) by krogh et al . , did not show any difference after prp , corticoid or physiological liquid infiltration in the context of epicondylitis over a period of three months . however , patients who received prp infiltration started to improve , while those who received corticoid infiltration ( resulting in fast improvement in pain in the very short term ) , indicated the progressive return of pain . recently , in their randomised and controlled study ( level 1 ) of 230 patients , mishra et al . after 12 weeks , there was no difference between the two groups in terms of vas when extending the wrist against resistance and the patient - rated tennis elbow evaluation ( prtee ) self - assessment score . at the end of the 24 week follow - up period , significantly positive clinical changes were observed in the prp group of patients . to summarise , although studies with low levels of proof on the use of prp for lateral epicondylitis currently appear to be favourable , the small number of series with a high standard of proof are still contradictory and more randomised controlled studies are still needed . , ( level 1 ) compared the value of two prp infiltrations in 39 patients affected by tendinopathy of the rotator cuff , compared to two dry needle insertions guided by ultrasound , over a period of six months . after the first intervention however , after the second infiltration of prp , a clinical improvement ( in pain and mobility of the shoulder ) was observed in comparison with the control group . in their randomised , controlled study ( level 1 ) , kesikburun et al . , assessed the effect in 40 patients of an injection of either prp or physiological liquid . after 12 months , no difference could be observed between the two groups in terms of pain , quality of life , handicap and range of motion of the shoulder . to summarise , the very small number of studies with a high standard of proof on the use of prp for tendinopathies of the rotator cuff currently remain contradictory . up to now patellar tendinopathies the prospective , randomised and controlled ( level 1 ) study by almeida et al . , specified that prp improved healing at the puncture site ( patellar tendon ) required for ligamentoplasties ( kenneth - jones type ) of the anterior cruciate knee ligament . after four weeks , the patients were able to gradually restart their sporting or recreational activities . an improvement in victorian institute of sport assessment for patellar tendinopathy ( visa - p ) scores , vas and pain during daily activities was seen in both groups , but more significantly so for the patients who had never had invasive treatment before prp infiltration . , report on the positive development and return to prior athletic form within six months of a 23-year old elite athlete ( level 4 ) who had received multidisciplinary treatment including prp infiltration for patellar tendinopathy . in their longitudinal study ( level 3 ) of 46 patients who had received three prp infiltrations two weeks apart , with a minimum monitoring period of up to 36 months ( average of 49 8 months ) , filardo et al . , demonstrated a positive evolution two months after infiltration , continuing at six months and until the end of the follow up period . patients with bilateral problems and whose symptoms had lasted the longest , however , evolved less well . in their randomised and controlled study ( level 1 ) , vetrano et al . , assessed the effect of prp over three treatments with focal shock waves in 46 patients suffering from patellar tendinopathy . however , after six and twelve months of follow up , the prp patients presented better progress than the group treated with shock waves . as part of their randomised and controlled study ( level 1 ) , dragoo et al . , compared the effect of prp infiltration to the introduction of a dry needle guided by ultrasound in 23 patients . although evolution at 12 weeks is better in the prp group , this beneficial effect dissipates over time and , at 26 weeks , no difference could be seen between the two groups . charousset et al . , followed 28 high level athletes ( level 4 ) who received three consecutive infiltrations of prp under ultrasound control . at the end of the two year follow - up period , the subjects reported improvements in their symptoms and function , enabling them to get back to their earlier athletic condition more quickly . moreover kaux et al . , followed 20 patients suffering from chronic patellar tendinopathy in a longitudinal cohort study ( level 3 ) following standardised prp infiltration , obtained by an apheresis machine combined with progressively intense sub - maximal eccentric physical exercise . evolution at six weeks and three months showed a significant reduction in pain and self - assessed scores , as well as in pain during maximal eccentric effort applied to the quadriceps , but no imaging improvements . two longitudinal studies ( level 3 and 4 ) relating to post - infiltration physical therapy have been published . however , the protocol used by kaux et al . , begins eccentric sub - maximal physical therapy earlier than that of van ark et al . at the end of the physical therapy treatment , which is supervised by a physiotherapist , report that three patients ( level 4 ) who had already received prp infiltration for unresponsive patellar tendinopathy , experienced an increase in painful symptoms leading to an inability to take part in sport , but also in increase in the thickness of the tendon and , in one patient , osteolysis of the patellar pole . , also observed an osteolysis of the distal pole of the patella in one patient who had worsening pain and thickening of the patellar tendon after one infiltration of prp ( level 4 ) . kaux et al . , also published ( level 4 ) the case of a particularly exuberant inflammatory reaction in a type-1 diabetic patient following a prp infiltration for patellar tendinopathy . to summarise , studies on the use of prp for patellar tendinopathies currently appear to be favourable , but high standard of proof studies remain fairly contradictory . calcaneal tendinopathies over a period of three months , silvestre et al . , followed 32 patients ( level 3 ) with achilles tendonitis who had received ultrasound - guided prp infiltration . only four patients did not see any clinical improvement and were operated upon . in their four - year longitudinal study ( level 3 ) of 27 patients suffering from chronic achilles tendinopathy , following three prp infiltrations two weeks apart , filardo et al . the patients who had suffered for the longest had more difficulties in returning to sporting activity . finally , murawski et al . , conducted a retrospective study ( level 4 ) on 32 patients over six months . at the end of the follow - up period , the seven others experienced no improvements in their pain and were operated upon . to summarise , studies on the use of prp for calcaneal tendinopathies currently appear to be positive . however , no high standard of proof studies have taken place since the unfavourable study by de vos in 2010 which did not report any improvement after an infiltration of leukocyte rich prp compared to placebo ( saline injection ) . by releasing different platelet growth factors , prps are a new treatment for chronic tendinopathies : the ease of use , the relatively low cost , and the low invasive nature of the treatment are complementary arguments . up to now care should be taken , however , to ensure that any undesirable side effects are actually published . this treatment has been used for several years in different surgical specialities with favourable clinical results . despite the effectiveness of prp on tissue regeneration in vitro and in animals , the rare controlled , randomised and blind studies which exist appear to contradict one another . would this be different for certain voluminous tendons ( patellar ) compared to others ( epicondylian , rotator cuff , etc . ) ? however , as already observed in previous reviews on prp , the more recent studies can not conclude that prp is really efficient in the treatment of tendinopathy , even if most of the low level studies remain very encouraging . moreover , many of the authors of the articles reporting success of prp application ( essentially with leukocytes rich prp ) have conflicts of interest . a percutaneous tenotomy using a needle ( guided by ultrasound ) could also be carried out during the infiltration . moreover , studies appear to be difficult to compare because there is no consensus on preparatory methods , qualitative characteristics of prp ( volume , platelet concentration , presence of leukocytes and erythrocytes , the infiltration technique , or the post - injection therapy protocol ) . indeed , different techniques collate different platelet volumes and concentrations : lab techniques which have been used in the past from an analytical perspective can be distinguished from recent commercial techniques . currently , the optimal concentration of platelets used in the treatment of tendinous lesions , has not yet been validated . however , the literature raises the following elements : a platelet concentration less than 10 platelets/l ( three to four times the blood platelet concentration ) would be optimal , while a concentration higher than 12.10 platelets would have a paradoxical effect of inhibiting collagen synthesis a total lack of white blood cells , which are likely to slow down healing due to the early occurrence of a higher local inflammatory reaction and the presence of pro - inflammatory factors ( cytokines and metalloproteinases ) likely to damage the extracellular matrix . the lack of red blood cells because these , once lysed , release various free radicals , similar to those of leukocytes , also likely to damage neighbouring tissues . anti - inflammatories are forbidden from day -10 to day + 21 , due to their inhibitory effect on prp action . it appears that studies where eccentric physical therapy takes place report better results than those with rest or classic rehabilitation techniques ; in fact , the prps trigger a healing process , subsequently developed through the vector of eccentric forces . the ioc coordinated the drafting of three articles on prp confirming the medical - sporting value of this treatment . a few minutes after infiltration , prps diffuse beyond the infiltrated area and the lesion site , singularly questioning the value of ultrasound - guided infiltrations . experimentally , prp , through the release of different growth factors , stimulates tendinous healing . up to now , even if prp seems to be safe and efficient to treat chronic tendinopathies non - responsive to classical conservative treatments , further high standard of proof series should enable this promising technique to be standardised and to optimise the clinical results .

docking can play a key role in addressing a number of important problems such as protein - function prediction or drug - lead identification and optimization . the first is pose generation , which starts with the determination of the position , orientation , and conformation of a molecule as docked to the target s binding site . the second stage is scoring , which predicts how strongly the docked pose of such a putative ligand binds to the target . while pose generation is relatively well handled by current algorithms , the inaccuracies of current scoring functions still constitute the main barrier to achieving reliability in docking . indeed , despite intensive research over more than two decades , accurate prediction of the binding affinities of large sets of diverse protein ligand complexes remains one of the most important open problems in computational bioscience . three classes of scoring functions have emerged over the years : force field , knowledge - based , and empirical . for the sake of efficiency , scoring functions do not attempt to simulate certain physical processes that influence the process of binding . this has an impact on their ability to rank - order and selects small molecules by predicted binding affinity . thus , two major sources of error in scoring functions arise from their limited description of protein flexibility and the implicit treatment of solvent . instead of scoring functions , other computational methodologies based on molecular dynamics or monte carlo simulations can be used to model protein flexibility and desolvation upon binding . in principle , a more accurate prediction of binding affinity than that from scoring functions is obtained in those cases amenable to these techniques . however , such expensive free energy calculations are not feasible for the evaluation of large numbers of protein ligand complexes , and their application is generally limited to predicting binding affinity in series of congeneric molecules binding to a single target . in addition to these two enabling simplifications , there is a third factor in scoring function development that , despite its importance , has received little attention until recently . each scoring function assumes a predetermined functional form relating the variables that describe the complex , which may also include a set of weights that are fitted to experimental or simulation data , and its predicted binding affinity . such a relationship typically takes the form of a sum of weighted physicochemical contributions to binding in the case of empirical scoring functions or a reverse boltzmann methodology in the case of knowledge - based scoring functions . as previously discussed , the inherent drawback of this approach is that those complexes not conforming to this strong modeling assumption will be predicted poorly . as an alternative to these classical scoring functions , nonparametric machine learning can be used to capture implicitly the binding interactions that are challenging to model explicitly . by not imposing a particular functional form for the scoring function , the collective effect of intermolecular interactions in binding can be directly inferred from experimental data . indeed , this unconstrained approach had to result in performance improvement given sufficient data , as it is well - known that the strong assumption of a predetermined functional form for a scoring function constitutes an additional source of error ( e.g. , imposing an additive form for the considered energetic contributions ) . on the other hand , recent experimental results have resulted in novelties in the definition of molecular interactions such as the hydrogen bond and the hydrophobic interaction , implying that previously proposed expressions for these energetic contributions might need to be revised accordingly . while a few classifiers exploiting x - ray crystal structural data for discriminating between binders and nonbinders of a protein target have been presented , it is only recently that machine learning for nonlinear regression has been shown to be a particularly powerful approach to build generic scoring functions . this approach has been highlighted as very promising for the improvement of scoring functions . indeed , a growing number of studies showing the benefits of machine learning scoring functions have been presented . however , these initial models are relatively coarse in the description of the complex , and thus the question remains as to whether the incorporation of additional chemical information relevant for binding would improve performance further . here , we investigate the impact of a more precise chemical characterization of the protein this includes the use of structural interaction fingerprints , using atom and interaction type definitions from the credo structural interactomics database . we show that the new version of rf - score performs much better than classical scoring functions on the same test set . the rf - score performed best when describing a complex using a 12 distance cutoff between atom pairs , suggesting that there is a minor contribution from long - range atom pairs . in the light of the improved performance obtained and considering the uncertainty introduced by the static nature of crystal structures , we discuss the role of interatomic distance cutoffs and binning as well as protonation states in binding affinity prediction . as a byproduct of this systematic battery of numerical experiments , the most accurate scoring function to date on a widely used pre - existing benchmark is presented . an important conclusion of our study is that a more chemically precise description of the protein ligand complex does not generally lead to more accurate prediction of binding affinity . we discuss four factors that may contribute to this result : modeling assumptions , codependence of representation and regression , data restricted to the bound state , and conformational heterogeneity in data . each complex was described by a vector of integer - valued descriptors or features . three description schemes were implemented : the element scheme uses the combination of the element symbols of the interacting atoms to classify the interaction , e.g. , c c or n o . the fingerprint of this scheme has a position for each pairwise combination of element symbols , and the directionality is preserved ; i.e. , n o is distinct from o n . here , all of the heavy atoms commonly observed in pdb complexes ( c , n , o , f , p , s , cl , br , i ) are considered . the sybyl scheme uses sybyl atom types instead of the element symbols to define the range of considered protein ligand atom pairs . these atom types permit deconvoluting the element into hybridization state and bonding environment . for instance , instead of having a single c element atom type , the sybyl scheme considers the following subtypes : c+ , c1 , c2 , c3 , cac , and car ( a description of sybyl atom types can be found at http://www.tripos.com/mol2/atom_types.html ) . the latter leads to 36 distinct c c descriptors in the sybyl scheme in contrast to a single c c descriptor in the element scheme . the credo scheme uses structural interaction fingerprints ( sifts ) to encode protein ligand interactions . here , interatomic pairs are categorized as interactions if particular geometrical and atom type constraints are satisfied . atom types were defined through a set of smarts patterns that are completely customizable through a configuration file . the atom types of atoms belonging to standard amino acids as well as nonstandard binding site residues that occurred in the used test sets were precalculated because determining them on the fly was not feasible with the open babel toolkit . these atom types were stored in a separate configuration file and can therefore be easily changed by the user . the determination of standard and weak hydrogen bonds required the protonation state to be known , and there the complexes to rescore must be already protonated . twelve different contact types are encoded in sift , of which four are solely distance - based . the latter are covalent bonds , van der waals clashes and contacts , and finally proximal interactions . the other eight feature contact types are hydrogen bonds , weak hydrogen bonds , halogen bonds , ionic , metal complexes , aromatic , hydrophobic , and carbonyl . the definition of these including the source of the smarts definitions and other constraints are described in the original credo publication with the following exception : the carbonyl interaction type has since then been implemented on the basis of the ab initio molecular - orbital calculations of allen et al . who have shown that carbonyl appendix a3 in the supporting information summarizes the exact classification scheme for all interactions that was used for the sift descriptor . once the scheme is selected , descriptors are generated by counting interatomic pairs between protein binding sites and their ligand molecules . these possibly interacting atoms were assigned a specific interaction type if their distance was within a distance threshold and if a combination of possible atom type and geometry constraints was satisfied . the software to calculate these three description schemes uses the open source chemistry toolkit open babel in version 2.3.0 ( through python bindings ) and the scipy library . the molecular structures of protein targets and their ligands comprising the used data sets from pdbbind are read - in separately . interatomic contacts for a given distance cutoff are determined using the kdtree structure in the scipy.spatial module with the atomic coordinates as input ( the kdtree is an efficient space - partitioning algorithm that limits the search space for interatomic contacts in order to prevent expensive all - by - all searches ) . the interacting atoms are then analyzed depending on the descriptor and the appropriate position on the fingerprint incremented . this descriptor - generating software is also capable of binning the identified interatomic pairs into arbitrary distance ranges . for each normal feature on a descriptor , a number of columns equal to the number of distance bins are created . using a distance cutoff of 6 and a bin size of 1 for example would create six bins for each feature : from 0 to 1 , 12 , and so on . the correct bin for each interatomic pair that has to be incremented the complete source code of the software that was used to generate the described results was released at https://bitbucket.org/aschreyer/rfscore under the mit license . rf - score uses random forest ( rf ) as the regression model . a rf for regression is an ensemble of p regression trees randomly generated from the same training data . in building these trees , rf determines the best split at each node of the tree from a subset of randomly selected features of size mtry ( the recommended value of this control parameter is a third of the number of features ) . note that although the selected features are generally different at each node , the same mtry value is applied to each node across the p trees of the forest . here , we operate the rf with the default value p = 500 , as its performance does not generally improve significantly beyond this threshold . the performance of each tree on predicting the out - of - bag ( oob ) set , here protein ligand complexes that were not in the bootstrap sample used to train that tree , collectively provides an internal validation measure of rf . further details about rf and its application to build rf - score can be found in ref ( 23 ) . the pdbbind benchmark has become a de facto standard in the validation of scoring functions . on the basis of the refined set from the 2007 release of the pdbbind database , it comprises 1300 diverse protein ligand complexes with high quality structural and binding data ( the protonation states of both proteins and ligands were already calculated by these authors ) . from this refined set , cheng et al . constructed a test set , named the core set , with 195 diverse complexes spanning more than 12 orders of magnitude in measured binding affinities . the pdbbind benchmark consists of testing the predictions of scoring functions on the core set , whereas the remaining 1105 refined set complexes are used as the training set . a discussion on the composition and suitability of this benchmark can be found in refs ( 23 ) and ( 29 ) . we start this section with a concise description of the approach to predicting binding affinity using machine learning . this process starts with the characterization of each protein ligand complex as a set of intermolecular features or descriptors relevant to binding affinity prediction . the sketch in figure 1 shows an example of how descriptors are generated from the x - ray crystal structure of a complex ( pdb : 2p33 ) . for example , a descriptor could be defined as the number of times that protein nitrogen and ligand oxygen are separated by less than a distance cutoff ( dcutoff ) . as in previous studies , nine atom types commonly observed in pdb complexes were selected by considering atomic number only ( c , n , o , f , p , s , cl , br , i ) to give rise to 81 protein ligand atom pairs which are considered as descriptors ( this descriptor scheme is named here element ) . sketch of the process of characterizing a protein ligand complex ( pdb : 2p33 ) as a set of structure - derived descriptors ( c.c to i.i ) . the discontinuous green lines connect the ligand chlorine atom with all protein carbon atoms within the distance cutoff represented by the green sphere , with the number of these pairs giving value to the c.cl descriptor . once the descriptor scheme ( i.e. , considered atom types , binning , and cutoff ) is chosen , the next step is to select a source of curated structural and interaction data suitable for training and testing scoring functions . the pdbbind database is an excellent choice for this purpose , with the additional advantage that a large number of scoring functions have already been benchmarked on a common pdbbind test set , which permits comparing new developments against the state of the art . moreover , some scoring functions have not only been tested on this common data set but also calibrated on the same training set ( further details can be found in the methods section ) . this is important to avoid the often large bias introduced by using a different training set for each scoring function ( such bias makes comparisons among scoring functions unreliable , even if compared on the same test set ) . therefore , we will be focusing here on these common training and test sets . last , a regression model is needed to predict the binding affinity of test set complexes from the structural and interaction data in the training set . here , we build upon rf - score , a machine learning scoring function using rf for regression . rf is typically tuned using a single control parameter ( mtry , which controls the number of features that are considered for the split at each tree node ) and may be subjected to a feature selection strategy intended to remove descriptors with low information content as a way to improve performance ( that is , in addition to the common practice of removing all those descriptors that have zero values across training complexes ) . as usual , predictive performance is measured as the difference between predicted and measured binding affinity across test set complexes . full details on descriptors , data , and regression protocols can be found in the methods section . top : descriptors are generated from two nonoverlapping data sets with 1105 and 195 complexes for training and testing , respectively . bottom : training random forest to learn the nonlinear relationship between this atomic - level description of the complex and its binding affinity ( pkd or pki ; pkd / i denotes both without distinction ) . the resulting scoring function ( rf - score ) is used to predict the binding affinity of the test set . we have seen that not only can a protein ligand complex be described in various ways , but also the process of building a predictive model from these descriptors involves a number of choices . in a way , the overall process of training a scoring function can be regarded as a quest for finding an optimal combination of these design variables . an exhaustive evaluation of all possible combinations is impractical , as this would involve a prohibitively large number of rf training runs ( 2 runs , one for each possible subset of m features , even if we fix mtry to its recommended value ) . thus , we were forced to assume the independence of these design variables and searched for the optimal value of each variable in a sequential manner as explained in the next subsections . the first question we addressed was which distance cutoff leads to the best performance on the independent test set ( henceforth referred to as simply the test set ) . different distance cutoffs have been previously used in the literature , some as large as 12 ( pmf ) and 15 ( fresno ) . here , we addressed this question empirically by generating element descriptors for four cutoffs ( 6 , 9 , 12 , and 15 ) , which gave rise to four different numerical characterizations of the training set with their corresponding test set counterparts . thereafter , rf was calibrated on each of these training set versions and the resulting model used to predict the binding affinity of the corresponding test set complexes . as scoring function calibration is a stochastic process , a slightly different model is obtained with a different random seed . to assess the variability in rf prediction due to this factor , we repeated the training 10 times for each cutoff , each time with a different random seed . such assessment is needed to establish whether the improvement in prediction is due to using another distance cutoff or just comes from variability in model calibration ( this procedure is more accurate than basing the analysis on a single model calibration as has been the case so far ) . lastly , we considered three commonly used metrics for quantifying the difference between predicted and measured binding affinity across the test set of protein ligand complexes : rp ( pearson s correlation coefficient ) , rs ( spearman s correlation coefficient ) , and sd ( standard deviation in log kd / i units ) . test set performance of rf - score with element descriptors for each of the four distance cutoffs ( 6 , 9 , 12 , and 15 ) . performance is measured as the difference between observed and predicted binding affinity in the test set using three metrics : pearson s correlation coefficient ( rp ; left plot ) , spearman rank - correlation coefficient ( rs ; middle plot ) , and standard deviation ( sd ; right plot ) . ten models are built from each of these four versions of the training data sets ( 6 , 9 , 12 and 15 ) , each time using a different random seed ( the boxplot summarizes the performance on the test set achieved by each of the 10 models ) . results showed that the best median performance , i.e. , that with the highest correlations and lowest standard deviation , is obtained with the 12 cutoff in all three performance metrics . it is worth noting that optimizing the distance cutoff only led to a modest performance improvement ( + 0.017 in median rp and 0.05 log k units in median sd ) . the element descriptors used in the previous experiment constitute a coarse representation of the complex . distinguishing between atoms of the same element in different local environments leads to a more chemically precise characterization ( e.g. , deconvoluting the occurrence counts of a carbon carbon intermolecular pair into pairs that specify the hybridization state of both atoms ) , and thus the resulting model would in principle be expected to perform better . to test this hypothesis further , one could also incorporate additional information into the descriptors such as the angle between hydrogen bond donors , acceptors , and hydrogen atoms as well as covalent and van der waals radius . these are the credo descriptors , which measure the abundance of a range of intermolecular interactions such as hydrogen bonds , hydrophobic interactions , or van der waals clashes . using the same training and test set , each description scheme gives rise to a different set of features that are used to characterize every complex . the performance of each of these three description schemes is presented in figure 4 . test set performance of rf - score using the optimal 12 interatomic distance cutoff with element , sybyl , and credo descriptors . interestingly , the model based on credo descriptors obtained much lower performance than that using sybyl and element descriptors . these results hint at a trade - off between the predictability and interpretability of the model , which we will discuss later in this paper . the strength of the interatomic interactions that collectively form the noncovalent intermolecular bond depends on the separation between the interacting atoms . therefore , it is reasonable to think that partitioning the descriptors into a number of interatomic distance bins should lead to a model with more predictivity . consequently , we generated element descriptors with a 12 cutoff , i.e. using all the optimal values , for six bin sizes ( a 12 bin size with a 12 cutoff simply corresponds to the case without binning , which was previously shown in the first boxplot in figure 4 and the third boxplot in figure 3 ) . figure 5 shows the results for each bin size , where the best median performance is achieved by models with lower bin sizes ( 1 , 2 , and 3 ) , representing a moderate improvement with respect to the model based on single - binned descriptors ( 12 ) . the experiments were repeated using the same bin sizes but now with sybyl and credo descriptors instead of elements descriptors ( the maximum cutoff for a credo interaction type is 4.5 , all other atom pairs in this description scheme are labeled as proximal ) . it was observed that the performance was not as high as that with element descriptors ( the best median performance for sybyl was rp = 0.779 , rs = 0.771 , and sd = 1.59 , whereas that for credo was rp = 0.739 , rs = 0.742 , and sd = 1.68 ) . test set performance of rf - score with element descriptors and 12 interatomic distance cutoff using six different bin sizes ( 1 , 2 , 3 , 4 , 6 , and 12 ) . the best median performance is achieved by models with lower bin sizes ( 1 , 2 , and 3 ) , representing a modest improvement with respect to the model based on single - binned descriptors ( 12 ) . in addition to exploring the impact of different ways to describe the complexes , we also applied basic feature selection strategies intended to remove sparse features that increased the complexity of the model without improving performance . here , the sparsity ( spr ) of a descriptor is defined as the average number of occurrence counts per training complex . in previous versions of rf - score , only features with sparsity higher than the zero threshold ( spr = 0 ) , i.e. , those that are nonzero for at least one training complex , were considered . here , we also considered two additional sparsity thresholds ( spr = 1 and spr = 2 ) . we conducted this experiment for the three best bin sizes in figure 5 ( 1 , 2 , and 3 ) , which had spr = 1 as the optimal value on all three bin sizes . figure 6 presents the results for the best bin size across the three spr values ( 2 ) . test set performance of rf - score with element descriptors , 12 cutoff , and 2 bin size using three values of the feature selection threshold ( spr ) . best median performance is obtained by spr = 1 , which corresponds to only considering descriptors that have an average of at least one atom the latter represents a moderate improvement with respect to the models using descriptors with spr = 0 and spr = 2 . last , we have been using the recommended value for the rf mtry parameter so far . however , interval validation strategies can be used to select an optimal value for mtry . one of these strategies is called out - of - bag ( oob ) validation and essentially consists of training the model for each possible value of mtry and selecting the model that best performs on the internal validation set ( a subset of the training set , as further explained in the methods section ) . figure 7 shows that this model selection strategy carries a small improvement in performance at the cost of much higher computational expense in model selection ( one rf training run per considered mtry value ) . test set performance of rf - score with element descriptors , 12 cutoff , 2 bin size , and feature selection threshold ( spr=1 ) for the recommended mtry ( model selection=0 ) and mtry selected by oob validation ( model selection=1 ) , which requires 123 times more training than just using the recommended setting ( as many rf training runs as features were selected to describe each complex ) . this systematic battery of numerical experiments led to the new scoring function rf - score::elem(c12,b2)_spr1_oob ( rf - score::elem - v2 for short ) . as we have seen , the descriptors come from partitioning occurrence counts of each element atom type pairs into six interatomic distance bins of 2 size , and the rf model is built with the 123 descriptors that are sufficiently dense ( spr = 1 ) using the internally validated mtry value ( mtry = 14 in this case ) . figure 8 shows the predictive power of rf - score::elem - v2 on the test set . rf - score::elem - v2 predicted versus measured binding affinity on the independent test set ( 195 complexes ) . pearson s correlation coefficient rp = 0.803 , spearman s correlation coefficient rs = 0.797 , standard deviation sd = 1.54 log kd / i units , and root mean square error rmse = 1.53 log kd / i units . this plot can be visually compared to those for the best performing scoring functions in cheng et al.s figure 6 . performance comparisons on the same test set are presented here in figure 9 . in terms of efficiency ligand complexes in 0.01 s ( all of the computation in this study was carried out with a single processing core intel core i72920xm at 2.50 ghz with 16 gb ram ) . in addition , the time to generate these features for the 195 complexes was 8 s , and hence this is the most expensive part of the calculation . therefore , the average time to score one protein ligand complex is about 0.04 s if the features have not been calculated before , which makes rf - score suitable to rescore a high number of docking poses in virtual screening applications . the predictive power of rf - score::elem - v2 was also compared against that of a wide selection of scoring functions on the pdbbind benchmark . by using a pre - existing benchmark , the danger of constructing a benchmark complementary to the presented scoring function is avoided . it also has the advantage of ensuring that previously tested scoring functions were provided with optimal settings by their authors . several of the scoring functions tested in the pdbbind benchmark have different versions or multiple options . however , for the sake of practicality , only the version / option of each scoring function that performs best on the pdbbind benchmark was reported by cheng et al . in addition to these 16 scoring functions , we also tested a more recent function called imp::rankscore . figure 9 reports the performance of all scoring functions on the test set , with rf - score::elem - v2 obtaining the best performance with rp = 0.803 ( the performance of the original version of rf - score is also included ) . in contrast , classical scoring functions tested on the same test set obtained a lower rp spanning from 0.216 to 0.644 . this trend was also observed with the other two performance measures ( rs , sd ) . it is worth noting that the root - mean - square error of the free energy of binding on such a diverse test set is just 2.1 kcal / mol . performance of 18 scoring functions on the pdbbind benchmark as measured by pearson s correlation coefficient ( rp ) , spearman s correlation coefficient ( rs ) , and standard deviation of the difference between predicted and measured binding affinity ( sd ) . the three plots on the right visually show the relative predictive power of rf - score ( x signs ) against that of the other 17 scoring functions ( + signs ) . nha is the performance of a linear regression model with the number of heavy atoms of the ligand as only variable ( this baseline is shown as a horizontal discontinuous line in the plots ) . when introducing a scoring function , only the scoring function built with the random seed that provides the best performance we have followed here a more precise way to assess performance differences between scoring functions by comparing median performances from a set of independent trials . moreover , in order to address the question of how significant is the reported improvement over the original version of rf - score , we have trained and tested the original rf - score using 10 different random seeds . thereafter , we have repeated the process , using the same random seeds , with the new version of rf - score . lastly , we carried out a two - sample t test for each performance measure to find out that all differences are statistically significant ( rp p value= 6.0 10 , rs p value=1.5 10 , and sd p value = 3.8 10 ) . performance comparison between the original version and the new version of rf - score . the new version of rf - score performs much better than classical scoring functions on the same test set . in fact , this performance gain must be actually larger in most cases since only rf - score and x - score , among all scoring functions represented in figure 9 , use training sets that do not overlap with the test set . having training complexes in the test set artificially enhances the performance of a scoring function , as it is not exclusively predicting unseen complexes but merely reporting the lower training error of those overlapping complexes . indeed , adding a third of the test set to the training set makes rf - score::elem - v2 s rp rise from 0.803 ( no overlap between training and test sets ) to 0.872 ( 65 overlapping complexes ) . clearly , the same training and test set must be used when comparing scoring functions , but unfortunately this has not been required in recent community benchmarks . furthermore , it could be argued that there is something particular about the training / test partition selected by cheng et al . in the pdbbind benchmark . this partition was chosen to compare rf - score against the best scoring function in that study under exactly the same experimental conditions . an experiment to investigate this question was already carried out in the original rf - score paper ( appendix a4 in the supplementary data of that paper ) and further discussed in a subsequent commentary ( see figure 1 therein ) . the performances of rf - score for 25 randomly generated training / test partitions with the same sizes as the benchmark partition ( 1105/195 ) were calculated . the experiment demonstrated that there is a minor difference in rf - score performance between the benchmark partition and the median of these 25 alternative partitions . while our study focuses on generic scoring functions , we would like to briefly comment on how rf - score would perform on subgroups of the test set ( e.g. , complexes whose proteins belong to the same family ) . clearly , the better the performance of rf - score over another scoring function on the full test set , the more test subsets will be better predicted by rf - score . to illustrate this , appendix a1 presents the performance of the new version of rf - score ( rf - score::elem - v2 ; full test set rmse = 1.54 ) and rf using credo intermolecular interaction features ( rf - score::credo ; full test set rmse = 1.72 ) on the four subsets resulting from partitioning the test set by binding affinity ranges . appendix a2 presents another experiment where two small subsets of 23 complexes are generated , one containing those with the most similar ligands and the other subset with the most dissimilar ligands in terms of chemical structure . rf - score::elem - v2 outperforms rf - score::credo in all subsets but the one with the most dissimilar ligands , where rf - score::credo performs slightly better . these results illustrate the fact that , despite rf - score::elem - v2 generally performing better , there could be a few complexes ( e.g. , ligand - bound structures of a particular target ) where other scoring functions perform better . we intend to study this issue further in the future . on the other hand , it is noteworthy that rf - score performed best when describing a complex using a 12 distance cutoff between atom pairs , a distance well beyond direct interatomic contacts . the improvement over rf - score with a more common 6 cutoff is , however , modest ( + 0.017 in median rp , + 0.003 in median rs , and 0.05 log k units in median sd ; see figure 3 ) . this result suggests that there is a minor favorable contribution from atom pairs separated by a distance between 6 and 12 over the 1300 considered complexes . such long - range contributions to protein ligand binding affinity have been attributed to the electronic properties of the binding site and ligand being affected by all protein atoms and also to long - range electrostatics interactions . increasing the noncovalent cutoff to 12 has also been found beneficial in protein folding and dna molecular dynamics simulations . it seems therefore that rf - score is able to capture long - range effects implicitly to some extent . the main reason why rf - score works much better than classical scoring functions at predicting binding affinity of most complexes is due to the circumvention of the strong assumption of a predetermined functional form . all classical scoring functions consist of a sum of typically nonlinear terms with respect to selected interatomic distances , such as van der waals terms in empirical scoring functions or particular atom for instance , the scoring function consortium ( sfc ) , in a concerted effort between 10 pharmaceutical companies and academic institutions , generated an empirical scoring function ( sfcscore ) , which by the time of its development was clearly superior to most of the then available scoring functions . very recently , one of the leading sfc authors has demonstrated that , by using random forest regression instead of sfcscore s additive functional form and keeping all other modeling choices unaltered ( training data , test data , and descriptors ) , performance rises from rmse = 1.84 to rmse = 1.56 ( 0.683 to 0.788 in the case of rs ) . this is a very large improvement for a single modification in a generic model , especially taking into account that scoring functions are highly optimized due to intense work over the years in this area . another study demonstrating that assuming an additive functional form is detrimental for the performance of empirical scoring functions is by kinnings et al . as force - field and knowledge - based scoring functions make the same assumption , these studies strongly suggest that a machine - learning version of other classical scoring functions will also result in significant improvement . another important conclusion of our study is that a more precise chemical description of the protein ligand complex does not generally lead to more accurate prediction of binding affinity ( see figure 4 ) . in the first study , li et al . present a scoring function tested on exactly the same test set as us , with a much larger training set that includes ours and the use of a very precise description consisting of 50 calculated descriptors falling into nine interaction categories : van der waals , hydrogen - bonding , electrostatic , pi - system , metal ligand bonding , desolvation effect , entropic loss effect , shape matching , and surface property matching ( table 1 on page 593 of li et al.s paper ) . li et al . obtained sd = 1.63 and rs = 0.779 ( table 4 at page 597 of li et al.s paper ) , whereas rf - score originally obtained sd = 1.58 and rs = 0.762 on the same test set . interestingly , these authors referenced rf - score but did not include it in the comparison or comment on why its performance was better in some performance measures despite using much simpler descriptors and less data for training . zilian and sotriffer used the same training set , test set , and regression model as rf - score . the only difference between their scoring function and ours is in the 63 used descriptors , which was one of the outcomes of the industry these descriptors include the number of rotatable bonds in the ligand , hydrogen bonds , aromatic interactions , and polar and hydrophobic contact surfaces , among others ( a full list can be found in table 1 of page 398 of the original sfc paper ) . their best scoring function achieved rmse = 1.56 and rs = 0.788 ( table 1 of zilian and sotriffer s paper ) , which is slightly better than the original version of rf - score ( rmse = 1.58 and rs = 0.762 ) . if the modeling assumptions implied in the calculation of chemical properties were generally accurate , we should have seen many scoring functions performing much better than rf - score thanks to using a more precise chemical description . but we have actually seen the opposite in these two independent studies , once we compare the performances achieved by li et al . ( sd = 1.63 and rs = 0.779 ) and zilian and sotriffer ( rmse = 1.56 and rs = 0.788 ) to that of the new version of rf - score ( sd = 1.54 , rmse = 1.54 and rs = 0.797 ) on the same diverse test set . the new version differs from the original version of rf - score in that features are distance - dependent but still do not explicitly incorporate calculated protonation states . we discuss next four convoluted factors that may contribute to this result : modeling assumptions , codependence of representation and regression , data restricted to the bound state , and conformational heterogeneity in data . the first factor is that more precise descriptors often mean making modeling assumptions that introduce additional error . for example , the protonation state of an atom needs to be estimated in order to assign its sybyl type , but the local change in ph induced by hydrogen bond donors / acceptors in nearby residues and water molecules is usually not incorporated into scoring functions for binding affinity prediction . similar arguments can be constructed regarding the calculation of donor - hydrogen - acceptor angles to perceive hydrogen bonds . the question remains as to how large the impact of this error is compared to that of not considering protonation states at all ( the element descriptor scheme ) . the second factor , often neglected , is the optimality of problem representation ( description scheme ) for the applied solution construction method ( regression technique ) . from a purely chemical perspective , deconvoluting elemental atom types into their various hybridization states constitutes a more precise description of the complex . however , this scheme also results in a higher number of features and thus more sparse features . the latter are detrimental for random forest regression because as many data as possible are needed to characterize the interaction between each pair of atom types , best achieved by minimizing the number of different types defined . in practice , the definition of atom types must reflect a compromise between these two conflicting objectives , so as to ensure that the features are backed up by sufficient data to be statistically as well as chemically meaningful . this situation gives rise to a trade - off between the predictability and interpretability of the model , which is not uncommon in regression problems and has also been observed here ( see figure 4 ) . for the sake of efficiency , scoring functions only exploit the information contained in the bound state of the complex , as represented by a crystal structure . however , binding affinity also depends on the energetic contributions from ligand and protein desolvation as well as induced fit upon binding . the third contributing factor is therefore the uncertainty about how well a particular description of the bound complex is also describing the complex just before desolvation and induced fit takes place . we speculate that descriptors whose values change less during the binding process might be more suitable for predicting binding affinity using only data about the bound state . for instance , element descriptors do not change much in general during the binding process , as a fixed cutoff will include roughly the same protein and ligand atoms just before and after binding . in contrast , protonation states will generally change significantly upon binding because of desolvation . the last contributing factor comes from the uncertainty arising from the fact that the crystallographers deposit a single structural model in the pdb while several different models may fit the electron density equally well . the conformational heterogeneity of a complex ( i.e. , several bound states are possible for this complex , at least within experimental uncertainty ) means that different sets of descriptors would be generated for exactly the same binding affinity . access to the multiple structural models of a complex that are significantly different at the binding site level is likely to be helpful in deciding how to best address this issue . in particular , it would be interesting to investigate whether combining the predictions from each structure is a better strategy than simply predicting from the deposited structure . our finding that binding affinity can be better predicted when calculated protonation states are not explicitly incorporated into the scoring function will be certainly seen as a controversial result by most molecular modellers . we are providing next an intuitive explanation for this result . in machine learning nomenclature , the chemical description of complexes constitutes a data representation . representations present an opportunity to incorporate domain knowledge into the problem , which in principle can help to disentangle the different explanatory factors for variation of the predicted variable ( binding affinity here ) and thus lead to better performance by simplifying the regression problem . however , as domain knowledge is affected by confounding factors and implemented with various degrees of efficacy , it is entirely possible to obtain better performance by incorporating less domain knowledge ( i.e. , introducing less noise ) and hence relying more on pure inference from the data . in our problem , binding affinity is experimentally determined in solution along a trajectory in the codependent conformational spaces of the interacting molecules , whereas the structure represents a possible final state of that process in a crystallized environment . consequently , very precise descriptors calculated from the structure are not necessarily more representative of the dynamics of binding than less precise descriptors . this means that a more precise description will not necessarily lead to a better prediction of binding affinity , as it has been proven here using random forest . because the information content of a set of variables is independent of the adopted regression model , the use of an alternative regression technique should lead to the same conclusion , although this point is still to be confirmed experimentally . we can not stress enough that we are not making any claim about the importance of protonation for pose generation in docking . pose generation and rescoring are different problems , and so are the objectives that the corresponding scoring functions must fulfill . in summary , we have seen that one can be easily fooled by uncertainty when investigating more accurate scoring functions . given the unavoidable uncertainty , we believe that rigorous and systematic numerical studies are the most reliable way to make progress in predicting intermolecular binding affinity . we hope that the availability of the rf - score software ( links are provided in the methods section ) will encourage experts in the area to try to perform better on the pdbbind benchmark using alternative chemical descriptions as a way to investigate this issue further . the code permits reproducing the results obtained by rf - score::elem - v2 and can also be used as a template to test alternative regression techniques implemented in r. without any modification , the rf - score software can be employed to rescore ligands in crystal structures or docking poses . there is a range of applications in which more accurate prediction of binding affinity of a complex would be very useful , some of them new such as replacing force - fields in molecular dynamics simulations . other applications include structure - based virtual screening and lead optimization . in fact , applying a simpler variant of rf - score::elem - v1 to prospective virtual screening has already been found to excel at discovering innovative inhibitors of antibacterial targets . very recently , rf - score::elem - v1 has been incorporated into an easy - to - set large - scale docking web server ( http://istar.cse.cuhk.edu.hk/idock ) to carry out virtual screening of up to 17 million purchasable molecules from the zinc database , which should be upgraded soon to rf - score::elem - v2 .

predicting the binding affinities of large sets of diverse molecules against a range of macromolecular targets is an extremely challenging task . the scoring functions that attempt such computational prediction are essential for exploiting and analyzing the outputs of docking , which is in turn an important tool in problems such as structure - based drug design . classical scoring functions assume a predetermined theory - inspired functional form for the relationship between the variables that describe an experimentally determined or modeled structure of a protein ligand complex and its binding affinity . the inherent problem of this approach is in the difficulty of explicitly modeling the various contributions of intermolecular interactions to binding affinity . new scoring functions based on machine - learning regression models , which are able to exploit effectively much larger amounts of experimental data and circumvent the need for a predetermined functional form , have already been shown to outperform a broad range of state - of - the - art scoring functions in a widely used benchmark . here , we investigate the impact of the chemical description of the complex on the predictive power of the resulting scoring function using a systematic battery of numerical experiments . the latter resulted in the most accurate scoring function to date on the benchmark . strikingly , we also found that a more precise chemical description of the protein ligand complex does not generally lead to a more accurate prediction of binding affinity . we discuss four factors that may contribute to this result : modeling assumptions , codependence of representation and regression , data restricted to the bound state , and conformational heterogeneity in data .

Introduction Methods Results Discussion

the first is pose generation , which starts with the determination of the position , orientation , and conformation of a molecule as docked to the target s binding site . indeed , despite intensive research over more than two decades , accurate prediction of the binding affinities of large sets of diverse protein ligand complexes remains one of the most important open problems in computational bioscience . in principle , a more accurate prediction of binding affinity than that from scoring functions is obtained in those cases amenable to these techniques . however , such expensive free energy calculations are not feasible for the evaluation of large numbers of protein ligand complexes , and their application is generally limited to predicting binding affinity in series of congeneric molecules binding to a single target . each scoring function assumes a predetermined functional form relating the variables that describe the complex , which may also include a set of weights that are fitted to experimental or simulation data , and its predicted binding affinity . such a relationship typically takes the form of a sum of weighted physicochemical contributions to binding in the case of empirical scoring functions or a reverse boltzmann methodology in the case of knowledge - based scoring functions . as previously discussed , the inherent drawback of this approach is that those complexes not conforming to this strong modeling assumption will be predicted poorly . as an alternative to these classical scoring functions , nonparametric machine learning can be used to capture implicitly the binding interactions that are challenging to model explicitly . by not imposing a particular functional form for the scoring function , the collective effect of intermolecular interactions in binding can be directly inferred from experimental data . indeed , this unconstrained approach had to result in performance improvement given sufficient data , as it is well - known that the strong assumption of a predetermined functional form for a scoring function constitutes an additional source of error ( e.g. on the other hand , recent experimental results have resulted in novelties in the definition of molecular interactions such as the hydrogen bond and the hydrophobic interaction , implying that previously proposed expressions for these energetic contributions might need to be revised accordingly . while a few classifiers exploiting x - ray crystal structural data for discriminating between binders and nonbinders of a protein target have been presented , it is only recently that machine learning for nonlinear regression has been shown to be a particularly powerful approach to build generic scoring functions . this approach has been highlighted as very promising for the improvement of scoring functions . however , these initial models are relatively coarse in the description of the complex , and thus the question remains as to whether the incorporation of additional chemical information relevant for binding would improve performance further . here , we investigate the impact of a more precise chemical characterization of the protein this includes the use of structural interaction fingerprints , using atom and interaction type definitions from the credo structural interactomics database . in the light of the improved performance obtained and considering the uncertainty introduced by the static nature of crystal structures , we discuss the role of interatomic distance cutoffs and binning as well as protonation states in binding affinity prediction . as a byproduct of this systematic battery of numerical experiments , the most accurate scoring function to date on a widely used pre - existing benchmark is presented . an important conclusion of our study is that a more chemically precise description of the protein ligand complex does not generally lead to more accurate prediction of binding affinity . we discuss four factors that may contribute to this result : modeling assumptions , codependence of representation and regression , data restricted to the bound state , and conformational heterogeneity in data . the definition of these including the source of the smarts definitions and other constraints are described in the original credo publication with the following exception : the carbonyl interaction type has since then been implemented on the basis of the ab initio molecular - orbital calculations of allen et al . here , we operate the rf with the default value p = 500 , as its performance does not generally improve significantly beyond this threshold . the performance of each tree on predicting the out - of - bag ( oob ) set , here protein ligand complexes that were not in the bootstrap sample used to train that tree , collectively provides an internal validation measure of rf . on the basis of the refined set from the 2007 release of the pdbbind database , it comprises 1300 diverse protein ligand complexes with high quality structural and binding data ( the protonation states of both proteins and ligands were already calculated by these authors ) . we start this section with a concise description of the approach to predicting binding affinity using machine learning . this process starts with the characterization of each protein ligand complex as a set of intermolecular features or descriptors relevant to binding affinity prediction . sketch of the process of characterizing a protein ligand complex ( pdb : 2p33 ) as a set of structure - derived descriptors ( c.c to i.i ) . the pdbbind database is an excellent choice for this purpose , with the additional advantage that a large number of scoring functions have already been benchmarked on a common pdbbind test set , which permits comparing new developments against the state of the art . this is important to avoid the often large bias introduced by using a different training set for each scoring function ( such bias makes comparisons among scoring functions unreliable , even if compared on the same test set ) . last , a regression model is needed to predict the binding affinity of test set complexes from the structural and interaction data in the training set . here , we build upon rf - score , a machine learning scoring function using rf for regression . rf is typically tuned using a single control parameter ( mtry , which controls the number of features that are considered for the split at each tree node ) and may be subjected to a feature selection strategy intended to remove descriptors with low information content as a way to improve performance ( that is , in addition to the common practice of removing all those descriptors that have zero values across training complexes ) . full details on descriptors , data , and regression protocols can be found in the methods section . bottom : training random forest to learn the nonlinear relationship between this atomic - level description of the complex and its binding affinity ( pkd or pki ; pkd / i denotes both without distinction ) . the resulting scoring function ( rf - score ) is used to predict the binding affinity of the test set . thus , we were forced to assume the independence of these design variables and searched for the optimal value of each variable in a sequential manner as explained in the next subsections . here , we addressed this question empirically by generating element descriptors for four cutoffs ( 6 , 9 , 12 , and 15 ) , which gave rise to four different numerical characterizations of the training set with their corresponding test set counterparts . thereafter , rf was calibrated on each of these training set versions and the resulting model used to predict the binding affinity of the corresponding test set complexes . lastly , we considered three commonly used metrics for quantifying the difference between predicted and measured binding affinity across the test set of protein ligand complexes : rp ( pearson s correlation coefficient ) , rs ( spearman s correlation coefficient ) , and sd ( standard deviation in log kd / i units ) . performance is measured as the difference between observed and predicted binding affinity in the test set using three metrics : pearson s correlation coefficient ( rp ; left plot ) , spearman rank - correlation coefficient ( rs ; middle plot ) , and standard deviation ( sd ; right plot ) . these are the credo descriptors , which measure the abundance of a range of intermolecular interactions such as hydrogen bonds , hydrophobic interactions , or van der waals clashes . these results hint at a trade - off between the predictability and interpretability of the model , which we will discuss later in this paper . the strength of the interatomic interactions that collectively form the noncovalent intermolecular bond depends on the separation between the interacting atoms . using all the optimal values , for six bin sizes ( a 12 bin size with a 12 cutoff simply corresponds to the case without binning , which was previously shown in the first boxplot in figure 4 and the third boxplot in figure 3 ) . the best median performance is achieved by models with lower bin sizes ( 1 , 2 , and 3 ) , representing a modest improvement with respect to the model based on single - binned descriptors ( 12 ) . in addition to exploring the impact of different ways to describe the complexes , we also applied basic feature selection strategies intended to remove sparse features that increased the complexity of the model without improving performance . here , we also considered two additional sparsity thresholds ( spr = 1 and spr = 2 ) . best median performance is obtained by spr = 1 , which corresponds to only considering descriptors that have an average of at least one atom the latter represents a moderate improvement with respect to the models using descriptors with spr = 0 and spr = 2 . one of these strategies is called out - of - bag ( oob ) validation and essentially consists of training the model for each possible value of mtry and selecting the model that best performs on the internal validation set ( a subset of the training set , as further explained in the methods section ) . test set performance of rf - score with element descriptors , 12 cutoff , 2 bin size , and feature selection threshold ( spr=1 ) for the recommended mtry ( model selection=0 ) and mtry selected by oob validation ( model selection=1 ) , which requires 123 times more training than just using the recommended setting ( as many rf training runs as features were selected to describe each complex ) . this systematic battery of numerical experiments led to the new scoring function rf - score::elem(c12,b2)_spr1_oob ( rf - score::elem - v2 for short ) . figure 8 shows the predictive power of rf - score::elem - v2 on the test set . in addition , the time to generate these features for the 195 complexes was 8 s , and hence this is the most expensive part of the calculation . therefore , the average time to score one protein ligand complex is about 0.04 s if the features have not been calculated before , which makes rf - score suitable to rescore a high number of docking poses in virtual screening applications . the predictive power of rf - score::elem - v2 was also compared against that of a wide selection of scoring functions on the pdbbind benchmark . by using a pre - existing benchmark , the danger of constructing a benchmark complementary to the presented scoring function is avoided . several of the scoring functions tested in the pdbbind benchmark have different versions or multiple options . in addition to these 16 scoring functions , we also tested a more recent function called imp::rankscore . performance of 18 scoring functions on the pdbbind benchmark as measured by pearson s correlation coefficient ( rp ) , spearman s correlation coefficient ( rs ) , and standard deviation of the difference between predicted and measured binding affinity ( sd ) . the three plots on the right visually show the relative predictive power of rf - score ( x signs ) against that of the other 17 scoring functions ( + signs ) . when introducing a scoring function , only the scoring function built with the random seed that provides the best performance we have followed here a more precise way to assess performance differences between scoring functions by comparing median performances from a set of independent trials . to illustrate this , appendix a1 presents the performance of the new version of rf - score ( rf - score::elem - v2 ; full test set rmse = 1.54 ) and rf using credo intermolecular interaction features ( rf - score::credo ; full test set rmse = 1.72 ) on the four subsets resulting from partitioning the test set by binding affinity ranges . such long - range contributions to protein ligand binding affinity have been attributed to the electronic properties of the binding site and ligand being affected by all protein atoms and also to long - range electrostatics interactions . the main reason why rf - score works much better than classical scoring functions at predicting binding affinity of most complexes is due to the circumvention of the strong assumption of a predetermined functional form . all classical scoring functions consist of a sum of typically nonlinear terms with respect to selected interatomic distances , such as van der waals terms in empirical scoring functions or particular atom for instance , the scoring function consortium ( sfc ) , in a concerted effort between 10 pharmaceutical companies and academic institutions , generated an empirical scoring function ( sfcscore ) , which by the time of its development was clearly superior to most of the then available scoring functions . very recently , one of the leading sfc authors has demonstrated that , by using random forest regression instead of sfcscore s additive functional form and keeping all other modeling choices unaltered ( training data , test data , and descriptors ) , performance rises from rmse = 1.84 to rmse = 1.56 ( 0.683 to 0.788 in the case of rs ) . another study demonstrating that assuming an additive functional form is detrimental for the performance of empirical scoring functions is by kinnings et al . as force - field and knowledge - based scoring functions make the same assumption , these studies strongly suggest that a machine - learning version of other classical scoring functions will also result in significant improvement . another important conclusion of our study is that a more precise chemical description of the protein ligand complex does not generally lead to more accurate prediction of binding affinity ( see figure 4 ) . present a scoring function tested on exactly the same test set as us , with a much larger training set that includes ours and the use of a very precise description consisting of 50 calculated descriptors falling into nine interaction categories : van der waals , hydrogen - bonding , electrostatic , pi - system , metal ligand bonding , desolvation effect , entropic loss effect , shape matching , and surface property matching ( table 1 on page 593 of li et al.s paper ) . the only difference between their scoring function and ours is in the 63 used descriptors , which was one of the outcomes of the industry these descriptors include the number of rotatable bonds in the ligand , hydrogen bonds , aromatic interactions , and polar and hydrophobic contact surfaces , among others ( a full list can be found in table 1 of page 398 of the original sfc paper ) . their best scoring function achieved rmse = 1.56 and rs = 0.788 ( table 1 of zilian and sotriffer s paper ) , which is slightly better than the original version of rf - score ( rmse = 1.58 and rs = 0.762 ) . if the modeling assumptions implied in the calculation of chemical properties were generally accurate , we should have seen many scoring functions performing much better than rf - score thanks to using a more precise chemical description . we discuss next four convoluted factors that may contribute to this result : modeling assumptions , codependence of representation and regression , data restricted to the bound state , and conformational heterogeneity in data . from a purely chemical perspective , deconvoluting elemental atom types into their various hybridization states constitutes a more precise description of the complex . this situation gives rise to a trade - off between the predictability and interpretability of the model , which is not uncommon in regression problems and has also been observed here ( see figure 4 ) . for the sake of efficiency , scoring functions only exploit the information contained in the bound state of the complex , as represented by a crystal structure . the third contributing factor is therefore the uncertainty about how well a particular description of the bound complex is also describing the complex just before desolvation and induced fit takes place . we speculate that descriptors whose values change less during the binding process might be more suitable for predicting binding affinity using only data about the bound state . in machine learning nomenclature , the chemical description of complexes constitutes a data representation . representations present an opportunity to incorporate domain knowledge into the problem , which in principle can help to disentangle the different explanatory factors for variation of the predicted variable ( binding affinity here ) and thus lead to better performance by simplifying the regression problem . in our problem , binding affinity is experimentally determined in solution along a trajectory in the codependent conformational spaces of the interacting molecules , whereas the structure represents a possible final state of that process in a crystallized environment . this means that a more precise description will not necessarily lead to a better prediction of binding affinity , as it has been proven here using random forest . because the information content of a set of variables is independent of the adopted regression model , the use of an alternative regression technique should lead to the same conclusion , although this point is still to be confirmed experimentally . in summary , we have seen that one can be easily fooled by uncertainty when investigating more accurate scoring functions . given the unavoidable uncertainty , we believe that rigorous and systematic numerical studies are the most reliable way to make progress in predicting intermolecular binding affinity . we hope that the availability of the rf - score software ( links are provided in the methods section ) will encourage experts in the area to try to perform better on the pdbbind benchmark using alternative chemical descriptions as a way to investigate this issue further . there is a range of applications in which more accurate prediction of binding affinity of a complex would be very useful , some of them new such as replacing force - fields in molecular dynamics simulations .