This article was the winner of the triennial Casey Holter Memorial Prize awarded by the Society for Research into Hydrocephalus and Spina Bifida, 2004.This essay explores the link between the limbic/hypothalamic systems within the complex conditions of hydrocephalus and myelomeningocele. Acknowledging the neuroanatomical and neuroendocrine risks inherent in the developing brains of these individuals, we focus on the converging components of temperament, cognition, and language. Behavioral characteristics have long been one such category for easy generalities. As modern investigators study the underlying keys to cognitive and behavioral disabilities, theories related to cortical development and differentiation have offered insightful possibilities ..48]].Developmental disruptions such as those related to MM can also provide "spin-off" developmental deficits related to anatomy and physiology arising from structures that form the limbic system and hypothalamus. Similarly, the potential exists for the developing HC, with its cerebrospinal fluid blockage within and around the brain, to impact the architecture of the emerging limbic system and its neuronal projections.For example, vacuolization and degeneration of neurons in the hippocampal formation have been observed both within hydrocephalic rabbits and humans . DendritClinical evidence from patients, and recent brain imaging studies suggest lesions of the right hippocampus result in spatial memory deficits while left lesions impact verbal memory ,8,52. Thresponse to the perceived frustrations associated with functional deficits, which come into play daily and hourly. What underlies and mediates the style of behavioral responses? In other words, how does the child's temperament phenotype become an integral part of the clinical management, and neurophysiological understanding of learning differences among children with MM?Anatomical and central molecular dynamics involved with these language and memory deficits remain critically important areas for future research. Equally intriguing are those – or other – molecular substances and pathways that mediate the yin) and the accompanying active, light, heavenly force (yang). The great 2nd century C.E. Roman physician, Galen, conceived a system of balanced equilibrium of four basic humors, which allowed the physician clues as to the nature of an illness by his awareness of personality traits. This system, based on yellow or black bile and phlegmatic or sanguine natures, represented a culmination of prior ideas  and provided the understanding of the relationship of temperament to health and function for more than a millennium [This task of describing individual differences among individuals within a society is ancient in its origin. Early Chinese tradition offers a view of human nature differences based on a dark, female, earthly force . The examiner, familiar with children with MM, recognized the triggers and the responses before her. By careful explanations, negotiating cues to assure Annie of her successes in the testing, and agreements for "breaks" as needed, Annie calmed, warmed to the tasks at hand, and completed the testing successfully in the allotted time.The developmental pediatric team was recently notified by the family that Annie was experiencing episodes at school of skin flushing, sweating, sensations of nausea, and emotional stress. On one occasion, urinary incontinence accompanied the episode. These symptoms were eventually noted to be related to those (infrequent) occasions when the teacher felt it necessary to enter a note onto Annie's personal folder for parent review. Beginning hints of school avoidance prompted the parents' concern.Academic challenges and barriers as in the story of Annie are quite common among youth with MM. Layers of seemingly separate but simultaneous factors – cognitive, memory, language, temperament, physical – can elevate even daily and mundane tasks into sources of stress not experienced by the typical classmate. Recent studies of youth with MM and shunted HC, ages 5–12, demonstrated clusters of findings different from those classically described by Chess and Thomas  or by Kagan ("inhibited" or "uninhibited"). Children with MM in these studies were reported to be significantly different from normative profiles in five areas: 1) adaptability – less; 2) distractibility – more; 3) approach – guarded; 4) persistence on task – less; and 5) predictability – less . Given tth century, only recently have physiologic variables and temperament categorical descriptors been paired for critical comparisons. These investigations stem from relatively firm notions about the anatomy and physiology of the central nervous system. Researchers have tracked differing patterns of social response, and encoding of emotional memories to the limbic system, and particularly amygdala [While the focused study of the biological basis for temperament dates to mid-20amygdala ,64. The amygdala ,65. Prelamygdala . PotentiThese limbic structures – and their vulnerability in the developing brain affected by MM – offer fertile ground for clinical exploration in the broader study of cognition, memory, and temperament. The segregation of emotion, temperament, language, and cognitive performance is difficult to accomplish in the typical child; it is decidedly more complex in the child with MM and HC who has co-existent ventriculomegaly from early gestation, mid-brain variations, and cerebellar differences.What then are the capacities and activities of these central nervous system components – the limbic and hypothalamic systems – that are typically relied upon for daily function, and which might have significant impact on those outward actions and responses which we categorize as temperament? Emotional triggers, goal direction for problem solving, assimilation of information from the various senses  – all of these relate to the anatomy and associated function of the limbic system , and the closely related hypothalamic region. Evidence from human and animal studies indicates that the amygdala intervenes between the regions concerned with the somatic expression of emotion  and the neocortical areas concerned with conscious feeling .Similarly, the hypothalamus is a central player in the integration of information. For the specific areas throughout the neocortex to finalize and actualize those processes described as "executive functioning", regulation of stimuli from the environment and peripheral systems of the body is aided through hypothalamic function. Some actions might well be expected to impact behavior we characterize as components of temperament.The anatomy of the hypothalamus is important when considered along with the variations commonly noted in our individuals with ventriculomegaly and Chiari malformations. It lies on the ventral surface of the cortex. The third ventricle divides the hypothalamus and lies in close proximity to hypothalamic projections . DisturbThe effects of HC on neuronal pathology and function in these regions have been demonstrated in both human studies and animal models ,49,50,67Annie's tale is not unusual for our children with MM and shunted HC. Clinicians and researchers alike have experienced stories of lesser or more severe impairments. For the parents and teachers working daily with children such as Annie, an understanding of the normal process of neuro-development leads to clearer understanding and a better differentiation from the abnormal. The research and subsequent writings of T. Berry Brazelton have provided ready information to parents of typically developing children by blending the Gesellian milestones of development with the temperament concepts as outlined by Carey, Chess, and Thomas . The intany progress could be made in better understanding her relative academic and learning strengths. The autonomic dysfunction, manifested in her excessive sweating, flushing, and urinary incontinence in school, only served to heighten both the emotional and physical symptoms.For the classroom, identification of stressors and methods to minimize autonomic over-response can be as critical to successful learning as the understanding of memory strengths, or language problems. As in our story, Annie demonstrated persistence to task, willingness to approach novel situations, positive mood, and the ability to adapt to the testing situation – all temperament domains with which she has struggled. The "goodness of fit" between Annie and the examiner was critical before The differences in temperament profiles among children with MM are unlike those in other diagnostic cohorts of children with special health care needs ,71. HughLearning, for the child with MM, certainly takes place beyond the confines of preschool and classroom. Negotiating physicians, curious strangers, medical emergencies, educational diagnosticians, environmental barriers, and other stressors is a continual process. Raising a happy, self-reliant, and resilient child is the dream of parents. Perrin  has suggT.S. Eliot wrote,We shall not cease from explorationAnd the end of all our exploringWill be to arrive where we startedAnd know the place for the first time.As we continue, on behalf of children and youth with MM, to explore and better understand the cognitive and learning differences within the group, the developmental variations related to underlying central nervous system structures may continue to hamper the human tendency to categorize. The modal profile may not be singular.As assessments of strengths and differences continue; as descriptors of quality of life are derived; as personality variances are accounted for; as curricula and "home programs" are constructed, the bio-psycho-social model increasingly becomes the clear route for professional and family partnership. For the researcher trying to join un-connected dots into a cohesive whole, we, like Eliot, may need to return to the early components long-standing in the evolution of the brain and its function. The ancient limbic and hypothalamic systems leave indelible tell-tale hints that should not be left behind in our quest for understanding and raising the resilient child.MM, myelomeningocele; HC, hydrocephalusThe author(s) declare that they have no competing interests.Both authors contributed equally to this work. All authors read and approved the final manuscript.


We honor Theo Hellbrügge's acclaimed endeavors in the rehabilitation, or rather the prehabilitation of handicapped children. So far, he has focused on obvious handicaps, and we trust that he will include concern for everybody's silent handicaps in the future by screening for abnormal variability inside the physiological range. Therein, we introduce cis- and trans-years, components of transdisciplinary spectra that are novel for biology and also in part for physics. These components have periods, respectively, shorter and longer than the calendar year, with a counterpart in magnetoperiodism. Transyears characterize indices of geomagnetic activity and the solar wind's speed and proton density. They are detected, alone or together with circannuals, in physiology as well as in pathology, as illustrated for sudden cardiac death and myocardial infarction, a finding calling for similar studies in sudden infant death syndrome (SIDS). As transyears can beat with circannuals, and depend on local factors, their systematic mapping in space and time by transdisciplinary chronomics may serve a better understanding of their putative influence upon the circadian system. Longitudinal monitoring of blood pressure and heart rate detects chronome alterations underlying cardiovascular disease risk, such as that of myocardial infarction and sudden cardiac death. The challenge is to intervene in a timely fashion, preferably at birth, an opportunity for pediatricians in Theo Hellbrügge's footsteps. The discovery in biology of far-transyears, 15–20 months in length , is in kFigure For discussion by transdisciplinary nomenclature committees, terms in English are emphasized. With advice by Prof. Robert Sonkowsky, proposed Latin equivalents are added for vanishing classicists. Essentially, "ad-transannual" means "a little longer than a year"; "ad-cisannual" means "a little shorter than a year"; "transior-annual" means "much longer than a year"; and "citerior-annual" means "much shorter than a year". Some specific limits that seem reasonable in the light of available physical and biological evidence are given in the scheme. The single syllable 'ad' is preferred to the 2-syllable 'prope', 'juxta', 'propter', 'minus' (paired with 'plus') or the 3- or 4-syllable 'proprior', 'proximus', 'vicinus', or propinquus'. While to a purist among grammarians the coinages adtransannual and adcisannual may seem preposterous (a word constituting itself an illustration of cumulative prefixes) precisely because of the piling on of prefixes, there are also other precedents in Late Latin such as exinventio ("discovery") and perappositus ("very suitable/apposite"). Normal assimilation of 'd' to 't' and 'c', respectively, may then result in the spellings and pronunciations "attransannual" [at-trans-annual] and "accisannual" [ak-sis-annual] acceptable as English pronunciation, notably by speakers with native romance languages, who may face difficulty with the near and far as added prefixes.Difficulties may stem from the fact that analyses usually provide estimates in frequency (not period) terms, and from the criterion of 95% CIs that may not be available. We need to allow for situations when, because of too-wide (or unavailable) CIs, we can diagnose only a candidate trans- or cis-annual component, when 95% CIs of τ overlap the limit distant from the year. By the same token, we may not be able to specify near or far, e.g., because of the brevity of the series. In other words, we cannot say whether we have a near- or a far- trans- or near- or far- cis-year, when there is an overlap by 95% CIs with the corresponding finer limits, shown on the scheme Figure .For the case of "circannual", we again go by 95% CIs rather than by the point estimate. In the circannual case, the 95% CI overlaps the 1-year estimate under usual conditions, bearing in mind that under unusual, e.g., constant conditions, circannuals are also amenable to free-running, in which case the 95% CI may no longer cover 1 year but will have to be tested further for non-overlap with the pertinent environmental cycle in the case of a biologic cycle and vice versa for non-overlap of a natural environmental cycle with an anthropogenic cycle. In the trans- or cis-annual case, the 95% CI does not cover the 1-year period under usual conditions, i.e., cis- or trans-annuals can be asynchronized rather than desynchronized. Strictly speaking, circannual cannot be an overall term, but almost certainly, whatever committees may decide, it will be (mis-)used as such. "Far-" and "near-", "cis-" and "trans-" and "citerior-" and "transior-" annual are hyphenated here only to indicate their derivation and need not be written with hyphens. We propose using circannual, transannual or cisannual and their refinements, only operationally as a function of periods and their 95% CIs. Matters of synchronization, desynchronization or asynchronization may then possibly emerge from the context of a given situation and from further testing.Trans- and cis-years lead to a novel chrono-helio-geobiology, awaiting application of the tools of transdisciplinary chronomics. It has been a challenge to look at circadians for the past half-century, but knowledge concerning them will not be completely useful before we answer another set of questions based on the evidence in Table Table Of interest are great geographic/geomagnetic differences insofar as no transyears, only calendar-yearly components, were detected in 3 locations, while in 3 other locations, transyears were present, in two of these, with a coexisting calendar-yearly component, with nearly equal prominence, while in Minnesota, only a transyear was thus far detected. A clarification of the roles played by local as well as global influences could also be based on transyear vs. calendar-yearly amplitude ratios when both components are present, which, however, is not the case in 4 of 6 locations. There is the challenge of developing eventual countermeasures.But first, we seek a clue as to why, for SCD in Minnesota, the prominence of the transyear exceeds by far any seasonal, thus far undetected influence of the harsh environmental temperature change in its mid-continental climate in the summary of 5 consecutive years, and why, in Arkansas and the Czech Republic, the transyear's prominence is about the same as that of the seasons, and why it seems to be absent in 3 other locations and furthermore why in MI the prominence (gauged by the amplitude) of the calendar year is so far greater than that of the transyear (by contrast to the case of SCD). Systematically collected data from different areas of the world will open a new chapter in transdisciplinary science, with particular pertinence at the extremes of extrauterine life, in natality as well as in mortality.Optimization of the about-yearly spectral region may also be considered, along with Hufeland's consideration of the daily routine in studies aimed at prolonging high-quality life . Notablypar excellence and professor emeritus of social pediatrics at the University of Munich, continues actively as a mentor of the specialty he founded [Beyond 85 years of age, Theodor Hellbrügge, chronopediatrician  founded -9. Our e founded ,10-14 in founded -58, many founded ,52,53,55 founded -19 to a  founded .par excellence, which could benefit from chronomics, the resolution of time-structural (chronome) alterations in the physiological range. Accordingly, chronobiologists honored Theo at a meeting on "Time structures – chronomes – in child development", leading to a proceedings volume of 256 pages [Theo himself turned in the interim to the care of children with obvious disabilities. He continues with concerns about them to detect early alterations for timely remedies, a preventive task 56 pages . On the 56 pages . The amp56 pages . About 256 pages . These a56 pages , but are56 pages . In conj56 pages  and also56 pages  (Table 1In his own recent words , Theo alTo continue in his words , in pracBy 1960 at Cold Spring Harbor  and agaiTheo Hellbrügge's contributions illustrate a solidly founded now widely distributed conceptual structure resting on a productive life's work available again in his own words . A few gWith one of his colleagues , we can • some in ethology as a method to account for the development of children,• mother-infant-interactions as a decisive requisite of social development, the topic of the last symposium he sponsored in October 2004• preverbal communication, as a condition for early speech promotion, especially for infants with impaired hearing,• the plasticity of the infant's brain as a neurobiological basis for early health promotion,• enriching integration of infant and child as part of a socially intact community,• preventive medical-check ups aiming at an early diagnosis of abnormality,• earliest diagnosis of risks as a condition of PREhabilitation – which he called rehabilitation, to gain a financial niche for his actions in existing laws.Hellbrügge's conference on chronomes  showed a


The proteasome is a large multiprotein complex that degrades unwanted cellular proteins. The mechanisms that control this protein-eating machine are being uncovered Inside eukaryotic cells there is a massive protein complex called the proteasome whose raison d'être is to remove unnecessary proteins by breaking them down into short peptides. The proteasome is thus responsible for an important aspect of cellular regulation because the timely and controlled proteolysis of key cellular factors regulates numerous biological processes such as cell cycle, differentiation, stress response, neuronal morphogenesis, cell surface receptor modulation, secretion, DNA repair, transcriptional regulation, long-term memory, circadian rhythms, immune response, and biogenesis of organelles . With thWith so many proteins to target for degradation, the activity of the proteasome is subject to multiple levels of regulation. In the overwhelming majority of cases, selected proteins are first “labeled” by the addition of several copies of a small protein tag called ubiquitin and are thus targeted for degradation in the proteasome . The ubiInterestingly, ubiquitination is a reversible process. Even when a protein has been tagged with ubiquitin, its fate is not sealed—specific hydrolytic enzymes called deubiquitinases can remove the ubiquitin label intact . By deubThe proteasome itself is made up of a multiprotein core particle (CP) where proteolysis occurs and a separate multiprotein regulatory particle (RP) that recognizes and prepares substrates for degradation by the CP. A base subcomplex of the RP is pivotal in anchoring polyubiquitin chains during this process, either directly or via auxiliary ubiquitin-binding proteins . The basAn intriguing evolutionary and structural relationship between the proteasome lid and an independent complex, the COP9 signalosome (CSN), may shed light on their respective roles in regulated protein degradation. Both are made up of eight homologous protein subunits that contain similar structural and functional motifs. While a lot is still unknown, the CSN appears to mediate responses to signals  in a manner that is intimately linked to the ubiquitin–proteasome system. This is accomplished, for instance, by suppressing ubiquitin E3 ligase activity or interacting with various components of the pathway . In partSurprisingly, the proteasome itself harbors intrinsic deubiquitination activity . Moreove+/JAMM motif garnered great attention. The trademark of the MPN+/JAMM motif is a consensus sequence E—HxHx(7)Sx(2)D that bears some resemblance to the active site of zinc metalloproteases. Members of this family were predicted to be hydrolytic enzymes, some of which are specific for removal of ubiquitin or ubiquitin-like domains from their targets  and the auxiliary deubiquitinating enzymes (situated in the regulatory particle) is clear-cut: the latter cleave between ubiquitin domains, while the core proteolytic subunits process the target protein itself . HoweverOnce bound to the proteasome, a polyubiquitinated substrate can be unfolded by the RP and irreversibly translocated into the CP. It has been proposed that long polyubiquitin chains commit a substrate to unfolding and degradation by the proteasome, whereas short chains are poor substrates because they are edited by deubiquitinating enzymes, resulting in premature substrate release . ExtendeIdentifying Rpn11 and Csn5 as members of a novel class of metallohydrolases immediately elevates them into promising “drugable” candidates. Undoubtedly, the molecular structures deciphered by the groups of Deshaies  and Bycr


Applied epidemiologists are increasingly working in areas of insecurity and active conflict to define the health risks, suggest feasible means to reduce these risks and, monitor the capacity and reconstruction of the public health system. In 2001, The Carter Center and the United States Institute for Peace sponsored a conference within which "Violence and Health" was discussed and a working group on applied epidemiology formed. The group was tasked to describe the skills that are essential to effective functioning in these settings and thereby provide guidance to the applied epidemiology training programs.We conducted a literature review and consultation of a convenience sample of practitioners of applied epidemiology with experience in conflict areas.The health programs designed to prevent and mitigate conflict are in their early stages of implementation and the evaluation measures for success are still being defined. The practice of epidemiology in conflict must occur within a larger humanitarian and political context to be effective. The skills required extend beyond the normal epidemiological training that focuses on the valid collection and interpretation of data and fall into two general categories: (1) Conducting a thorough assessment of the conflict setting in order to design more effective public health action in conflict settings, and (2) Communicating effectively to guide health program implementation, to advocate for needed policy changes and to facilitate interagency coordination. These are described and illustrated using examples from different countries. In 2004 it is estimated that there are 95 violent conflicts worldwide ,2. The pIn February 2001 the Carter Center and the United States Institute for Peace (USIP), in collaboration with CARE, Emory University and the Centers for Disease Control and Prevention (CDC), sponsored a meeting on "Violence and Health". The goals of the meeting were to determine the impact of violent conflict on public health and to advise public health training programs on means to enhance the work of public health professionals in settings of violent conflict.During the meeting a specialty workgroup for "training public health care professionals" was formed and asked to focus on applied epidemiologists by describing their role in conflict prevention, mitigation, and documentation. Applied (or field) epidemiology was initially created by the US Centers for Disease Control and Prevention in the 1950s as a post-doctoral training program . Over tiIncreasingly, applied epidemiologists are recruited to areas of insecurity and conflict to define the health risks, suggest feasible means to reduce these risks, and monitor the capacity and reconstruction of the public health system -22. ThisBecause of the increasing demand for applied epidemiologists in conflict, the workgroup leaders reviewed the relevant literature and sought further input from applied epidemiologists and public health practitioners experienced with conflict settings to better understand training needs in this setting. The literature review and discussions focused on the role of applied epidemiologists in violent conflict and what skills are needed to function effectively. The literature review included MEDLINE and Google keyword searches for "war", "conflict", "complex emergencies" and "epidemiology" combined with either "conflict", "disaster" and "refugees". The examination included specific training and reference materials developed to support Health as a Bridge for Peace ,17. The Applied epidemiologists have an important role in conflict prevention, mitigation, and reconstruction, but to be effective in these circumstances their training must emphasize knowledge about international law, human rights, and complex emergencies ,17. In aThe first contribution of the applied epidemiologist in conflict is to insist that a thorough assessment of the situation be done as soon as possible. Without such insistence the rush to begin programs without adapting interventions to local circumstances will prevail with predictably poor results ,4,10. ThThe use of validated qualitative research methods is essential to the processes of building trust and performing high quality assessments. Many workgroup experts suggested including social and behavioural scientists with practical field skills from the beginning; if this is not possible then their techniques for collecting data in the field should be adopted. Using ethnographic skills increases the accuracy and validity of assessments and allows the development of a common vocabulary of key terms for communicating with local people, even when using a translator ,29. DespOnce there is agreement that an assessment will be done- either prior to, or  simultaneously with emergency interventions- the role of the applied epidemiologist is to plan and coordinate the collection of valid and useful data. ,20,26 ThThe description of previous health programs is one source of useful information for assessments that can be obtained before fieldwork commences. Did previous programs engender trust or suspicion? Did they work, and if so, why? This information can indicate a community's ability to work together or with outside agencies. In addition, if a new program resembles a past program, it will inherit the community's view of the past program, for good or ill. For example, in planning a maternal and child health program in Afghanistan with WHO, we determined that communities that previously had good experiences with girls' schools were much more willing to consider other projects that educated or benefited girls (SMM). Thus, maps from the previous ministries of education and health showing the location of these schools provided initial leads that were followed up by interviews with community members.Rapid survey methods are most often used to describe the current needs of a population in conflict ,20. They• baseline health status, risks and determinants • availability, quality, and use of health services • access to health care• the security of a situation , and• changes in the population (e.g. migration) and the conflict (i.e. location of the front lines) ,21.Field experience in previous conflicts enables the epidemiologist to know what data are most important to assist in determining risks and designing and assessing interventions in a specific situation. For an assessment to be useful, it must have clear objectives and resist the constant pressure to add more questions . NegotiaDespite the potential shortcomings of rapid surveys, systematically collected data that are appropriately interpreted are superior to anecdote. Additionally, by emphasizing and clearly documenting what sources of data are used to build a conclusion it can be re-evaluated over time. In 1990, patient logs compiled from multiple NGO-supported clinics within Afghanistan revealed that the majority of patient visits were for routine primary care services and not war-related injuries as was assumed. The result was a significant revision of the training curriculum for Afghan medics and physicians in more than 15 non-governmental health service programs (SMM).Many of the challenges in generating useful, high-quality data can be addressed with innovative data collection methods and judicious interpretation . In confThere are many examples of innovative ways to select a sampling frame in the conflict setting . Global In war, obtaining a denominator is very challenging ,26,31,32Accurate counts of births, deaths, and disease are also affected by social and political instability . ConflicThe epidemiologist will have to assess the level of access to health-care services for military and non-military personnel, particularly women, children, the elderly, and those with chronic medical conditions ,11,26. DAssessing the data from disease reporting systems is a core function of epidemiologists. If these systems are intact and can describe trends of priority diseases or conditions over time, maintaining them might be worth the effort ,22. TypiApplied epidemiologists believe that the value of epidemiologic science must also be measured by whether and how it is used to create effective public health action . ApplyinPracticing epidemiology in conflict involves working in rapidly changing circumstances and uncertain security. Formal epidemiologic assessments and public health actions in war can only proceed where the environment is "permissive". The security situation for health workers, civilians and programs, as well as community participation and ownership of health programs, must be continuously monitored. Selected parameters that reflect the level of security, participation, demographics and disease should be discussed between partners and security agencies and re -evaluated frequently . Health Acute and chronic war settings, while superficially similar, represent different challenges to the epidemiologist and can significantly affect the type of intervention selected . In warsA chronic war setting (e.g. Afghanistan) is more challenging ,23. KnowAll aspects of communication are more difficult during conflict ,26. Pre-Ongoing consultation with communities is necessary for the integrity of programs and to monitor security. Epidemiologists can improve the quality of the information they receive and disseminate by developing alliances with partners who have a communication infrastructure in place- and people travelling into areas that are restricted or insecure. For example, contacting agencies that work in agriculture, health care, education, land-mine removal, and food provision about sharing or coordinating field staff that monitor and supervise projects can be helpful. Jointly planning the efficient use of field staff from all types of programs using checklists, and other tools to help the non-technical visitor be able to bring back useful information and support local communities can help these communities feel less isolated, reduce waste and increase the data sources about health programs and problems.Workgroup members suggested that journalists may be potential sources of useful information, insofar as they have access to restricted areas. They may be able to:• report on health-related activities or risks ,• provide reliable communication with all sides and help establish ceasefires for the provision of health services , and• help control rumours by defining means to check information early and responding to misinformation quickly ,26.The different goals of the media and public health and the rapid turnover of journalists, however, may limit useful cooperation.Epidemiologists in Colombia's Centro de Referencia Nacional sobre Violencia (CRNV) used national media coverage to highlight political homicides. They published information regarding deaths along one particular river in Colombia to raise awareness of the level of violence occurring there. This data-based approach drew less political resistance while focusing public attention on the deaths ,40.Effective communication strategies should target policy-makers from local to international levels. Epidemiologists can be an effective voice for public health if they maintain their credibility with policy-makers. Sometimes the most important role for the epidemiologist is to act as a witness to describe the local situation to an international audience of policy-makers and to advocate for action ,27,41,42A less rapid but possibly more respected method for bringing awareness of local health and social issues to national and international levels is publishing research in credible journals. Peer-reviewed work can be a tool to facilitate negotiations between governments, NGOs and communities, and to influence international and national level policy-makers.Effective advocacy for public health includes the ability to use communication skills such as advocacy, consensus development, and negotiation to promote population health to policy-makers ,28,42,44During conflict, health and information services that were once unified under a single health ministry can become fragmented with parts falling under the aegis of government, those resisting the government, or nongovernmental organizations ,31. AcceMany workgroup members felt that negotiation skills were essential when working with multiple aid groups or the main parties in the armed conflict. For example, consensus building is useful when the health problems are not well defined and diverse groups must collectively identify and solve them. At the simplest level this includes the skill of running meetings and enhancing a group's utility and the understanding that disagreement might not necessarily represent failure. The goal is to help all parties understand that improving health meets the interests of all groups and to develop complimentary strategies. Too often decisions are rushed and participants pushed aside, leading to unsuccessful outcomes and future difficulties working together . When diTo facilitate the coordination of health services, the epidemiologist needs to be familiar with the many agencies working in the target area, including governmental, nongovernmental, and United Nations agencies. The diversity of groups may make it difficult to establish a shared view of the situation, much less a collaborative plan of action. Additional obstacles to coordination may stem from competition for resources, lack of information or true differences in their philosophy about humanitarian aid and their ability to deliver it. Donors in particular can create a powerful force for coordination if they agree on priorities and processes. International donors can influence policy-makers by providing resources and technical support conditional on their commitment to the resolution of violent conflict . It is tApplied epidemiologists can use their skills and position to promote positive population health policies and programs to address inequities that exacerbate conflict and violence ,2,4. In Providing health resources, including epidemiological support, should be part of a much larger diplomatic and humanitarian strategy. In fact, when a larger framework and commitment are missing, the health activities are unlikely to result in lasting changes and only increase the risks to the entire population. ,47. EpidEpidemiologists need to advocate for focused field research on conflict resolution and violence prevention, and to evaluate the success of health programs in conflict ,25,36. IWell-documented epidemiologic methods to assess conflict settings combined with ongoing re-assessment of the assumptions for interventions will greatly facilitate health program evaluation. In addition, measures of success for health programs need to reach much further into cultural competence, economic development, and social well-being. Based on the results of these studies more effective and cost-effective methods for public health response could be implemented in the future. With the number and intensity of current armed conflicts there is even greater urgency to begin this work.The knowledge and skills described here are not typically part of epidemiology training. Yet they do fit with the epidemiologist's mission of gathering data and using it to improve the lot of populations. The materials developed to date to train health personnel working in war zones need more specificity and case examples. However, whether epidemiologists are able to respond to war largely depends on the availability of appropriate additional training and modelling by teachers and practicing epidemiologists ,23. EducHowever well-intentioned the science and programs of epidemiology, we must be vigilant that epidemiology benefits public health, that it is not used to contribute to the prolongation of conflict and that it does not become part of it ,22-25,33We should all strive for a time when, through the efforts of public health workers and others, war too will be eliminated.- Jimmy Carter [y Carter


Cryptic structural abnormalities within the subtelomeric regions of chromosomes have been the focus of much recent research because of their discovery in a percentage of people with mental retardation (UK terminology: learning disability). These studies focused on subjects (largely children) with various severities of intellectual impairment with or without additional physical clinical features such as dysmorphisms. However it is well established that prevalence of schizophrenia is around three times greater in those with mild mental retardation. The rates of bipolar disorder and major depressive disorder have also been reported as increased in people with mental retardation. We describe here a screen for telomeric abnormalities in a cohort of 69 patients in which mental retardation co-exists with severe psychiatric illness.We have applied two techniques, subtelomeric fluorescence in situ hybridisation (FISH) and multiplex amplifiable probe hybridisation (MAPH) to detect abnormalities in the patient group.A subtelomeric deletion was discovered involving loss of 4q in a patient with co-morbid schizoaffective disorder and mental retardation.The precise region of loss has been defined allowing us to identify genes that may contribute to the clinical phenotype through hemizygosity. Interestingly, the region of 4q loss exactly matches that linked to bipolar affective disorder in a large multiply affected Australian kindred. The isolation of unique DNA probes from the sub-telomeric regions of all chromosomes has opened up a field of cytogenetics research that was previously inaccessible to conventional karyotyping protocols . Since tFISH, using a commercially available set of subtelomeric probes is the most commonly used screening technique ,20. VariDISC1/DISC2 [DIBD1 [GRIA3 [Psychiatric disorders such as schizophrenia (SCZ) and bipolar affective disorder (BPAD) are relatively common in the general population and there is much evidence for a genetic component to susceptibility (for a review see ). HoweveC1/DISC2 , DIBD1 [2 [DIBD1  and GRIA1 [GRIA3 . The chrThe risk of schizophrenia and affective disorders in patients with idiopathic mild mental retardation is significantly raised and it is well established that schizophrenia is three times more common in this group than the general population and that there is a strong familial element . Both biLocal research ethics permission was obtained for this study. The patients were initially ascertained through computerised psychiatric clinical case-registers that allowed us to identify adults with dual diagnosis of psychosis and mental retardation. A specific psychiatry service exists in Scotland to meet the needs of patients with mental retardation who also suffer from psychiatric disorders and initial clinical diagnoses were confirmed by consultation between the relevant specialist clinician involved and the research team member who is also a specialist in the psychiatry of mental retardation (WM). Confirmation that IQ fell within the mild range of mental retardation was obtained from case records.th Edition  and a referral diagnosis of co-existing schizophrenia or major affective disorder were studied. Parental samples were not available in many cases due to the age of the probands. This cohort is a subset of 74 originally ascertained subjects: 5 were removed because of aneuploidy (2 cases of 47(XXX)) or after more thorough psychiatric evaluation. One discounted subject with only mental retardation possessed a 6q subtelomeric deletion as determined by several MAPH probes (data not shown). The lifetime version of the Schedule for Affective Disorders and Schizophrenia (SADS-L ) along w (DSM-IV ). Diagno . In addition, 5 subjects were diagnosed as having a unspecified functional psychosis .None had co-existing Down Syndrome or Fragile X disorder. A breakdown of the patients into their clinical categories and methodology of screening is presented in table DNA was extracted from venous blood samples (10 mls) of all patients by standard methods using Nucleon BACC2 kits (Nucleon Biosciences). 1 mg/ml dilutions were prepared for MAPH.All 57 MAPH samples were tested in triplicate using the subtelomeric screening set described previously . All samBlood samples from a subset of the patients were cultured in Peripheral Blood Medium (Sigma) for 72 hours. After colcemid treatment for one hour, lymphocytes were lysed and fixed in methanol:acetic acid (3:1). Fixed metaphase material was dropped onto microscope slides. Each slide was hybridised by three fluorescently labelled probe mixes  under separate coverslips. All 15 mixes covering every subtelomeric region for a patient could thus be analysed on 5 slides. Images were captured on a Zeiss Axioskop2 microscope coupled to a Macintosh G4 computer running SmartCapture2.1 software . Five metaphases were scored for each probe mix.-3, corrected for multiple observations). It is unlikely that this copy number change (4q-) defined by MAPH represents an irrelevant polymorphism; no similar change was found on analysis of 83 unrelated control individuals, giving an upper (95% confidence) limit of 1.6% for the frequency of this variant.Complete accord was seen in the 12 instances where both screening methodologies were used. The FISH approach did not detect any subtelomeric abnormalities . However, MAPH identified a subject with a loss of one copy of the 4q subtelomeric region . P values for boundary probes , p < 5 × 10-5 for deletion and p > 0.05 for normal dosage. This 4q deletion encompasses a region of annotated genomic DNA of approximately 3 Mb. The transcript map of this region is not yet completely defined  offers a model of how a cryptic deletion associated with schizophrenia has highlighted several candidate genes for future study -35.DISC1 in psychiatric patients with (non-subtelomeric) chromosomal rearrangements. Therefore, the selection of the screening technique should be determined by the number of cases to be studied and the nature of the abnormalities expected.We used two methods to screen for subtelomeric changes in our cohort of patients. The MAPH technique has proved a fast and accurate method for determining copy number changes in the human genome and represents a cost-effective route to the screening of large numbers of patients. In addition, the disorders that can be studied by this approach are limited only by the design of suitable primers so that screening for both single and contiguous gene disorders is feasible. MAPH has also been proved to be a simple method to map deletion breakpoints with greater resolution than FISH. The subtelomeric FISH approach is a more demanding approach because of the patient sample preparation, the requirement for specialised microscopy equipment, the cost of the commercial probe sets and the labour involved. Nevertheless, FISH has some advantages: the FISH approach is the only way to detect balanced chromosome rearrangements such as inversions and translocations. Other studies of subtelomeric regions in mental retardation subjects have identified such chromosomal aberrations. In addition, FISH has been a vital technique for identifying disrupted genes such as de novo origin. However, psychiatric illness has not been diagnosed in other members of the family. We cannot, therefore, formally link the presence of the deletion with mental retardation and/or psychiatric illness in the patient. The annotation of transcripts at 4q35.2 is currently an active area of research . Consent was not forthcoming to determine whether the deletion was of parental or FRG1, for facioscapulohumeral muscular dystrophy. The 4q patient does not show any of the typical features of this disorder but this can be explained by the fact that copy number does not appear to be critical for the onset of the disorder [The subtelomeric region of 4q is also interesting because it contains a candidate gene, disorder . Rather,disorder .Of the 69 patients with clear co-morbidity, one (1.4%) possessed a single copy subtelomeric deletion. This frequency is in line with those from studies of individuals with mental retardation alone . As moreThe identification of the precisely delimited 4q deletion may contribute to the mapping of the susceptibility gene for psychiatric illness at this locus. The finding of bipolar affective disorder linkage to this region suggests that, in this case at least, the schizoaffective and mental retardation components to the co-morbid phenotype may be discrete and genetically separable in the manner of other contiguous gene disorders. If it is assumed that either component of the clinical phenotype is caused by haploinsufficiency then examining the comparative expression levels of candidate genes in normal, bipolar-linked and 4q deleted lymphoblastoid cell lines might provide a quick route to the identification of causative genes. Alternatively, gene association studies may be required to identify the candidate psychiatric illness gene at 4q35. The high rate of recombination in subtelomeric DNA means that a higher density of genetic markers will be required to establish linkage or association reliably; conversely, once detected, the high rate of recombination will allow high resolution fine mapping of significant loci.None declared.BSP participated in the design of the study and the FISH analysis and drafted the manuscript. EJH and JALA carried out all MAPH assays and associated statistical analyses. MPM carried out blood culture and FISH analysis. WJM conceived the study and was responsible for the generation of all clinical data, and DHRB and DJP participated in the study design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


European scientists are pressing for the creation of an independent body to fund European research - driven by the pursuit of scientific excellence Science looks set for a fundamentalist revival within the European Union. Its leading proponents are taking advantage of unprecedented political upheaval—as ten new Member States accede to the Union—to press their case for funding of basic research that is driven solely and independently by investigators themselves in the pursuit of excellence.The broad thrust of their appeal calls for the setting up of a new agency, most commonly referred to as a ‘European Research Council’. The ERC could be an entirely new organisation or a new division within an established body, run by a small staff able to draw on the best expertise available. It would administer a new fund from EU coffers, tagged the European Fund for Research Excellence, that would be valued modestly, initially at least, at much less than half of the EU's existing budget for research. Most importantly, dispersal of that fund would reflect the wishes of eminent peer reviewers, assessing competitive bids in search of the best science, rather than the judgements of Eurocrats, looking for the most politically and economically expedient solutions and operating on a lead time of two years or more.juste retour, or fair reward, in which Member States traditionally seek to recover grants at least equal to their contributions to the EU pot . ‘Research activities at national and [European] Union level must be better integrated and co-ordinated to make them as efficient and innovative as possible, and to ensure that Europe offers attractive prospects to its best brains,’ concluded the EU leaders, eager to reverse the flow of trained talent abroad, notably to North America. All appropriate means, they added, ‘must be fully exploited to achieve this objective in a flexible, decentralised and non-bureaucratic manner’.Two years later, at the European Council in Barcelona, the EU leaders went one step further by defining the target more precisely. ‘In order to close the gap between the EU and its major competitors,’ they said, ‘overall spending on R & D and innovation in the Union should be increased with the aim of approaching 3% of GDP by 2010. Two-thirds of this new investment should come from the private sector.’The scale of the challenge is illustrated by the latest figures for R & D expenditure, published in February by the Statistical Office of the European Communities (Eurostat). The EU's estimated R & D spending in 2002 was 1.99% of GDP, still far short of the US (2.80%) and Japan (2.98% in 2000), and a long way from the target of 3%.Emphasising the UK's uneasiness about the EU's escalating enthusiasm for a regional science base, the Royal Society  poured scorn on the ‘ambitious’ GDP target by noting how the UK alone would have needed an extra £11 billion in 2000, or more than 60% of total spending on R & D, to lift its ratio of 1.85% to the 3% target. The Royal Society also noted how public funding of R & D in the EU matches that in the US and Japan, with the disparity among GDP ratios reflecting the differentials in private investment in R & D, over which the EU has little control.Nevertheless, the challenge could not be ignored. According to Bob May, professor of mathematical biology at the University of Oxford, president of the Royal Society, and former UK Chief Scientist, such initiatives might be ‘driven more by political expediency than common sense, but the moment you see that train beginning to roll, there's a chance to do something useful with it’.Among the leading proponents of an ERC is Bernard Larrouturou, director general of the National Centre of Scientific Research (CNRS) in Paris, France. For Larrouturou, a biomathematician currently engaged in streamlining the organisation, the changes at the European level are a breath of fresh air. However, he is not convinced that funded investigators should expect to exclude Commission strategists entirely from their lives. The scientific community should lead an ERC, says Larrouturou, ‘but I do not like the idea that this should be completely under the guidance and wisdom of the scientific community with no strategy guidance. You cannot ask for 1 or 2 billion Euros every year and say there will not be any strategy and [that it will be done solely] on this basis of excellence.’ And Larrouturou distances himself from the idea that basic and applied research can be treated separately because this suggests, wrongly he says, a conflict between the two.On these issues, Larrouturou moves onto some common ground with John Taylor, former director general of Research Councils UK and now chairman of Roke Manor Research, a UK subsidiary of Siemens, the German electronics group. Research Councils UK oversees spending of Britain's national research councils . Interactions across disciplines and between scientists and technologists ‘are not helped by making artificial distinctions between this kind of research and that kind of research,’ says Taylor. ‘The distinctions I make are much more between top-down and bottom-up.’While Taylor is a joint architect of one proposal to create an ERC, he remains unconvinced that the research funding system is broken, especially from the UK's perspective, and needs to be fixed. Nor is he convinced that EU funds for an ERC will not affect national R & D budgets. ‘I'm middle of the road,’ he says. ‘Much greater collaboration is good. It has to be a slow process, with all the different cultures involved. Collaboration on various areas of science is an excellent way to go, provided you don't try to organise it from the top and legislate for it all to happen in a particular way and to a particular timescale. Excellence is key.’Taylor's cautions reflect his experience of the EU's Framework Programme and his reservations that any initiative from Brussels can be free of red tape. ‘If you want to do research, then you can't lay out beforehand all the answers you're going to get,’ he says. ‘And if you try to get people to stick rigorously to a plan, then you get a lot of silly things going on. If you try to form very complex bureaucratic organisations to do the research, you get a lot of delays and so on, so things are not very timely.’But the Framework Programme's failures need not spell disaster for the fledgling funding council, insists Lennart Philipson, former director general of the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, and now an emeritus professor at the Karolinska Institute in Stockholm, Sweden. Drawing on his 11 years as head of EMBL, until 1993, Philipson recalls how ‘pan-European peer review was the best method for distributing the funds of EMBL and EMBO [European Molecular Biology Organization]’. The continuing high status of the two organisations, he says, is testimony that the system works. In fact, EMBO is mentioned as a possible incubator for an ERC, in spite of its specialisation.Other proponents of the proposed research changes in the EU include 45 Nobel Laureates from Europe or of European origin, who headed a petition organised by EMBO. The European Life Scientists Organization (ELSO) organised another. Its president, Kai Simons, also the Director of the Max Planck Institute for Cell Biology and Genetics in Dresden, Germany, says research funding in Europe is just not working. ‘It's not geared for basic research—it has other aims,’ he notes. EU funds are ‘not grants, they are contracts with in-built milestones that have nothing to do [with basic research]. Basic research doesn't work like that.’The evaluation and peer review system is falling apart, continues Simons. He says that the best people are not interested in peer reviewing a system that doesn't work: ‘You're not attracting the peer reviewers that you need to maintain quality.’ But at last, acknowledges Simons, someone in Brussels is listening. ‘In the past two years there has been enormous progress.’Within a month of the Barcelona Council in 2002, the European Science Foundation (ESF), which brings together the funding agencies of 29 countries and acts as a bridge to Brussels, had formed a High Level Working Group to review the case for an ERC and how it might operate. The group, chaired by Sir Richard Sykes, Rector of Imperial College, London, United Kingdom, reported a year later, in April 2003. It endorsed the creation of an ERC as ‘the cornerstone for the ERA and the key approach to developing a locus for…long-term fundamental curiosity-driven research judged on the basis of excellence and merit’. The Sykes group also proposed, controversially, an enhanced ESF as the most effective medium for establishing an ERC swiftly.‘Some people say that the ESF has no experience in funding large amounts… for research,’ acknowledges Enric Banda, director general of the Catalan Research Foundation in Barcelona, Spain, who finished a five-year term as the ESF's chief executive at the end of 2003 and is credited with ‘waking up’ the foundation. ‘But certainly if you create a new [organisation], that's the same thing. So the ESF is in a good position because its member organisations are the funding agencies.’Bertil Andersson, who was a member of the Sykes Group before taking over from Banda at the ESF in January, also stakes the ESF's claim to nurture a fledgling ERC. But he accepts that any one of the respected national funding agencies, such as the German Research Foundation (DFG), or even a specialist body, such as EMBO, could do the job. ‘We don't need a new skyscraper in Brussels, but a lot of… peer review and running of the ERC could be done by existing bodies.‘Compared to soccer, we have only the national leagues—we don't have the Champions League [the league of Europe's best teams],’ says Andersson. There is no competition for basic research grants across national boundaries in Europe, he insists. ‘The Swedish league is exciting, but the Champions League is more exciting.’In the meantime, while the Sykes group was still deliberating, the Council of the EU appointed another group of experts to evaluate the case for an ERC. Chaired by Federico Mayor, former director general of the United Nations Educational, Scientific, and Cultural Organization (UNESCO), the ERC Expert Group also delivered its verdict—a resounding endorsement—within 12 months.‘The first and main task for the ERC should be to support investigator-driven research of the highest quality selected through European competition,’ concluded the Mayor report, published in December 2003. ‘In doing so, the ERC should create and support nodes of excellence in European universities and research institutions, strengthening the knowledge-base that underpins economic, industrial, cultural and societal development, and thereby stimulating European competitiveness and innovative capacity at all levels.’While few disagreed with the Mayor report's sentiments, the absence of a detailed analysis exposed underlying tensions over the rationale for an ERC. In the UK, in particular, some scientists seemed concerned that their mature and respected system for funding research risked dilution.‘The British have always had doubts about what goes on in Europe,’ notes Kai Simons. ‘They always think that they can do it better. But the big problem for the British is that they are also too small to fund a new innovative area,’ he says. ‘Of course, we can do it without Britain, but they are an important part of Europe and it would be sad if they're not part of it.’The agnostic John Taylor, who was a member of the Mayor group, recalls his early reservations when the group convened. ‘I'm way beyond the euphoria; I'm into practical pragmatics,’ he notes. ‘My major input into the whole thing has been to get them to “get real” instead of just philosophising. They've been using the sort of, dare I say it, Gallic approach… of thinking about the reasons why, and the philosophy, and not thinking about what you would actually do.’Taylor dismisses the notion that wariness of the ERC is representative of a general antipathy in Britain towards European integration. ‘What we're saying is that science in the UK is not yet well-funded enough to say we would rather do this [the ERC] instead of the things that we're already trying to get done in the UK scene.’Anticipating the Mayor report's publication, the Royal Society quickly pulled together a detailed background paper late last year that identified ‘a number of problems that need resolution, although not necessarily through the establishment of any major new institutions within Europe’. An addendum followed in March, in direct response to the Mayor report. That addendum highlighted what it saw as the paucity of solid evidence in the Mayor report and, in some cases, the confusing data in the report's case for an ERC.On balance it looked as though the Royal Society, and as such the British science establishment as a whole, had weighed the disadvantages of an ERC as greater than its advantages, but Bob May is quick to refute this charge. ‘My vision and the Royal Society's vision of the ERC is that it will fund the very best science,’ he insists. ‘The Mayor committee itself was really good people who'd produced basically a good report…. I'm basically in favour of this European Research Council… provided it can be set up properly, which is by no means certain.’For May, and other scientists on the continent, the ERC offers a real chance to redress the balance of fortune in favour of young scientists. ‘The way to encourage science is to get the best people and set them free to express their creativity while they are young, which means bring them into the best laboratories—don't let them get entrained in hierarchies of deference to second-rate people,’ says May.juste retour, whether they're serving some industrial purpose, it will just try to fund the best science. But I hope increasingly the best projects will involve collaborations, as they do in Britain, collaborations among institutions within Europe.’‘The most important single thing to create one Europe in science is a flexible postdoctoral programme that gets the best young people wherever they are and lets them go to the best places,’ enthuses May. An ERC will then foster those collaborations, he forecasts. ‘It won't ask whether they're Council of the European Union – Ruling organisation , and not to be confused with the European Council (see below). It comprises ministers from governments of the Member States, which have varying voting powers led by France, Germany, Italy, and the UK.Euro (€) – Common European currency launched on 1 January 2002 in 12 participating Member States .European Commission – Executive organisation, mainly based in Brussels, run by 20 Commissioners and around 24,000 civil servants.European Council – Body that brings together leaders of Member States to define broad policy objectives for the EU's six main institutions . Meets twice a year in the Member State holding the Council's presidency, which changes every six months.European Parliament – Elected organisation, based in Strasbourg, France, that rules the EU  and will have 732 Members after the accession of the ten new Member States in May 2004.European Research Area – Commissioner Philippe Busquin's vision for the future of research in Europe, and the main focus of the 6th Framework Programme. It aims to achieve ‘scientific excellence, improved competitiveness and innovation through the promotion of increased co-operation, greater complementarity and improved co-ordination between relevant actors, at all levels’.European Union – Evolving political, social, and economic union of an increasing number of European countries, or Member States. First proposed in 1950 during rehabilitation after the Second World War and formally created by the Maastricht Treaty in 1992. Grew from six nations in 1951  to nine in 1973 , to ten in 1981 (addition of Greece), to 12 in 1986 , to 15 in 1995 , with a total population of 380 million people . Ten more countries  join in May 2004, which will lift the EU's population to 450 million people. Bulgaria and Romania are due to join in 2007, which will add another 50 million people.Framework Programme – The EU's principal mechanism for funding research in Member States, proposed by the Commissioner for Research (Philippe Busquin) and adopted by the Council and Parliament. Framework Programmes have four-year budgets but cover five-year periods, so consecutive programmes overlap, and are prescribed two years before they begin. The 6th programme (FP6) is worth €17.5 billion  and runs from the beginning of 2003 to the end of 2006.Juste retour (fair reward) – Claim made by Member States for rewards at least equal to their share of the cost of any programme or initiative; critics say it promotes bureaucracy and uncompetitiveness.


Defensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics.We find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes.Complexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association. One obvious reason for these gaps is that the appropriate regions are enriched in sequences that are not tolerated by the cloning systems. The second possibility is that even if clones are available and amenable for sequencing, their sequences cannot be unambiguously aligned due to gap flanking segmental duplications. Generally, those duplicons are defined by >90% sequence identity and lengths of >1 kb and about 87% of all human ones are longer than 50 kb . The coding sequences of the gene copies differ and in some cases the reading frame contains premature termination codons. Blat search of the type III repeat subunit revealed three paralogs in the human genome. Two of these are located on chromosome 8p23.1 at about 12 Mb (flanked by repeats of type II), the third at 12p13.31. The paralogs are slightly rearranged in comparison to the subunits of III.1-5.Located adjacent to DEF clusters b .We manually inspected seven clones covering Pan troglodytes, ptr) whole genome shotgun (WGS) working draft , but trace data inspection indicates the presence of several different haplotypes. Additionally, ptrDEFB108 and ptrDEFB109p are not covered by any chimpanzee WD sequences. As an alternative to the WD approach, we sequenced for ptrDEF cluster b three BAC clones. Examination of SNPs in overlapping regions (104 kb) of the three clones [GenBank:AC150655], [GenBank:AC150656], [GenBank:AC150657] revealed three different haplotypes originating from one chimpanzee. The detected aa changes in human and chimpanzee defensins are illustrated in ptrDEFB108 is a pseudogene, since also the start codon ATG is changed into GTG.In order to compare the human chromosome 8p23.1 DEF region to the orthologous locus in our closest relative, we both employed the chimpanzee  to 1:7 (proband 1). Interestingly, this haplotype is also found in the trace archives of chimpanzee and baboon. Furthermore, ratios of the individual haplotypes of DEFB104 as well as of DEFB4 indicate different DEF cluster b numbers in the four individuals. While proband 3 bears five copies, proband 4 most probably harbors eight copies or multiples thereof.In order to determine individual DEF copy numbers we PCR-amplified a 500 bp fragment of ls Table . Three ode facto gap, it better reflects the region in the sense of a „human genome reference“, since the clones harboring copies of DEF clusters b derive from three libraries  and may therefore represent up to five alleles (library RP13 is represented by only one clone). Our assembly also reflects better the diversity of all available sequence data of this chromosomal region: 27 out of 32 finished clones are incorporated into the tiling path. The remaining five clones cannot be included in the assembly according to our quality criteria and therefore must be regarded as parts of additional copies or alleles. Furthermore we point out that the identification of a third copy of the DEF cluster b in the 360-kb-contig does not represent an allele of clusters b1 or b2 derived from an alternative library / donor, since besides four SCb clones one RP11 clone is incorporated. With respect to the RP11 library of which most of the DEF cluster b covering clones derive we conclude that at present sequence information of at least five variants of the cluster is available from a single individual: cluster b1 ; b2 (clones 16 – 20); b3 (clone 30); b4 (clone 11) and b5 (clone 12). All these results are in agreement with the reported interindividual variability of DEF cluster b genes ; green/yellow signals) and CTB-415D8 [GenBank:AF228730]; red signals) visualized by FISH on metaphase chromosomes according to standard protocols ,53. MetaClick here for fileResolving LCR type II and III "pairs" on chromosomes at approx. 900 band stage. Probe CTB-415D8 [GenBank:AF228730]; LCR type II and III) generates two clearly separated FISH signals at 4p16 and 8p23, respectively : In contrast, probe SCb-561b17 [GenBank:AF238378]; LCR type I) yield a single signal at 8p23, solely , that is co-localized with the telomeric signal of probe CTB-415D8 [GenBank:AF228730]. Signals with lower intensity are indeterminable in this picture.Click here for fileptrDEF cluster a) are deduced from the chimpanzee WD and might therefore include sequencing errors. Those for ptrDEFB1, ptrDEFB107, ptrDEFB105, ptrDEFB103, ptrDEFB4 and ptrDEFB108 (DEF cluster b) are derived from high quality BAC sequences and the appropriate traces were visually inspected. The gray shadow indicates the motif of six cystein residues (except for DEFB107 with only five cysteins).DEF aa sequences with highlighted residues (bold) different between human and chimpanzee. Boxes: human aa – human position – chimpanzee aa. All aa positions refer to the following human protein accessions: DEFA6 = [GenBank:NP_001917]; DEFA4 = [GenBank:NP_001916]; DEFA1 = [GenBank:NP_004075]; DEFA5 = [GenBank:NP_066290]; DEFB1 = [GenBank:NP_005209]; DEFB107 = [GenBank:AAM93909]; DEFB105 = [GenBank:NP_689463]; DEFB103 = [GenBank:NP_061131]; DEFB4 = [GenBank:NP_004933]; DEFB108 = [GenBank:AAN33116]. Aa for the chimpanzee orthologs ptrDEFA6, ptrDEFA4, ptrDEFA1, ptr novel defensin similar to DEFA4, ptrDEFA5 and ptrDEFB1 , individual loci may have incorporated variable numbers  of additional b clusters in either orientation. The proposed duplicon consists of two inverted LCRs flanking a DEF cluster b (top/bottom). The orientation of any DEF cluster b can change either by inverted duplication/crossover (i) or homologous recombination within inverted LCRs . Moreover, the proposed genomic structure indicates that even in a 'minimal' DEF locus one or both DEF clusters may be deleted due to homologous recombination between direct LCR copies (Δ). Sequence features of the most distal LCR  as well as LCRs type II (rectangles) are shown. Click here for file


The main goal in analyzing microarray data is to determine the genes that are differentially expressed across two types of tissue samples or samples obtained under two experimental conditions. Mixture model method (MMM hereafter) is a nonparametric statistical method often used for microarray processing applications, but is known to over-fit the data if the number of replicates is small. In addition, the results of the MMM may not be repeatable when dealing with a small number of replicates. In this paper, we propose a new version of MMM to ensure the repeatability of the results in different runs, and reduce the sensitivity of the results on the parameters.Hyp mice. In each study, the proposed algorithm successfully selects genes closely related to the disease state that are verified by biological information.The proposed technique is applied to the two different data sets: Leukaemia data set and a data set that examines the effects of low phosphate diet on regular and The results indicate 100% repeatability in all runs, and exhibit very little sensitivity on the choice of parameters. In addition, the evaluation of the applied method on the Leukaemia data set shows 12% improvement compared to the MMM in detecting the biologically-identified 50 expressed genes by Thomas et al. The results witness to the successful performance of the proposed algorithm in quantitative pathogenesis of diseases and comparative evaluation of treatment methods. Recently, microarray technology has provided the means for simultaneous screening and analysis of thousands of genes. Although an enormous volume of data is being produced by microarray technologies, the full potential of such technologies cannot be accessed without the ability to sift through the noisy signals to obtain useful information. The large data sets produced by microarray technology have resulted in the need for reliable, accurate, and robust methods for microarray data analysis. A major challenge is to detect genes with differentially expression profile across two experimental conditions. In many studies, the two sample sets are drawn from two types of tissues, tumours or cell lines, or at two time points during the course of a biological processes.The computationally simple methods used for such analysis, including the methods of identifying genes with fold changes (such as the popular Log-ratio graphs) , are knoThe major disadvantages of the above-mentioned methods, especially the MMM, include the lack of repeatability of the results under different runs of the algorithm, and the sensitivity of the algorithm on parameter initialization. A reliable microarray analysis method must be reproducible and applicable to different data sets under different experimental conditions. More specifically, an accurate microarray processing method is expected to pinpoint, with a relatively high degree of accuracy and robustness, genes with elevated expression levels that are related to the experimental condition in all runs. The main objective of this paper is to design and test an extension of the MMM whose results are reproducible, more biologically meaningful, and significantly less sensitive to the models' parameters.The paper is organized as follows. In Algorithms section, a review of the MMM and its recent extensions, Mod2MMM, together with the detailed description of the proposed method are given. In Results and Discussion section, the K5M algorithm is first applied to the well-studied Leukaemia data set  that is Yij as the expression level of gene in array i , where the first j1 and last j2 arrays are obtained under two conditions. A general statistical model for the resulting data is:We start this section with a brief review of the existing MMM based techniques. Consider Yij = ai + bixj + εij     (1)xj = 1 for 1 ≤ j ≤ j1 and xj = 0 for j1 + 1 ≤ j ≤ j1 + j2. In addition, εij is a random error with mean 0. From the above formulation, it can be seen that ai + bi is the mean expression level of the first condition, and ai is the mean expression level of gene i in the second condition. The method requires that both j1 and j2, the number of data sets for each experiment condition, be even.Where ai is a random permutation of a column vector that contains j1/2 1's and j1/2 -1's and bi contains j2/2 1's and j2/2 -1's.The t-test statistic type scores (2) and (3) are calculated on the pre-processed data. Here, f0 and f are estimated as in (4) and (5), respectively. The null distributions, f0 and f, are estimated directly in a nonparametric model for gene expression data.Since the data are not assumed to be normally distributed, the distribution functions φ symbolizes the normal density function with mean μi, variance Vi, and the mixing proportions πi define the linear combination of the normal basis function. We use Φg0 to represent all unknown parameters {: i = 1, ..., g0 } in a g0-component mixture model. The number of normal basis functions, i.e. g0 can be estimated by the EM algorithm, which maximizes the log-likelihood function of (6) to obtain the maximum likelihood estimation of .Where K iterations, the EM algorithm is expected to find the local maxima for all unknown parameters. It is recommended to run the EM algorithm several times with various random starting parameters and choose the final estimate as the one resulting the largest log-likelihood [Within kelihood . As ment for different g0 's, the algorithm selects the sub-optimal g0 corresponding to the first local minimum of BIC or AIC [After finding the optimized C or AIC .vg0 is the number of independent parameters in Φg0. Then, the algorithm uses the resulting g0 as the number of normal functions to fit f0. The same procedure is performed to estimate the sub-optimal number of normal functions to estimate f. As mentioned above, with the fixed number of normal functions, the parameters of functions f and f0 are iteratively updated for a number of iterations. When the iterations are terminated, the likelihood ratio is estimated based on the final estimations of f0 and f:where LR(Z) = f0(Z) / f(Z)     (9)s, if LR(Z) <s, then the gene is identified to have significantly altered expression in two experiments. It is possible to determine the rejection region numerically, i.e. for any false positive rate α, the threshold value s = s(α) can be calculated from the following integral:A bisection method  with a Bα = 0.01 is often used as the genome wide significant level, so the gene-specific significance level is: α* = α/(2n) Recently a new modification of the MMM algorithm, Mod2MMM hereafter, was introduced [z may be different from the null distribution of Z, which can lead to invalid inference. The modified algorithm starts with the assumption that j1 ≥ 2 j2 [z and Z as you can follow in appendix1.In literature of microarray processing, troduced . This me1 ≥ 2 j2 , and conj1 ≥ j2 but j1 < 2 j2, j1 observations drawn under condition one are split into two equally-sized parts to calculate , vi(1a) and , vi(1b) respectively. To calculate  and vi(2) about j1/2 observations are drawn under condition two. While this modification can address the differences in the distributions of f and f0, the stability of the parameter estimation step still remains a major problem.For the cases where z  may be different from the null distribution of the statistics Z (as defined below). This assumption can potentially lead to invalid inference. A modified version of the MMM (Mod2MMM hereafter), introduced in [zi may not be independent. This method addresses the issue by constructing new zi and Zi variables as will be discussed later.The main difference between the conventional MMM and its recent extensions are that the conventional MMM disregards the fact that the true distribution of duced in , assumesA concern over all existing MMM based methods (including Mod2MMM) that greatly affects the results is associated with the way mixed distributions are estimated. In the MMM, Expectation Maximization (EM) algorithm  is oftenz by using mixture of normal distributions in a stable and reliable way. The following observations made in the experimental study of the MMM for gene expression analysis were the main motivations for the proposed changes to the MMM:In order to address the stability and reproducibility of the MMM, we propose a new modified approach for the MMM that estimates the distribution function of 1 The observed variations in the parameter estimation process in some versions of the MMM can be attributed to the algorithm's attempt to iteratively update the means and variances of the normal distributions using often noisy data. In experimental studies, often the direct observation of the data reveals specific points where centers (means) can be positioned and the scattering patterns that can give reliable estimates on the variance of each cluster. However, the iterative updating of model parameters with noisy data and based on some random starting points often misses the true optimal points and even creates variations and fluctuations in parameter estimation in many runs.f and f0. This in turns results to lists of differentially expressed genes in different runs. More specifically, a set of two typical runs of the algorithm on the same data set can result to two lists that are very different both in number of the genes as well as the exact genes picked up by the algorithm. In our study of the conventional MMM and Mod2MMM, two runs with the same algorithm (on the same data) resulted to lists whose size vary between 50 and 200.2 Even when variations do not occur, two runs of the algorithm can result to significantly different estimations of 3 The literature of other areas of research utilizing normal basis function for estimation including neural networks indicates that in order to have more robustness in different runs and have reproducible results, the means and variances of the basis functions must be estimated and fixed during the iteration on the coefficients . This isz is regarded partially as a clustering problem, i.e. the means and variances of the normal distributions are estimated as the prototypes of a clustering step. Specifically, if z is distributed in a one-dimensional space, wherever there is a mass of z, there is a cluster with mean μi and variance Vi, which are identified by the members of that cluster.4 Based on the observations mentioned above, in our proposed method, finding the distribution of f0 and f based on K normal distributions. While the algorithm can use K-means to find the optimal values of means and variances, the coefficients πi 's need to be optimized using an optimization process such as the EM.Hence applying a clustering method is capable of estimating the means and variances of each normal distribution. The key is to use a simple clustering technique such as K-mean to estimate the mixture distributions Based on the above discussion, the proposed algorithm can be described in the following two steps:Step 1: Using BIC, find the sub-optimal number of normal distributions for both f and f0 (as described above). These optimal numbers are then used as the number of clusters in K-means technique.Step 2: Using K-means clustering technique, for both f and f0 find the best mean μi and variance Vi for all clusters.Step 3: With the obtained values of μi, Vi and using the EM algorithm, iteratively update the values of the optimized πi for all clusters (both f and f0), i.e.f and f0. The MMM creates the likelihood ratio (LR) statistics plotted in Figure K = 4 forms the LR statistics plotted in Figure K = 2 results to the LR plot of Figure A reasonable number of clusters is expected to be obtained from the first step of the algorithm, and the estimation results of the two bellow data sets in Tables πi 's, in each run, it is expected that the number of identified differentially expressed genes fluctuate slightly. However, as indicated above, since the K- means clustering algorithm is known to a robust method, and considering the fact that in the EM estimation process, only a linear estimation is performed, it is expected that the robustness of the proposed algorithm be much more than the other version of the MMM based algorithms. This observation, as have been shown before, is supported by our experimental results. In addition, our experimental indicate that the most expressed genes are identified in all runs or the algorithm and in each run one or two new genes with less expression ratio are added to this set.It is worth mentioning that due to the random initialization in K-means and the random initialization of the coefficients In this section, first the two applications and their corresponding data sets are described and then the results produced by the proposed method (i.e. K5M) is compared with the other MMM based methods on two data sets. The detailed description of the methods is given in MMM & its recent extensions Section.α = 0.01 (according to Benferroni adjustment used in the MMM based methods). Each sample in the dataset is pre-processed as in [In this section, we apply the nonparametric MMM method with and without the proposed modifications to the Leukaemia data presented in . The objed as in , by subtThomas et al  used knoThe comparison of the result obtained by the K5M with those of the MMM and the Mod2MMM is summarized in Table K = 3 identifies the total of 185 differentially expressed genes, while with K = 4 the total of 58 genes are identified, however; the 58 genes found with K = 3 are the most expressed genes among 185 genes found by K = 4. This result shows the consistency of the K5M method.As the BIC suggested the optimum number of clusters K = 4 for the MMM, the K5M is applied with K = 4 also. Running K5M with different number of clusters leads to the different but reasonably similar results. As the number of the clusters increase, the number of expressed genes decreases. Table In order to further compare the performance of the MMM and K5M on the leukaemia data, The ROC curve is plotted based on False Positive rate and True Positive rate of the data set calculated as in [Hyp mouse. Hyp mice have a mutation of the Phex gene [Phex gene block renal adaptation to low phosphate diet. Hyp mice have a primary osteoblast defect and defects in vitamin D metabolism. Parabiosis experiments on normal and Hyp mice have revealed that there is an intrinsic osteoblast defect in Hyp mice rather than an intrinsic renal abnormality. Hyp kidneys transplanted into normal mice reabsorbed phosphorus at normal levels. Kidneys transplanted from normal mice into Hyp mice began phosphate wasting in the Hyp mice.The following study is the main application for which the proposed method was specialized and therefore is described in more details. Hypophosphatemia is a common X-linked metabolic bone disorder in human. Hypophosphatemia results from phosphate wasting in the renal tubules. Phosphate that is normally reabsorbed from the urine is excreted. It appears that elevated levels of FGF-23 activate the excretion of phosphorous by the kidneys. Previous studies have demonstrated an impairment of the high- affinity, low capacity Na+ dependent phosphate co-transport system . The maihex gene ,9. The dHyp mice should indicate the systems involved in the phosphorus homeostasis. In an attempt to identify these genes, nutritional experiments were performed on normal and Hyp mice [[Hyp mice were placed on low phosphate diets for 3 – 5 days. Tissue samples from the kidneys of test and control mice were collected. 16 samples were analyzed using Affymetrix GeneChip mouse U74A arrays- 4 samples for each experiment state. The mRNA of 12,488 genes was analyzed. Two GeneChip microarrays were done for each diet for normal mice and three microarrays for each diet for the Hyp mice for a total of 10 arrays.The mechanism that leads to the excessive excretion of phosphorous is unknown. On a low phosphate diet a normal mouse will activate systems to conserve phosphate by increasing re-absorption. The genes activated in the normal mouse on the low phosphate diet, and the genes with differential expression between normal and yp mice [,8-14 andyp mice . NormalHyp were fed a control (1.0% P) or low phosphate (0.03% P) diet for five days. The four group experiments are shown in Table To investigate this, 5-week-old normal and In this study, we consider the gene expression signal less than 100 as noise caused by the microarray machine, and in the pre-processing step we ignored the genes whose expression signals in both conditions are less than 100. The following two specific goals are considered in this study:1. To identify the genes in whose mRNA expressions are altered by low phosphate diet in normal mice.Hyp mutation on this response, i.e. identifying the genes in Hyp condition that are differentially expressed across the normal and low phosphate diet experiments.2. To determine the effect of Hyp dataset includes five samples for each group. In order to make the number of data samples even, we used four samples of each group. For this data set, since j1 = j2, the Mod2MMM cannot be applied. In MMM method, five mixture models are used to estimate f0 and f  with number of normal basis functions ranging from 1 to 5, i.e. The MMM algorithm was run several times and the run with maximum log-likelihood was chosen as the final model. Bayesian Information Criterion (BIC) [α = 0.01, and therefore the gene-specific significance level used here is calculated as:The on (BIC)  was usedα* = 0.01/(95.44 * 2) = 5 * 10-7s is obtained as s = 3 × 10-6.. Both the MMM and K5M were run 100 times. Figure Using bisection method , as discHyp condition on the most definitely expressed gene in normal and low phosphate diet microarrays. The ten most significant genes that are differentially expressed across the two experimental conditions, i.e. Normal Low Phosphate and Hyp Low Phosphate, are listed in table 6. As shown in the table 6, again eight genes are related directly to the kidney's function. These further witnesses to the capability of the proposed technique to discover the genes that are truly involved in the biological study.The ten most significant genes expressed by the low phosphate diet in the normal mouse identified by the MMM, and the ten most significant genes provided by K5M are represented in Table Hyp mice [In this paper, we proposed a technique to improve the repeatability, and robustness of the mixture model method by using the K-mean clustering method in estimating the distributions. Our proposed method finds the distribution of the variables partially based on a clustering procedure and an EM optimization process. The method is applied to analyze two microarray data sets, Leukaemia data set and a data set reflecting the effect of the low phosphate diet on regular and Hyp mice  data. ThMaryam Zaheri, and Ali A. Rad were in charge of writing the codes and programming aspects of the paper.Siamak Najarian and Javad Dargahi's primary role was to perform a literature review on mixture model techniques, identify the aspects of the method that need to be improved, and provide suggestions to address these shortcomings.Kayvan Najarian's primary roles were to design improvments to the algoritm , prepare and pre-process the data (for both datasets), partcipate in preperation of the Hyp dataset, define the Hyp problem interpret the results and finally write and edit the manuscript.The Mod2MMM makes a new z and Z based on the following formula:Where:And:


The purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results.There is a substantial discrepancy between in vitro we demonstrated that the in vivo response has only one maximum. For biphasic patterns of biological response in vitro both the bimodal and biphasic in vivo responses might be observed.For dose-response curve reaches a plateau in vivo responses might be predicted from dose-effect curves measured in vitro.As the main result of this work we have demonstrated that  In vitro bioassay is very useful in biomedical experiments. It has the potential to yield very important data about molecular mechanism of action of any biologically active compounds. However, the major challenge for such experiments is extrapolation to in vivo responses. Unfortunately, there is a substantial discrepancy between in vitro and in vivo experiments, and there is a paucity of work directed to prediction of in vivo response from in vitro bioassay. So, the purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results.in vitro experiments fall into three classes. They are:A survey of literature revealed that most cases of dose-effect curves for • monophasic response;• biphasic pattern;• bimodal or polymodal dose-effect curve.MONOPHASIC RESPONSE is the form most commonly reported in articles on in vitro bioassay. In these cases, with increasing dose of biologically active substance (BAS), the cellular response increases to a maximum (dose-response curve reaches a plateau). The most general schemes exhibiting this class of response can be classified as 3 classes:(I) BAS regulation of enzyme activity,(II) Ligand interaction with one type of receptor, and(III) Ligand interaction with negatively cooperative receptors.We will consider these three classes:(I): BAS might regulate enzyme activity. It might be:• substrate:E+S ←→ ES → E+P → cell response,     (scheme 1)E is enzyme, S is substrate, ES is enzyme-substrate complex, P is product. Cellular response is suggested to be proportional to product concentration.where Scheme (2) approximates the classic Michaelis scheme .A)• enzyme activator (E+S ←→ ES → E+P → cell responseE+A ←→ EA     (scheme 2)EA+S ←→ EAS → EA+P → cell response increasing,Scheme (3) is characteristic of many BAS. The majority of these groups are vitamins and minerals, which are known to be enzyme cofactors and serve to increase enzyme activity.I)• enzyme inhibitor For example, there is the large class of drugs, whose action can be described with the help of scheme (4). This class is called "inhibitors of angiotensin-converting enzyme". These drugs are commonly used for hypertension treatment and prevention .(II) Ligand interaction with one type of receptors:L+R ←→ LR → cell response     (scheme 4)L is ligand (BAS), R is receptor, LR is ligand-receptor complex.where Scheme (4) is "classic" receptor theory as described by Clark 1937) [7 [3].For example, kinetic schemes of such type were proved in the case of estrogen regulation of gene expression , apolipo(III) Ligand interaction with negative cooperative receptorsL+R ←→ LRL+LR ←→ 2R L→ cell response     (5)2R Lis complex ligand-receptor complexes.where Scheme (5) is characteristic for insulin receptors .Kinetic equations for schemes 1)–(5) are well known . They in–(5) are y = B*x/(1+A*x)     (6)and for scheme (5):y = B*x2/(1+A*x2)     (7)x is incoming signal (x is BAS concentration). For scheme (1) x is substrate concentration, for scheme (2) it is activator concentration, for scheme (3) it is inhibitor concentration, for schemes (4) and (5) it is ligand concentration. y is cellular response for the in vitro system. A and B are scaling coefficients.where The BAS concentration in the whole organism changes as a function of time according to equation (14) (see Methods.) i.e.x(t) = C(t) = 0[exp(-kelγt)-exp(-k1t)] C    (8)in vivo responses for monomodal in vitro dose-effect curves for schemes (1)–(5). We used illustrative values from works  for. (1987) .B value. This might be observed in cascade system of signal transduction and amplification. If x regulate intermediate z formation in biphasic way with B1, and z has biphasic response on y formation with B2, then if 1B<2B, summary dose-effect curve (y concentration from x) is bimodal  will require measurements of acute biological response at multiple concentration and time points. For longer-term responses requiring protein synthesis , it could be argued that such multiple time point studies would not be as important. However, in so far as long term biological responses are the consequence of critical initial events which may require threshold concentrations of free hormone, or repeated patterns of hormone exposure over prolonged periods  [B] ≈β [B    (16)B] is concentration of free BAS, [B0] is initial concentration of BAS. If β = 1 then BAS dose not conjugate with protein. If β = 0 then all BAS is in conjugate form.where β is constant ("degree of conjugation"),  C    (18)C(t) is BAS concentration in the blood compartment  is blood volume), C0 is seems initial BAS concentration (C0 = M/V).where