We will disseminate our evidence base, GIS models, and documentation through the project's Web server. The models will be human-readable in different ways to inform NHS GIS implementers, and it will be possible to also use them to generate the necessary template databases  for running the modelled applications. Our products and experience in this project will be transferable to address other national health topics based on the same principles. Our ultimate goal is to provide the NHS with practical, vendor-neutral, modular workflow models, and ready-to-use, evidence-based frameworks for developing successful GIS business plans and implementing GIS to address various health issues. NHS organisations adopting such frameworks will achieve a common understanding of spatial data and processes, which will enable them to efficiently and effectively share, compare, and integrate their data silos and results for more informed planning and better outcomes.EB-GIS4HEALTH UK aims at building a UK-oriented foundation evidence base and modular conceptual models for GIS applications and programmes in health and healthcare to improve the currently poor GIS state of affairs within the NHS; help the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, and requirements; and also foster the much-needed NHS-academia GIS collaboration. The project will focus on diabetes and dental care, which together account for about 11% of the annual NHS budget, and are thus important topics where GIS can help optimising resource utilisation and outcomes. Virtual e-focus groups will ensure all UK/NHS health GIS stakeholders are represented. The models will be built using Protégé ontology editor  Geocoding (street address matching or assignment of latitude and longitude) will be the basis for data linkage and analysis in the 21st century. The versatility of GIS supports the exploration of spatial relationships, patterns, and trends that otherwise would go unnoticed." – US Healthy People 2010 Objectives (item 23-3 – )"Geography plays a major role in understanding the dynamics of health, and the causes and spread of disease . The claToday's health planners aim at developing health policy and services that address geographic and social inequalities in health, and therefore should benefit from evidence-based approaches that can be used to investigate spatial aspects of health policy and practice, and evaluate geographic equity (or inequity) in health service provision .According to the US Federal Geographic Data Committee (FGDC), geographic location is a key feature of 80–90% of all government data . The samUnlike other resources like employees or funds, spatial data do not suffer any wear and tear from repeated use. On the contrary, reusing data increases the possibilities for improving the content quality of data collections and gaining new insights by linking and exploring the relationships between the different datasets that makeup the big picture .This implies the need to develop applications and not just focus on data. An overemphasis on data acquisition, without health sector-linked applications, will not provide any momentum for further development. Visualisation, modelling and analysing activities will be the focus of value-added services in the coming years ,5. Metho), a step reflecting the importance and growing use of GIS in health and healthcare research and practices.In 2003, the US National Library of Medicine added the term "Geographic Information Systems" (GIS) to its controlled vocabulary thesaurus known as MeSH . The inGIS offer a very rich toolbox of methods and technologies that goes far beyond the mere production of simple maps . From a community health perspective, GIS could potentially act as powerful evidence-based practice tools for early problem detection and solving. When properly used, GIS can be used to: inform and educate ; empower decision-making at all levels; help in planning and tweaking clinically and cost-effective actions, in predicting outcomes before making any financial commitments and ascribing priorities in a climate of finite resources; change practices; and continually monitor and analyse performance and changes, as well as sentinel events.Traditionally, two broad types of GIS applications can be distinguished which also reflect the two traditions in health geography , namely health outcomes and epidemiology applications, and healthcare delivery applications. There are also studies at the interface (overlap) between epidemiological and healthcare delivery applications, for example in relation to healthcare commissioning and needs assessment ,7,8.). GIS are also explicitly included in the National Electronic Disease Surveillance System (NEDSS) specifications and systems architecture of the US Centres for Disease Control and Prevention (CDC – ).The use of GIS in the UK National Health Service (NHS) can still be considered as an emerging technology . Despite all their potentials, GIS remain very much under-utilised in the UK NHS in mostly low-level, non-strategic tasks, and in a largely fragmented and uncoordinated way. Spatial data and GIS are still not mentioned in any main UK health information strategy or policy document ,8. In stAlthough multiple novel spatial statistical and GIS methods are potentially available, we still need to unambiguously determine which method(s) and data specifically should be used by practitioners for each specific health condition of interest, and whether the proposed methods are cost-effective and scalable in the context of UK/NHS settings .Moreover, several researchers have highlighted a gap between academic health-related applications of GIS and their everyday use within the NHS. Research undertaken in academia has certainly highlighted the benefits of spatial statistics and GIS approaches in mapping disease and in healthcare planning, but still needs to respond to NHS needs on the ground ,8. On thIn a recent major review paper by this author, the reasons behind the under-utilisation of spatial information and GIS in the NHS, as well as the causes of the gap between academic GIS research and the current everyday use of GIS in the NHS were investigated, and remedial recommendations were made . The reaHow could GIS be beneficial in optimal ways in the context of UK/NHS settings and needs? Any answer(s) or proposed solutions must be based on the best current evidence in the health GIS field.This author proposes to build, consolidate and disseminate a comprehensive evidence base for GIS applications in health and healthcare in the context of UK/NHS settings, and an associated set of evidence-based GIS programme and application models (EB-GIS4HEALTH UK). We will learn from national projects running in the US  and elsewhere, while ensuring that EB-GIS4HEALTH UK's output properly fits the UK health and healthcare agenda.[A systematic and critical review and consolidation are needed of the evidence for GIS for specific preventable, mitigable and treatable health conditions. A good model is the CDC "Guide to Community Preventive Services" . Topics . However, it should be noted that this particular handbook was conceived with the US healthcare system in mind and is thus more suited to US settings, though still useful as a model to follow.We propose to address a subset of the topics in the CDC Community Guide  during the three-year period of our project by conducting a focused review of GIS literature on the chosen topics, and then categorising the "nature of the scientific evidence" documenting whether GIS add any value to our understanding and management of the reviewed topics and/or the evidence that it would be feasible and cost-effective for the respective UK NHS and public health programmes tackling the reviewed topics to adopt GIS. Areas requiring further research will be also highlighted. A good example that comes to mind in this context is the 73-page "GIS for cancer" handbook titled "Using Geographic Information Systems Technology in the Collection, Analysis, and Presentation of Cancer Registry Data: A Handbook of Basic Practices" that was published by the North American Association of Central Cancer Registries In reviewing GIS literature for the above mentioned purposes, this author appreciates the fact that the set of definitions and criteria for reviewing evidence as used in the CDC Community Guide is not directly usable for reviewing currently available GIS literature due to the nature of the latter; a modified set of definitions and criteria will first need to be developed. Furthermore, we will organise virtual e-focus groups that bring together UK programme administrators, practitioners and the public to complement the expected gaps and deficiencies in current GIS literature.The desired GIS information outputs and ways of using them within the NHS will be determined for both diabetes and dental care. Datasets (inputs) and the appropriate processing methods required to reach the desired outputs will be identified driven by the best available evidence (from the literature and e-focus groups), and vendor-neutral GIS programme and application models or ontologies will be created accordingly. Any limitations of these applications and any associated possible data/analysis problems or errors will be also highlighted, along with techniques for recognising and reducing their negative impact on result interpretation and any drawn conclusions.We will also launch EB-GIS4HEALTH UK Web server to disseminate our results and reach out to the wide NHS audience and the public.et al, a substantial proportion of respondents from health authorities (90%) and trusts (74%) stated that a dedicated Web site giving advice on GIS matters for NHS organisations would be helpful in promoting and disseminating good practice examples of GIS use in healthcare [In a recent study by Higgs althcare . EB-GIS4d trusts 4% statedalthcare ,10.EB-GIS4HEALTH UK will establish links between real-world NHS practice and the growing body of health and healthcare GIS research produced by the academia and research communities to ensure quality GIS practice and innovative applications are developed and implemented in the UK health service.By putting strong emphasis on real-world, practical GIS scenarios in a UK context, and by being based on the current best evidence, EB-GIS4HEALTH UK will also provide a much needed contribution to future national GIS training and raising awareness campaigns. Such evidence-based training and raising awareness activities have been strongly recommended by different researchers to improve the current poor GIS state of affairs within the NHS .Raising awareness activities are also vital given the need to build business cases for the development of GIS within NHS organisations and to show the capabilities and "business benefits" of GIS to directors  Figure . EB-GIS4EB-GIS4HEALTH UK will provide semantically and procedurally consistent health and healthcare GIS application frameworks. This will help individual NHS organisations adopting such frameworks achieve a common understanding of data and processes (shared semantics), which in turn will enable them to efficiently and effectively share, compare, and integrate their data silos and results at local, regional and national levels for more informed planning, national comparisons, joined-up working, and better outcomes.EB-GIS4HEALTH UK also forms the basis of our vision of a next-generation intelligent tools specifically designed for the UK public health and NHS, and seamlessly weaved into everyday workflows and decision-making processes to enable users to focus and spend the larger part of their work time on what they want to achieve rather than on learning and overcoming the limitations of tools they are supposed to use to achieve their goals. These tools are part of our wider vision of a "national spatial data and information infrastructure and associated multivariate, GIS-enabled health surveillance services processing real-time or near-real-time data streams rather than just retrospective data" mentioned above and in .Such tools must be able to convey reasonable conclusions . The ideal tools also need to be fault-tolerant and capable of analysing and presenting assembled data in ways that facilitate only appropriate interpretations of integrated data. This can be achieved by using some form of user friendly, "intelligent", goal-oriented health GIS wizards based on robust statistical and epidemiological methods, so that only valid results and maps are produced, even when users attempt to select inappropriate settings for a particular analysis. The tools are also best designed and built to work in modular and nested fashions, so that they may be reused, linked and combined in different ways as needed to serve different scenarios and compound situations with little or no modifications (of the tools) .This vision definitely calls for a sound evidence base and comprehensive conceptual blueprints (the proposed EB-GIS4HEALTH UK evidence-based models) to drive the envisaged tools and wizards.An incremental approach has been widely recommended in the literature for programmes with a national vision like EB-GIS4HEALTH UK. Rather than addressing all the spectrum of health and healthcare topics that are amenable to GIS processing in one project, we selected only two health conditions to start with. Diabetes and dental care were chosen because of their importance and the huge burden they place on the NHS, and the facts that they affect various age and socio-economic groups and involve a wide spectrum of preventive and curative interventions, which make it possible to apply the GIS models we are proposing to develop for these two topics to many other health and healthcare topics with little modification.Diabetes has a significant impact on the UK health and social services. Around five per cent of total NHS resources and up to ten per cent of hospital in-patient resources are used for the care of people with diabetes . It has ). Of this latter sum, £35 million will be allocated to enable PCTs to improve access, choice and quality for patients, and £30 million will be directed to information technology to integrate dentistry within the national information technology programme. Regarding dental care, the cost of General Dental Services to the government in the year to March 2002 was £1.12 billion  . Under tProfiling service areas and needs assessment, dental healthcare commissioning, and improving/ensuring equitable access to dental services are all areas where GIS can make a positive difference. GIS have significant potential in examining spatial patterns in dental health, and in analysing patterns of registration and utilisation of dental services for different sectors of the community. GIS can reveal gaps in dental health provision, and thus help target resources and programmes to particular areas of needs. GIS can be also used to analyse the composition and spatial distribution of the dental workforce, and to inform the development and monitor the execution of programmes for attracting dental care professionals to work in under-served areas ,17,18., could also benefit from EB-GIS4HEALTH UK as a foundation resource to provide evidence-based training programmes to NHS staff in "GIS for health and healthcare" and foster the much-needed NHS-academia collaboration. NHSU currently does not have programmes covering this important area. As an aside, one might add that when the Director of the Medical Informatics programme that the US CDC runs for its new staff conducted a poll of 40 students on the area they most wanted more information about, top of their list was GIS ; and (3) the UK citizenry and communities who will be empowered to become more active partners in their healthcare, and will also benefit on the long run from improved health services and outcomes as a result of the introduction of well-founded spatial information management within the NHS through EB-GIS4HEALTH UK and other synergistic/follow-on projects in the future.These include: (1) the NHS and public health services in England (and the UK) – EB-GIS4HEALTH UK aims at adding the missing spatial information dimension to NHS organisations, and informing the development of successful GIS business plans for the health conditions under consideration; (2) the recently established NHSU, the corporate university for the NHS with Special Health Authority status – EB-GIS4HEALTH UK aims at building a foundation evidence base and conceptual models for GIS applications in health and healthcare in the context of UK/NHS settings to (i) improve the currently poor GIS state of affairs within the NHS; (ii) help the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, requirements, and NSFs ; and also (iii) foster the much-needed NHS-academia GIS collaboration.1. Organise e-focus groups of representatives of all stakeholders of UK/NHS health GIS to inform the development of all EB-GIS4HEALTH UK products.2. Review the literature and current UK Public Health/NHS data flows and practices of relevance, and build an evidence base of GIS applications in diabetes and dental care (the two topics chosen for this project) in the context of UK/NHS settings.3. Build modular GIS programme and application models for diabetes and dental care tailored to UK/NHS settings (driven by the evidence identified through objectives 1 and 2).4. Run a Web server to disseminate the project's evidence base and resultant GIS programme and application models for diabetes and dental care to the wide NHS audience and the public.5. Conduct a small-scale formative evaluation of EB-GIS4HEALTH UK server use and potential impact on NHS resource utilisation and improving health outcomes.We will gain insight about current Public Health/NHS (in England) data assets, flows and processes of relevance to EB-GIS4HEALTH UK through our NHS collaborators, and by identifying key contacts and organising virtual e-focus groups (see below). This insight will help us come up with a set of "localised" (UK oriented) questions that we will have to answer through our literature review and further e-focus group rounds., Transactions in GIS , International Journal of Geographical Information Science , CDC Public Health GIS News and Information , ESRI's HealthyGIS and ArcUser Online ( and ), etc. We will also hand search a range of textbooks on GIS applications in health and healthcare.We will formulate a search strategy to locate potential resources for our evidence base. This will also cover the so-called grey literature. Our search strategy will span MEDLINE/PubMed and other journal resources not listed in PubMed, e.g., Cartography and Geographic Information Science ; the US CDC National Public Health Performance Standards Programme (NPHPSP – ); the US Primary Care Service Area Project (PCSA – ) and the related Dartmouth Atlas project ; the "Mapping A Shared Vision of Hope: The American Indian/Alaska Native (AI/AN) GIS Diabetes Atlas", which was developed to assist in the analysis, design, and evaluation of AI/AN diabetes intervention and prevention programmes ; Georgia Medical Care Foundation Diabetes Quality Indicators project ; and CDC's data systems for oral health .We will also review the extensive online documentation of flagship UK and foreign projects and initiatives of relevance to our proposed research. These include, among others, the UK Department of Health NSFs We will review studies that used GIS or spatial methods in diabetes and dental care , practitioners and representatives of the public to (1) inform the project about NHS settings; (2) define and refine the key questions that decision makers would want to be able to answer with GIS for diabetes and dental care, and think explicitly about what data and methods should be used to answer those questions; (3) discuss the reviewed literature and guide us to any relevant literature we may have missed for our evidence base; (4) complement our review of the evidence by the group members' own experiences and consensus of opinions, especially in areas where the literature is lacking; and (5) provide iterative feedback on EB-GIS4HEALTH UK models, as these are developed and refined. The virtual e-focus groups will ensure that representatives of all stakeholders of UK/NHS health GIS are involved in formulating EB-GIS4HEALTH UK products, a very important ingredient of success ,20.The e-focus groups will take place online using threaded discussion technology on EB-GIS4HEALTH UK Web server. Simple audio-conferencing over the Internet, e-mail/e-mail list, and/or telephone may be also used if necessary. The project's team will moderate/facilitate and guide/coordinate the discussions by adopting a kind of Delphi process and/or "cluster analysis" approach .et al's set of categories they have recently used in a small-scale GIS literature review, with the following top classes/sub-classes among others: data collection method(s) ; spatial data analysis method(s) ; study scale ; resolution/geographic unit of analysis ; study purpose ; GIS software used; bias of study design/limitations; etc. . The metadatabase will include fields to record the strength of evidence and bottom-line summaries of the reviewed articles. EB-GIS4HEALTH UK models and their documentation will have cross-links to the underpinning evidence in the online metadatabase.A metadatabase of reviewed resources classified according to topics and applications will be created and populated. This will form EB-GIS4HEALTH UK searchable online evidence base (see below). The design of resource records in this metadatabase will be guided by the spirit of the matrix method for conducting and organising literature reviews . A classSuitable input from the virtual e-focus groups will be also included in the evidence base. Resources and findings of insufficient evidence quality will still be documented in the evidence base , but will not be used in developing EB-GIS4HEALTH UK models.. EB-GIS4HEALTH UK will take the concepts and methodologies of these projects one step further by developing evidence-based, vendor-neutral modular GIS programme and application models rather than mere data models or vendor-specific solutions.We will develop a general conceptual framework for EB-GIS4HEALTH UK models as outlined below, guided by Briggs' indicators methodology for environmental health hazard mapping ,23, the EB-GIS4HEALTH UK will feature two interrelated types of models: application models and programme models Figure .An application model comprises (1) data and/or input from other models to be processed; (2) processing methods and tools ; (3) desired information outputs and output visualisation methods; as well as (4) all valid interpretations/inferences that can be made from the model's output and ways of using them within the NHS. The best current evidence  will be used to formulate all EB-GIS4HEALTH UK models, and will form an integral part of their documentation.For any single broad health/healthcare topic covered in EB-GIS4HEALTH UK , modelled applications can include all or some of the following (among other possibilities): (1) monitoring and measuring NHS performance ; (2) surveillance/monitoring/early detection of health and disease patterns and trends in populations; (3) profiling target populations and health service catchment areas; (4) selection of appropriate target groups for public health interventions, optimising these interventions to match target group profiles, and measuring intervention success; and (5) needs assessment and optimising healthcare system planning and responses, including new healthcare facility siting and improving hospital bed availability.EB-GIS4HEALTH UK models will take into account the influence of non-medical determinants  on population health status, qualitatively relate these determinants to health outcomes, and consider their implications on healthcare service planning and delivery ,20. For A programme model is also an ontology that addresses a single broad health/healthcare topic, and comprises at least two application models linked together and interacting with each other in predetermined and purposeful ways towards a broader goal than that covered by a single application model. Application models may have uses in more than one programme ., a free ontology modelling tool from Stanford Medical Informatics with which this author has long experience [, including a Java-based Web application for sharing Protégé ontologies over the Web . Protégé is not alien to geographic information science; last year (2003) for example, a geographic information metadata (ISO 19115) ontology was developed at Drexel University, US, using Protégé  will be built using Protégé perience ,26. ProtA model's ontology/documentation will specify the model's version/date and release history, the programme(s) (for application models)/topic(s)/sub-topic(s) to which the model relates, the model's rationale and role, any alternative or related models/model sets (programmes), links to the underpinning evidence (from the literature/the project's evidence base), and a listing of all agencies/NHS bodies involved in the model's process(es) (and their exact roles).Data involved in the modelled applications will be identified and assessed regarding availability, quality, characteristics, and constraints in terms of the model in question. Whenever this is possible, EB-GIS4HEALTH UK models will strive to refer to and model readily available data already collected for other purposes, since implementing new data collection processes can have prohibitive costs, and healthcare workers have repeatedly demonstrated poor compliance with additional data collection and administrative tasks .For data that are not available but are "required" we will investigate and suggest the most efficient and effective way(s) for collecting and maintaining such datasets. During the course of the project, we will be identifying and communicating with the custodians of various datasets in the UK health sector and related sectors, as appropriate, to check the availability of required datasets to the UK NHS, note any constraints related to their release and use , and flag any need to review policies related to the release of such data. The results of all these data-related investigations will be included in the documentation of EB-GIS4HEALTH UK models.only valid analyses and visualisations of data.The spatial and related methods involved in processing the model's input to produce the desired outputs will be described in detail in the model. A reusable toolbox of "generic" methods will be identified to make this process easier for all the models we develop, e.g., "pattern spotters and testers" and "relationship seekers and provers" . All metThe model's information output(s), any units of measurements used in presenting outputs, and any specified output visualisation methods will be also described in detail.The area across which the model can be used  will be determined. Finally, the ways in which the model's output may be interpreted (in relation to the topic(s)/sub-topic(s) it covers) and linked to other EB-GIS4HEALTH UK models/programmes will be described. This includes determining what inferences can be made from apparent trends or patterns in the model's output and how such inferences can be used within the NHS, and any constraints on the interpretation of this output, due for example to data limitations or complexities in the relationships implied by the model.Data and methodological problems and limitations are not uncommon and a wide range of them has been well documented in the literature and must be anticipated and cared for . TechniqFor a programme model, the component application models will be listed and their relationships and interactions within the programme also documented Figure .EB-GIS4HEALTH UK knowledge-based models (ontologies) in Protégé will not only be human-readable and visualisable in a variety of ways (to inform NHS GIS service developers and implementers), but it will be also possible to use the models to generate the necessary template databases for running the modelled applications. Furthermore, the resulting ontologies could form the basis for the development of goal-oriented, user friendly and fault tolerant health GIS wizards and solutions in the future using software agents technology ,29, sincThe goal for EB-GIS4HEALTH UK application and programme models is to provide practical, vendor-neutral modular workflow models and ready-to-use, evidence-based frameworks for implementing GIS to address various health and healthcare topics within the NHS. NHS organisations adopting such frameworks will be able achieve a common understanding of data and processes (shared semantics), which in turn will enable them to efficiently and effectively share, compare, and integrate their data silos and results at all levels.We will launch EB-GIS4HEALTH UK public Web server (with it's own domain name) to disseminate our evidence base, GIS models and associated documentation/metadata, as well as other project documentation and recommendations. The server will also host the virtual e-focus groups. The server will run one of the many free content management and discussion board platforms available today to manage and make searchable all of the project's online components.We will advertise EB-GIS4HEALTH UK online service among target groups (NHS and academia) and the general public, and conduct a small-scale formative evaluation of its use and user-perceived utility of the project using an online user questionnaire, in addition to analysis of EB-GIS4HEALTH UK server transaction logs. This formative evaluation study will be carried during the third year of the project  with the goal to inform and guide any further development of EB-GIS4HEALTH UK or similar projects. The study will evaluate the potential impacts of EB-GIS4HEALTH UK on NHS resource utilisation and improving health outcomes, among other things, by surveying participants opinions. Evaluation results will be also available from EB-GIS4HEALTH UK public Web server.Research into ontology-driven geographic information systems (ODGIS) is very rapidly growing due to the many potential and unique advantages that ODGIS promise -36. We a and ). We conduct many complex analyses in health and healthcare to answer much more complicated questions (compared to other sectors), and there are many problematic issues surrounding our analyses like, for example, data confidentiality and Jacquez's famous "gee whiz" effect [This author also advises the use of Tomlinson's methodology to plan for the successful deployment of GIS within the NHS, but only at a later stage ,37,38. T.Besides being one of the main project beneficiaries, the UK citizenry (and indeed anyone connected to the World Wide Web) will have full access to EB-GIS4HEALTH UK public reports and fully documented products on the project's public Web server. Representatives of the public will be also involved in the project's e-focus groups (see above). Lay audience short summaries of the project's progress and expected benefits in a jargon-free language will be also published on EB-GIS4HEALTH UK public Web server (see Table  and ), but this has never resulted in any coherent national strategic adoption of GIS within the NHS. This is due to the fact that a successful national strategic implementation of GIS in the health sector is dependent upon many other very important ingredients besides the acquisition of hardware and software systems, and core digital geo-datasets [Most major GIS vendors and solution providers have been targeting the UK health sector for many years now . All national health GIS stakeholders must be involved in this process, including representatives of the general public (the ultimate beneficiaries). EB-GIS4HEALTH UK is just the start towards this goal. An incremental approach has been widely recommended in the literature for programmes with a national vision like EB-GIS4HEALTH UK. The experience gained at the end of this project will hopefully be transferable to further develop the covered topics , and for addressing other national health and healthcare topics not initially covered in this project, based on the same principles.Healthcare systems work differently around the world and this has knock-on effect on health GIS. When it comes to UK health GIS, we still need to go back to the design board, and to link/integrate what research has to offer into workflow models and "recipes" that are both usable and useful in everyday practice per se our ultimate goals. The actual purpose of this design/modelling exercise and its ultimate goals are optimising NHS resource utilisation and improving health outcomes by paving the way to the incorporation of the missing spatial information dimension in NHS organisations.It must be emphasised that the evidence base and conceptual GIS workflow models we are proposing to build in this project are not This paper presented an overview of a project we are proposing to carry out here in England, where the author is based. However, the same concepts, principles and approaches described in this paper can be also applied in other countries, especially where financial constraints are not a major issue. The task might not be as easy as replacing "UK" in this proposal with another country name, but the author believes that the paper has provided enough details and set an example to enable "quick starting" similar health GIS foundation projects in other countries.Our proposal also fits very well into the spirit of Mark Musen's philosophical paper on medical informatics as an academic discipline, in which he argued that "informatics involves the construction of ontologies that define the concepts relevant to different aspects of human experience and the elucidation of problem-solving methods that can solve specific computational tasks" .A detailed breakdown of human and other resources required to complete this project with a timeline of the main EB-GIS4HEALTH UK execution tasks distributed over a suggested three-year duration of the project are not provided in this manuscript, but are directly available from the author.).A slightly abridged version of this proposal has been submitted to, and very favourably reviewed by the UK Medical Research Council (MRC) during 2004, and received a final ALPHA-C banding , health, geography, public health, policy and practice, epidemiology, etc. It is therefore important to bear in mind that if the proposal is judged by criteria generally applicable to only one or two of these specialist areas, it might be found deficient and it would be quite inappropriate to do this".


Although feeding deficits have been reported in snakes and lizards following vomeronasal system disruption, no deficit has been previously reported in a mammal. We tested gray short-tailed opossums with items from four different food categories prior to occluding access to the vomeronasal organ. Preoperatively, opossums preferred meat to fruit or vegetables. Following occlusion of the nasopalatine canal, but not after control treatment, opossums failed to demonstrate food preferences. The vomeronasal system (VNS) is a nasal chemosensory system usually considered primarily a pheromone-detecting system -4 for re for re4 In snakes and lizards, in addition to its role in pheromone detection, the VNS is important for feeding behavior -7. To daMonodelphis domestica, are members of the Didelphidae or American opossums. The didelphids are the most ancient marsupial family and thought to be the origin of all other New World and Australian marsupials [M. domestica was recently introduced into laboratories [Gray short-tailed opossums, rsupials ,9. M. doratories  and has,ratories -13, theyad libitum.Six male and seven female gray short-tailed opossums, 12–16 months old, were used as test subjects in this study. The opossums were progeny of animals originally purchased from the Southwest Foundation for Biomedical Research  and were offspring of different parents. The opossums were housed individually in plastic cages (42 × 21 × 20 cm) with wood shavings as bedding, and provided with cylindrical plastic containers for nesting. The animals had dry fox food  and water available To determine which foods to use in the formal experiment, a preliminary test was conducted in which 24 different foods, representing meats, fruits and vegetables were presented to the opossums. Each opossum was presented with one food per day, selected at random, and its consumption of the food noted. The animals were allowed three minutes to investigate and eat the food, which was placed on an 8 × 20 cm cardboard tray at the front of the home cage. Each animal received two trials with each food. Of the 24 foods tested 16 were selected for the formal experiment based on the animals' observed preferences.3) of food from each food category, was placed in a dish.During testing the opossums were simultaneously presented with four foods one from each of the following food groups: fruits , meats , processed vegetables  and unprocessed vegetables . The animals were tested daily with one of 16 food combinations (4 foods × 4 categories). Four sterile Petri dishes, each 3.5 × 1.0 cm, were placed equidistant from each other and glued to an 8 × 20 cm cardboard tray. One piece  placed at a distance of 3 m from the test cage. Trays, dishes and unconsumed food were discarded after each trial. After testing, food troughs containing fox food were replaced in the animals' home cages. Fresh disposable gloves were used when handling dishes, trays and foods to prevent transfer of food odors.Videotapes of food trials were analyzed using a videocassette recorder (JVC SR-VS30U), BTV Pro 5.4.1 and "Videoanalyzer" software .For control surgery and occlusion of the nasopalatine canal, opossums were lightly anesthetized with Ketaset  and atropine sulfate  Access to the vomeronasal organ (VNO) was blocked with gel foam and Crazy Glue™  applied to the roof of the mouth, covering the opening to the nasopalatine canal. Cresyl violet crystals were added to the Crazy Glue to facilitate visualization of the block. Opossums were visually checked daily to insure that the VNO block remained in place. Control surgery consisted of placing Crazy Glue on the roof of the mouth to either side of the nasopalatine canal. Opossums were allowed two days to recover before postoperative testing.The VNO block remained in place for only one test day in all thirteen opossums. Therefore, pre-post operative and control comparisons were only made for trials identical in composition (foods tested) and order (position on the tray) to the first postoperative test day. Control postoperative trials were run as described above. All 13 animals were tested under both control and experimental  conditions, with the order of surgical treatment randomized.The first food selected and consumed on each trial was identified as the animal's food choice. Statistical analyses utilized a chi-square test of the frequencies of choices of each food type for pre-operative, control and experimental trials. Figure Preoperatively, opossums preferred meats to fruits and fruits to processed and unprocessed vegetables Figure . The mos2 = 10.08, df = 3, p < .02), postoperatively, no significant difference in food category preference was observed . During control trials the opossums continued to demonstrate significant food preferences . Meat was again, the most preferred food category (Figure Whereas the preoperative comparison trials on the thirteen opossums revealed significant preferences among the food categories (χOne deficiency of this study is that, because the opossums removed the glue blocks after the first day, we were unable to verify that the nasopalatine occlusion had, indeed, prevented access to the vomeronasal organ. However, we had previously  demonstrThis study suggests that without a functional VNS, the food preferences of gray short-tailed opossums are significantly impaired. Previous studies on the VNS of mammals have not addressed the issue of changes in feeding behavior. It remains to be seen whether other mammals, newly introduced into the laboratory, or bred over many generations in the laboratory, also demonstrate a food preference deficit when deprived of a functional VNS. Studies comparing wild populations with laboratory-reared populations might contribute information resolving this issue.It is not surprising that food preferences might be influenced by vomeronasal stimulation since the vomeronasal organ of many vertebrates, including opossums, is directly accessible from the oral cavity  via the MH conceived of and designed the experiment and wrote the manuscriptYD conducted the experiment, extracted the data from the videotapes and analyzed the data statisticallyIZ supervised YD in the conduct of the experiment and instructed YD in the experimental procedures and data analysis


The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide–MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former αβTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other αβTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins. A baculovirus expression library -- encoding peptides in the context of MHC molecules -- has been developed to identify and characterize unique peptide-MHC complexes that bind specific T cell receptors The identification of peptide epitopes associated with particular αβ T cell receptors (αβTCRs) is often still a bottleneck in studying T cells and their antigenic targets in, for example, autoimmunity, hypersensitivity, and cancer. A direct genetic or biochemical attack on this problem can be successful, especially with class I major histocompatibility complex (MHCI)-presented peptides. For example, tumor  and tranThe reward for the labor involved in identifying peptide epitopes directly can often be the identification of the protein source of the peptide, especially as the sequencing of the genomes of many organisms approaches completion. However, in many situations, rather than identifying this precise peptide epitope, it is sufficient to identify a peptide “mimotope.” Mimotopes can be defined as peptides that are different in sequence from the actual peptide recognized in vivo, but that are nevertheless capable of binding to the appropriate MHC molecule to form a ligand that can be recognized by the αβTCR in question. These peptides can be very useful for studying the T cell in vitro, for altering the immunological state of the T cell in vivo , for vacMimotopes can sometimes be identified in randomized peptide libraries that can be screened for presentation by a particular MHC molecule to the relevant T cell . Thus fab. The peptides in the library varied in five peptide amino acids known to be surface exposed and predicted to lie within the footprint of αβTCR interaction.In other applications, another powerful library method has been sequential enrichment/expansion of a displayed library of protein–peptide variants by direct ligand–receptor binding, e.g., using bacterial phage or yeast  and the lack of synchrony in viral infection and expression. To focus on cells bearing a particular level of IAb, we stained the cells simultaneously with the fluorescent αβTCR reagents and with an anti-IAb monoclonal antibody (mAb) that did not interfere with αβTCR binding. In this case, there was a direct correlation between the amount of surface IAb-p3K expressed by an individual insect cell and the amount of αβTCR bound . The infected cells were stained with fluorescent YAe-62 αβTCR and analyzed by flow cytometry. Although a distinct population of brightly fluorescent cells was not seen, the 1% of the cells with the brightest fluorescence were sorted, as were an equal number of cells that were very dully fluorescent   in-frame with the IAb signal peptide and with a 3′ termination codon  compensated for the absence of the asparagine sidechain.When bound to IA APC see B. Perhapb–peptide combination that bound the YAe-62 αβTCR. It is likely that many more would be identified if more clones were analyzed. Five sequences were found multiple times. Not unexpectedly, these were among those that bound the YAe-62 αβTCR most strongly. There was a 100-fold range in the intensity of αβTCR binding to the different IAb–peptide combinations, ranging from about 4-fold to 400-fold binding above that seen with a negative control peptide. One obvious property of these peptides stands out. There appeared to be a very strong selection for a basic amino acid at position 5. In 16 of 20 of the peptides, a lysine, arginine, or histidine was found at position 5, matching the lysine found in p3K. As a control, we sequenced random clones picked either from the original E. coli construction of the library (17 clones) or from the baculovirus library that expressed IAb–peptide well, but did not bind either αβTCR (11 clones). The frequencies of basic amino acids at p5 in these sequences were only 12% and 9%, respectively (data not shown).Consistent with the hypothesis that the αβTCR of YAe-62 would be more peptide promiscuous than that of B3K-06, we found 20 different peptide sequences among the analyzed clones that produced an IAb-binding peptides that were not bound by the YAe-62 αβTCR, but only two of 17 (11.8%) of the random E. coli plasmids. The amino acid in this position is only partially exposed on the surface and can contribute significantly to peptide–MHC interaction  of the YAe-62 αβTCR-selected peptides and three of 11 (27.2%) of the IAeraction . After action  at this position. Perhaps this indicates that, in general, larger sidechains can be inhibitory at this position, but again more data would be required to test this idea.b–peptide combinations that bound the YAe-62 αβTCR most strongly were also the ones that were able to induce IL-2 production from YAe-62. Among these, a number with the very highest apparent affinities stimulated YAe-62 better than did p3K. However, there was not a direct correlation between apparent affinity and the level of IL-2 production; i.e., several peptides that yielded complexes with IAb with about the same apparent affinity for the αβTCR nevertheless stimulated very different levels of IL-2 production from YAe-62. This may be related to the phenomenon of altered peptide ligands  for the αβTCR was already known. However, this method should be useful as well in identifying mimotopes for αβTCRs whose peptide epitope is not known, provided that suitable peptide anchor residues for MHC binding are known. One limitation of this display method as presented here is the size of the peptide library. The bottlenecks caused by the preparation of the library in an ligation  or in viligation . In prelligating  a randomb as the displayed MHCII molecule carrying the peptide library. However, using the same strategy, we have successfully displayed numerous other MHCII molecules, such as murine IEk and human DR4, DR52c, and DP2 (data not shown). While the leucine zippers that we included in this construct are not strictly required for expression of IAb, they have helped considerably in expression of some of these other MHCII molecules. Moreover, we . Given that baculovirus has been such a successful expression system for many different types of complex eukaryotic proteins that express or assemble poorly in E. coli, this method may have broad applications to other receptor–ligand systems.We have developed this method using IAover, we  and otheover, we  have shob–peptide combinations. Thus, depending on whether one was interested in high- or low-affinity ligands for the αβTCR, one could enrich for peptides with these properties directly during the screening of the library. Such an approach has been used with antibody , p3K (FEAQKAKANKAVD), and various alanine-substituted variants of p3K were synthesized in the Molecular Resource Center of the National Jewish Medical and Research Center , as were all oligonucleotides used in PCR and DNA sequencing. Automated DNA sequencing was also performed in this facility.b-p3K-reactive T cell hybridoma B3K-06 was produced from C57BL/6 mice as previously described . The IAescribed . The IAbLB-15.13  was usedb β gene (ΔIAβ) and the invariant chain (ΔIi) and that carried a transgene for the IAb β gene that was modified to insert sequence encoding pEα and a flexible linker between the signal peptide and the N-terminus of the β chain. These mice were immunized intravenously with 3 × 106 dendritic cells from ΔIAβ/ΔIi C57BL/6 mice. These cells had been retrovirally transduced  addition region were synthesized by PCR using baculovirus DNA as template. The fragments were used to construct an insect cell expression vector (pTIE1) on a pTZ18R  backbone with the hr5 enhancer at the 5′-end, followed by the IE1 promoter, a large multiple cloning site , and the IE1 poly(A) addition region. The complete sequence of the pTIE1 vector has been deposited in GenBank (see Supporting Information). DNA fragments encoding mouse ICAM and B7.1 were cloned between the XhoI and NotI sites of the multiple cloning site. Sf9 cells were transfected with a combination of the plasmids by the standard calcium phosphate method and cells expressing both molecules on their surfaces were cloned without selection at limiting dilution to establish the line Sf9-ICAM/B7.1.5) were added to microtiter wells containing either (1) saturating immobilized peptide–MHC, (2) 10 μg/ml peptide plus 105 LB-15.13 cells, (3) 5 × 104 Sf9-ICAM/B7.1 insect cells infected 3 d previously with baculovirus encoding a displayed peptide–MHC, (4) 106 spleen cells from IAb-pEα single peptide mice, or (5) 106 spleen cells from various knockout or MHC congenic mice. After overnight incubation the culture supernatants were assayed for IL-2 as previously described  at a molar ratio of 2.5:1 (biotin:antibody) for 4 h at room temperature. The reaction was quenched with 0.1 M lysine and the product dialyzed extensively against PBS. The resulting derivative contained about one biotin per molecule of mAb. The biotinylated mAb was complexed in excess with AlexaFlour647–streptavidin . The complex was separated from the free biotin–antibody using Superdex-200 size exclusion chromatography (Pharmacia). In preliminary experiments, the amount of soluble αβTCR required to saturate an aliquot of a large single batch of this reagent was determined. To prepare the multivalent αβTCR, the appropriate amount of soluble αβTCR was mixed with an aliquot of the fluorescent anti-Cα reagent overnight. For staining for flow cytometry, this mix was used without further purification. Each 100 μl sample contained approximately 2 μg of the fluorescent reagent plus 105 Sf9 insect cells. This mixture was incubated at 27°C for 1–2 h. The cells were then washed for analysis. The advantages of this method for preparing fluorescent multimers over using direct enzymatic biotinylation . For sorting, a MoFlo instrument was used .b on the surface of baculovirus-infected insect cells, modifications were made as described in b-p3K (b-binding peptides. The constructions were incorporated into baculovirus by homologous recombination using the BaculoGold system (PharMingen).For displaying IAin b-p3K . Other vE. coli transfer plasmid to replace the portion encoding p3K with sequence encoding enhanced GFP, flanked by sites for the restriction enzymes SbfI and CeuI. A PCR fragment was produced as described in b-binding peptide randomized at positions p2, p3, p5, p7, and p8, but identical to p3K at other positions. This sequence was flanked by sites for the restriction enzymes PstI and BstXI, such that the cohesive ends generated by these enzymes were compatible with those generated by SbfI and CeuI in the GFP-containing plasmid. Cloning the restricted fragment into this site regenerated a covalent peptide in-frame with the signal peptide and flexible linker of the IAb β chain . An estimated 3 × 104 to 10 × 104 independent transformants were obtained that were used to make a mixed plasmid preparation. This mixture was incorporated into baculovirus by homologous recombination as above. In order to assure a high efficiency of conversion of plasmids to virus, 1.5 × 107 Sf9 cells were cotransfected with 6 μg of the plasmid mixture and 1.5 μg of BaculoGold DNA.As described in hain see E. After http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the sequences described in this paper are B7.1 (AJ278965), baculovirus DNA (L22858), ICAM (X52264), and pTIE1 vector (AY522575).The GenBank (


Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency.in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS.To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications. Cytokines are a family of low-molecular weight proteins secreted by various cell types, with pleiotropic functions and constitute a tightly regulated network that plays a central role in the immune system. Cytokines, classified into different groups such as interleukins (IL), interferons (IFN), colony-stimulating factors (CSF), tumour necrosis factors (TNF), tumour growth factors (TGF) and chemokines are implicated in the differentiation, proliferation, migration and effector functions of immune cells. Interacting one with the others, they have polarizing effects on the target cells and are pivotal in tuning immune responses . TherefoCytokines are often produced locally so that the concentration of circulating cytokines in the plasma is usually low. Their half-life and turnover may vary complicating the delineation of informative cytokine profiles. Although transcription of messenger RNA is not strictly correlated to protein secretion and activity, detection of cytokine RNA by real time PCR is now considered a reference technique for analysis of small-size samples with high sensitivity . It can The already available techniques, which offer a so-called "absolute quantification" of the target cytokine mRNA, achieve quantification by reference to an external standard curve based on serial dilutions of a known amount of the corresponding cDNA . MoreoveIn the present study, we first show that the efficiency of the RT step depends on the target mRNA and on the experiments and that these variations have critical impact on the reliability of mRNA quantification. To overcome this, we describe here CyProQuant-PCR, a new technique for absolute measurement of cytokine mRNA based on an external RNA standard curve. Primer pairs have been designed for allowing amplification of a set of cytokine mRNA using the same conditions both in terms of thermocycling parameters and master mix components, a prerequisite for multiple cytokine mRNA measurements with high throughput.in vitro stimulated PBMC and its intra and inter-experimental variability were assessed. Finally, CyProQuant-PCR was used to quantify TGF-β1 transcripts in small blood samples from children with acute Plasmodium falciparum malaria.In the present paper, we describe i) the construction of the synthetic RNA standard, ii) the primer pairs specific for the following human cytokines IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, and for the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), the β2-microglobulin (β2-MG) and the ubiquitin-C (UBC) to be used as internal standards and iii) the conditions for efficient real time amplification of multiple cytokine specific mRNA. The technique was validated using Primer pairs were designed from published genomic sequences using Primer Express software (Applied Biosystems), except for the UBC and the YWHAZ genes for which the primers had already been described . When poT) and the maximum signal-to-noise fluorescence ratio (ΔRn) while minimising non-specific amplification (data not shown). Among the final concentrations of 50, 300 and 900 nM tested for each primer, the optimal final concentration was set at 900 nM for every primer. The primer pairs are shown in Table Primer concentrations were optimised to determine the minimum primer concentrations giving the lowest threshold cycle . The molecular mass of each RNA standard was calculated on the basis of its sequence and solutions ranging from 101 to 1012 copies of standard RNA were made.To generate the standard RNA corresponding to each target sequence, gene specific primers were fused in their 5' end to the RNA polymerase T7 promoter sequence. The PCR performed with these modified primers pairs lead to a larger PCR product with the T7 promoter sequences upstream and downstream from the specific amplicon. The T) against the logarithmic value of the starting RNA copy number for each dilution. Figure These serial diluted solutions were reverse transcribed and the cDNA amplified in duplicate to generate a standard curve by plotting the threshold cycle . The slope of the standard curves generated by these three ranges as well as the corresponding efficiency are shown in Table CyProQuant-PCR was compared to the classical approach based on a DNA standard curve for the quantification of TNF-α, IL-1β and IFN-γ transcripts. DNA templates for RNA transcription of TNF-α, IL-1β and IFN-γ were used to generate a range of concentration from 10 to 10In order to show that these intergenic differences were not due to interassay variation, we compared amplification efficiencies of TNF-α, β2-MG, MIF and UBC from i) a single cellular RNA sample, ii) a pooled RNA sample containing standard RNA for each of the targets and iii) a pooled DNA sample corresponding to the cDNA obtained from the pooled RNA sample containing standard RNA for each of the targets. Results obtained are summarised in Table Altogether, these data demonstrate that RT efficiency displays intergenic variations that are not due to interassay differences.RT efficiency has critical impact on transcript quantification as shown in Table Taken together, these results support the use of RNA as external standard for reliable and reproducible quantification of transcripts.Since RT-PCR efficiency varies, absolute mRNA quantification can only be reliably obtained if the external RNA standard and the cellular RNA are retro-transcribed and amplified with the same efficiency. This was secured by comparing the standard curves obtained after amplification of a range of 10X serial dilution of cellular RNA and β2-MG external RNA standard. The slopes obtained were of -3,307 and -3,305 for the cellular RNA and for the external RNA standard, respectively. This corresponds to efficiencies of 100,6% and 100,7% respectively (data not shown).in vitro conditions. Moreover, based on the approach recently described by Pachot et al.  were N is the number of bases composing the standard DNA. Stock solution of 10in vitro transcribed . The standard RNA generated was purified  and loaded on a 4% agarose gel for electrophoresis. The concentration of the standard RNA was determined spectrophotometrically at 260 nm. The molecular weight of the transcript was calculated by N*500-[(N-1)*175] were N is the number of bases composing the standard RNA. Stock solution of 1012 copies of standard RNA /3,85 μL were made in RNA storage solution , split in single-use aliquots and stored at -80°C in safe-lock tubes. External RNA standard range was made extemporaneously by 1:10 serial dilutions in water.Gene specific primers were fused on their 5' end to the sequence of the RNA polymerase T7 promoter to generate modified primers. These primers were used to amplify a gene specific PCR product flanked by transcription initiation sites. Five hundred nanograms of this construct were For CyProQuant-PCR assays, 100 ng of total cellular RNA from PBMC and serial dilution of external RNA standard were reverse transcribed simultaneously in a parallel procedure using Reverse Transcription TaqMan reagents  on a MasterCycler Gradient . The final volumes were set at 100 μL for the cellular RNA samples and 50 μL for the external RNA standard range. The thermocycling parameters were as follows: 25°C, 10 min.; 48°C, 60 min. and 95°C, 5 min. cDNA were immediately used for PCR amplification.T) against the logarithmic values of the starting RNA standard copy number.Reverse-transcribed standard RNA and cellular RNA were amplified simultaneously on the same PCR plate on an ABI Prism 7700 . An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix . Thermocycling conditions were 50°C for 2 min., 95°C for 10 min. and 40 cycles of . The sample target RNA copy numbers were calculated using SDS 1.9 Software . The baseline fluorescence was set manually to correct for differences in initial cDNA concentration and the threshold was positioned at a fluorescence level that was 10 times higher than the background signal. Target mRNA copy numbers in cellular samples were calculated based on a standard curve generated by SDS 1.9 Software  by plotting cycles at threshold . Results are expressed as pg/mL for one million living cells.Tests for significance were done using Stata software  by Kruskal-Wallis rank test.Results disclosed in this manuscript have been protected in French patent application FR0408645.in vitro stimulation experiments. DJ gave technical assistance. SL helped in RNA extraction of malaria samples. JCB introduced PB to molecular biology techniques and provided critical advices. BDA designed and conducted the study that yielded the malaria samples. OMP revised the manuscript and supported the work. CB conceived the strategy and coordinated the study. PB and CB drafted the manuscript. All authors read and approved the final manuscript.PB developed the entire technique. IV did the


IIβ and β3 transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the α subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins.Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the α and β subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin α Integrin receptors mediate cell-matrix interactions by altering the conformation of their intra- and extra- cellular domains, a process mediated by lateral separation of the transmembrane helices Integrins are major metazoan cell adhesion receptors that have the distinctive property of transducing signals across the plasma membrane in both directions. Intracellular binding of cytoskeletal components to integrin cytoplasmic domains activates the ligand binding competency of the extracellular domain . Furthermore, ligand binding to integrin extracellular domains is coupled to alterations in cytoplasmic domains that are linked to downstream signaling . The three-dimensional architecture of integrin extracellular domains as well as their rearrangement in activation have been revealed by crystal, nuclear magnetic resonance (NMR), and electron microscopic methods , 2003. N3 subunit TM-cytoplasmic domain fragment in dodecylphosphocholine micelles predict that the TM segment comprising residues Ile693 to Ile720 is largely α-helical   Figure . HoweverIIb and Pro691 to Gly702 of β3  specific to the αIIbβ3 complex and subjected to sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE). Because of the extensive chase, only mature, cell-surface αIIbβ3 with complex N-linked glycan was isolated, which can readily be distinguished from the lower Mr αIIb and β3 precursors with high mannose N-linked glycans (data not shown). When the two cysteines on the α and β subunits are spatially close and are oxidized during biosynthesis, they form a disulfide bridge that can be detected by the appearance of a covalently attached αβ heterodimer band in nonreducing SDS-PAGE with a concomitant decrease in the intensity of α and β monomer bands 3 (Cu-phenanthroline)  and the 12 β3 cysteine mutants (691C to 702C), i.e., a total of 120 different cysteine pairs. Transient transfection in 293T cells and CHO cells gave similar results. The disulfide bonding efficiency of all of these pairs is graphically summarized in IIb and Leu694 and Val695 in β3 (dashed line in 2), except that the efficiency of disulfide induction was slightly lower (data not shown). The third group, corresponding to the remaining 90 pairs, showed little or no intersubunit disulfide bond formation even after treatment with oxidant . Consistent with this, studies on dimerization of the glycophorin A TM domains have shown that cysteine substitutions are on average less disrupting than substitutions with any other hydrophobic residue (On both CHO-K1  and 293T residue . We concIIb band but not of the β3 band (IIb (data not shown). Furthermore, the same high-molecular-weight band was observed when α-W967C was cotransfected with any of the β3 cysteine mutants 2 tetramer. Notably, among the ten α and 12 β cysteine mutants used in this study, only α-W967C formed a homodimeric disulfide bond. This is consistent with the model of α–β TM domain association deduced here  motif in the αIIb subunit  formed a heterodimer on the cell membrane and appeared as an approximately 130-kDa band, slightly smaller than wild-type αIIb in nonreducing SDS-PAGE  No periodicity in disulfide formation was observed  Just as in αIIb*/β3, in αIIb"/β3, the helical periodicity of disulfide bonding was lost, as evidenced by the results with the β3 scanning mutants β-I693 to β-V700  As at 0 °C A and 5B.It is significant that a number of α*/β and α"/β cysteine-scanning mutants could form disulfide-bonded heterodimers during biosynthesis, but in contrast to α/β, none showed increased disulfide formation after oxidation at 0 °C. During biosynthesis at 37 °C, the membrane is fluid. Disulfide bond formation is catalyzed in the endoplasmic reticulum by disulfide isomerases, and because the redox balance is oxidizing in the endoplasmic reticulum, disulfide bond formation can covalently trap protein complexes that form only transiently. Therefore, a complex that would not be stable energetically by noncovalent interactions alone may nonetheless be stabilized by a covalent disulfide bond. This may particularly be the case for interactions between integrin TM domains, because the noncovalent association between the α and β subunits in the headpiece in the extracellular domain increases the probability of collision between the α and β subunit TM domains. If disulfide formation is the result of a stable noncovalent interaction between TM domains, it should occur at 0 °C when membranes are in a gel phase and proteins do not diffuse, as well as at 37 °C when membranes are liquid-crystalline and proteins diffuse. On the other hand, if disulfide formation is the result of transient interactions that are energetically unfavored, it should occur at 37 °C but not at 0 °C.To confirm the hypothesis that in α*/β and α"/β transient collision between TM helices can result in disulfide formation, Cu-phenanthroline oxidation was performed both at 0 °C and 37 °C. As described above, the α-G972C/β-L697C pair in the context of the wild-type receptor shows greatly increased disulfide bond formation upon oxidation by Cu-phenanthroline at 0 °C A, lane 5The above results were confirmed with the full-length α"/β receptor containing the Phe-Phe/Ala-Ala substitution see B and 5C.IIb which mediated α–α homodimerization in the wild-type receptor, underwent α–α homodimerization in the context of the activated α*/β receptor . The same reversal of basal ligand binding was also found for all constitutively disulfide-bonded α"/β pairs examined, including α"-I966C paired with either β-I693C, β-L694C, β-V695C, or β-V696C  and trimers (β3) in detergent micelles , respectively, as described by IIb cytoplasmic domain mutant receptors were made by introducing a stop codon at residue Gly991 to obtain αIIb1–990 (denoted α*), or by mutating G991/FFKR to GAAKR (denoted α"). Single amino acid substitutions to cysteine were made in αIIb, αIIb*, αIIb", and β3 in the positions indicated in the text. All mutants were made using site-directed mutagenesis with the QuikChange kit , and DNA sequences were confirmed before transfection of 293T cells using calcium phosphate precipitates, or CHO-K1 cells using Fugene transfection kit .Plasmids coding for full-length human α35S]cysteine/methionine for 1.5 h before adding chase medium containing 500 μg/ml of cysteine and 100 μg/ml of methionine, and cells were cultured 17 h overnight (2+/1 mM Mg2+ (106 cells in 100 μl). After chilling on ice for 5 min, 20 μM CuSO4/100 μM o-phenanthroline was added by 10-fold dilution from stock solutions, and cells were incubated on ice for another 10 min. Oxidation at 37 °C was similar, except cells were suspended at room temperature and after Cu-phenanthroline addition were incubated at 37 °C for 10 min. Oxidation was stopped by adding an equal volume of TBS containing Ca2+/Mg2+ and 5 mM N-ethyl maleimide. Cells were centrifuged and resuspended in 100 μl of TBS containing 1 mM Mg2+, 1 mM Ca2+, and 5 mM N-ethylmaleimide, and lysed by addition of an equal volume of 2% Triton X-100 and 0.1% NP-40 in the same buffer for 10 min on ice. Cell lysate was immunoprecipitated with 10E5 (anti-αIIbβ3-complex-specific mAb) and protein G Sepharose at 4 °C for 1.5 h. After three washes with lysis buffer, precipitated integrin was dissolved into 0.5% SDS sample buffer and subjected to nonreducing 7.5% SDS-PAGE and fluorography , 150 mM NaCl, 5.5 mM glucose, and 1% bovine serum albumin, and incubated with 1 mM Ca2+/Mg2+ or a combination of 1 mM Mn2+ and 10 μg/ml of activating mAb PT25–2. Then cells were incubated with FITC-conjugated fibrinogen with a final concentration of 60 μg/ml at room temperature for 30 min, Cy3-conjugated AP3 was added to a final concentration of 10 μg/ml, and cells were incubated on ice for another 30 min before subjected to flow cytometry. Binding of soluble fibrinogen was determined and expressed as the percentage of mean fluorescence intensity relative to immunofluorescent staining with Cy3-labeled AP3 mAb.Binding of fluorescein-labeled human fibrinogen was performed as previously described . To dete2+ or 1 mM Mn2+ and 100 μM GRGDSP peptide at room temperature for 30 min. Anti-LIBS mAbs  was added to a final concentration of 10 μg/ml, and cells were incubated on ice for 30 min before staining with FITC-conjugated antimouse IgG and flow cytometry. LIBS epitope expression was determined and expressed as the percentage of mean fluorescence intensity of anti-LIBS mAbs relative to the conformation-independent mAb AP3  and thus was chosen as the final model. Models for clusters 11 and 12 were kindly provided by the authors of Model building was performed using the NMR structure of glycophorin A TM dimer  as a template. The entire TM domains of αL server . For eac


Virtual reality (VR) technology is rapidly becoming a popular application for physical rehabilitation and motor control research. But questions remain about whether this technology really extends our ability to influence the nervous system or whether moving within a virtual environment just motivates the individual to perform. I served as guest editor of this month's issue of the Journal of NeuroEngineering and Rehabilitation (JNER) for a group of papers on augmented and virtual reality in rehabilitation. These papers demonstrate a variety of approaches taken for applying VR technology to physical rehabilitation. The papers by Kenyon et al. and Sparto et al. address critical questions about how this technology can be applied to physical rehabilitation and research. The papers by Sveistrup and Viau et al. explore whether action within a virtual environment is equivalent to motor performance within the physical environment. Finally, papers by Riva et al. and Weiss et al. discuss the important characteristics of a virtual environment that will be most effective for obtaining changes in the motor system. Virtual reality (VR) technology has been used for several decades for a variety of psychosocial interventions. But since the early 1990's there has been an explosion of laboratories and clinics promoting the use of virtual technology for physical rehabilitation . PresentVR describes a computer-generated scenario  with which the user can interact in 3 dimensions so that the user feels that he or she is part of the scene . CurrentMany people question why we don't just have subjects perform motor tasks in the real world. The answer to this question is that VR offers us the opportunity to bring the complexity of the physical world into the controlled environment of the laboratory. VR gives us the potential to move away from reductionism in science and towards the measurement of natural movement within natural complex environments. In general, VR allows us to create a synthetic environment with precise control over a large number of physical variables that influence behavior while recording physiological and kinematic responses . To thisThere are numerous strengths underlying the use of VR with rehabilitation ,12. AmonAnother question that has arisen at meetings and in the review of the papers for JNER is under what circumstances a computer generated environment should be considered virtual reality? Factors that differ among many of the laboratories claiming to use virtual reality and that also emerge amongst this group of papers include field of view, the presence of stereo vision, and real-time feedback of head position so that the scene can be updated to reflect natural movement of the visual world. There is evidence demonstrating that a transfer of training from the virtual to the physical environment is greater if the learner is immersed in the training environment . Perhaps


There is a large range of human skin color, yet we know very little about the underlying genetic architecture. Is the number of skin color genes close to five, 50, or 500? Diversity of human appearance and form has intrigued biologists for centuries, but nearly 100 years after the term “genetics” was coined by William Bateson in 1906, the genes that underlie this diversity are an unsolved mystery. One of the most obvious phenotypes that distinguish members of our species, differences in skin pigmentation, is also one of the most enigmatic. There is a tremendous range of human skin color in which variation can be correlated with climates, continents, and/or cultures, yet we know very little about the underlying genetic architecture. Is the number of common skin color genes closer to five, 50, or 500? Do gain- and loss-of-function alleles for a small set of genes give rise to phenotypes at opposite ends of the pigmentary spectrum? Has the effect of natural selection on similar pigmentation phenotypes proceeded independently via similar pathways? And, finally, should we care about the genetics of human pigmentation if it is only skin-deep?TP53 have helped to educate society about the risks of tobacco    However,e common , and itse common .More important than the ratio of melanin types is the total amount of melanin produced. In addition, histological characteristics of different-colored skin provide some clues as to cellular mechanisms that are likely to drive pigmentary variation . For theOf equal importance to what happens inside melanocytes is what happens outside. Each pigment cell actively transfers its melanosomes to about 40 basal keratinocytes; ultimately, skin reflectance is determined by the amount and distribution of pigment granules within keratinocytes rather than melanocytes. In general, melanosomes of African skin are larger and dispersed more widely than in Asian or European skin . RemarkaFor any quantitative trait with multiple contributing factors, the most important questions are the overall heritability, the number of genes likely to be involved, and the best strategies for identifying those genes. For skin color, the broad sense heritability  is very high , provideminimal , one of the more interesting findings was that skin reflectance appeared to be mainly additive. In other words, mean skin reflectance of “F1 hybrid” or “backcross hybrid” groups is intermediate between their respective parental groups.Statements regarding the number of human skin color genes are attributed to several studies; one of the most complete is by An alternative approach for considering the number of potential human pigmentation genes is based on mouse coat color genetics, one of the original models to define and study gene action and interaction, for which nearly 100 different genes have been recognized . SettingTYR, P, and MATP genes discussed earlier are well-known causes of albinism whose primary effects are limited to pigment cells  than synonymous (which do not change any amino acid) alterations. Data have been collected only for  regions . Thus, wCredit for describing the relationship between latitude and skin color in modern humans is usually ascribed to an Italian geographer, Renato Basutti, whose widely reproduced “skin color maps” illustrate the correlation of darker skin with equatorial proximity . More re3); when not otherwise provided by dietary supplements, deficiency for vitamin D causes rickets, a characteristic pattern of growth abnormalities and bony deformities. An oft-cited anecdote in support of the vitamin D hypothesis is that Arctic populations whose skin is relatively dark given their latitude, such as the Inuit and the Lapp, have had a diet that is historically rich in vitamin D. Sensitivity of modern humans to vitamin D deficiency is evident from the widespread occurrence of rickets in 19th-century industrial Europe, but whether dark-skinned humans migrating to polar latitudes tens or hundreds of thousands of years ago experienced similar problems is open to question. In any case, a risk for vitamin D deficiency can only explain selection for light skin. Among several mechanisms suggested to provide a selective advantage for dark skin in conditions of high UV irradiation  alleles. While specific answers will be different for every phenotype, there may be common themes, and some answers are better than none.