David Goldstein - US grants

The objective of this proposal is to participate in a multicenter collaborative clinical trial to determine whether strict control of blood glucose levels is effective in preventing or ameliorating the vascular complications of insulin-dependent diabetes mellitus. The central hypothesis to be tested is that the degree of altered blood glucose control is an important risk factor in the development and progression of chronic diabetic vascular complcations. Another hypothesis to be tested is that a therapy exists which will restore the metabolic state of the diabetic person to as close to normal as possible and that this therapy can be effectively and safely applied to a large number of individuals over a prolonged period of time. The specific aims of the proposal are to determine if patients treated with an intensive regimen (continuous subcutaneous insulin infusion) can achieve better glucose control than patients treated with a conventional regimen (two daily injections of insulin). In respect to the intensive therapy, we will determine (as part of the multicenter collaborative study) if high rates of patient acceptability are possible and if the treatment is safe. Two treatment groups (intensive vs conventional therapy) will be established and following an extensive baseline evaluation, patients will be monitored at regular intervals for 1) blood glucose control using the results of hemoglobin Alc determinations as well as other means, and for 2) pathologic endpoints such as retinopathy as determined by stereo color trtinal photographs. The study will proceed in three phases, planning of protocols, limited feasibility trial (two years) and if indicated, full scale trial. The proposed studies will provide important new information which will increase our understanding of diabetic vascular complications, and suggest rational approaches to treatment of patients with diabetes mellitus.

This project is in the Chemistry of Life Processes Initiative (COLP) and supported jointly by Chemistry and Biophysics. Fundamental studies of protein structure-function relationships will be carried out. Iso-cytochrome c from yeast is used to study how amino acid replacements and deletions affect key parameters such as electron transfer rates, binding strength, specificity to protein partners of cytochrome c, and protein stability. Detailed intracellelar measurements will be made for comparison with the in vitro work, using growth in lactate medium as a measure of relative specific activity. Specific mutations have been generated for each of the studies. Along with the experimental work, a variety of theoretical approaches are being developed to aid in the design and interpretation of experiments. This project is a continuation of that previously supported under the COLP Initiative.

The objective of this proposal is to participate in a multicenter collaborative clinical trial to determine whether strict control of blood glucose levels is effective in preventing or ameliorating the vascular complications of insulin-dependent diabetes mellitus. The central hypothesis to be tested is that the degree of altered blood glucose control is an important risk factor in the development and progression of chronic diabetic vascular complcations. Another hypothesis to be tested is that a therapy exists which will restore the metabolic state of the diabetic person to as close to normal as possible and that this therapy can be effectively and safely applied to a large number of individuals over a prolonged period of time. The specific aims of the proposal are to determine if patients treated with an intensive regimen (continuous subcutaneous insulin infusion) can achieve better glucose control than patients treated with a conventional regimen (two daily injections of insulin). In respect to the intensive therapy, we will determine (as part of the multicenter collaborative study) if high rates of patient acceptability are possible and if the treatment is safe. Two treatment groups (intensive vs conventional therapy) will be established and following an extensive baseline evaluation, patients will be monitored at regular intervals for 1) blood glucose control using the results of hemoglobin Alc determinations as well as other means, and for 2) pathologic endpoints such as retinopathy as determined by stereo color trtinal photographs. The study will proceed in three phases, planning of protocols, limited feasibility trial (two years) and if indicated, full scale trial. The proposed studies will provide important new information which will increase our understanding of diabetic vascular complications, and suggest rational approaches to treatment of patients with diabetes mellitus.

Acquiring a transmission electron microscope would greatly strengthen research opportunities in cell, molecular, and developmental biology at Wright State University. Students taking our research-oriented course in electron microscopy or doing a research project using electron microscopy will have the opportunity to use modern equipment that is flexible, reliable, and comparable to facilities available in commercial laboratories or major research institutions nationwide. Specific research opportunities for undergraduates include the projects outlined in this proposal: (1) the influence of gravity on plant growth and development, (2) cuticular and gut ultrastructure of Sarcoptes scabiei from susceptible and resistant hosts, (3) growth and differentiation of epidermal keratinocytes, (4) morphological adaptation of avian osmoregulatory organs to changing physiological demands, and (5) secretion of gonadotropin releasing hormone in the central nervous system.

Dehydration is a stressor that may be encountered by many organisms during their lifetimes, including the period of body growth and organ development. Dehydration induces changes in hormone levels, including prominently increases in levels of anti-diuretic hormone, and the restricted water inhibits general body growth. Together, these factors may influence the growth and the development of many organ systems. The research will focus particularly on the effects of dehydration during growth on development of the kidney, a principle target organ for anti-diuretic hormone. The PI will study this problem in bobwhite quail, a species in which the chicks must forage for themselves beginning shortly after hatching, and in which therefore there is a very real possibility of dehydration during the early life stages. Preliminary data suggest that the region of the kidney (the renal medulla) primarily concerned with water conservation during dehydration hypertrophies when the birds are chronically water restricted. Associated with this is an enhanced ability to concentrate the urine, which should contribute to enhanced water conservation. The proposed research will further explore the functional consequences of chronic dehydration, and in particular will examine the basis for the enhanced concentrating ability. Does this reflect changes in the rate of urine formation, changes in hormone levels, or changes in the cellular response to the hormone? How do morphological changes induced by dehydration correlate with changes in function? Are these changes reversible, or do stresses imposed during youth have consequences that continue into adulthood? These studies will help to define the physiological responses of animals to dehydration. They will also examine the relation between structure and function in the avian kidney, about which much remains unknown or speculative. More generally, these studies will examine the flexibility of an important homeostatic system, and will address the general issue of the importance of environmental stresses on developmental pathways. This approach should identify constraints in design and function of the avian kidney. This relates both to the ability of individuals to acclimate to changing environments, as well as to the ability of species to invade and adapt to different habitats.

At a unique point in the history of health care in this country, the University of Rochester Medical Center (URMC) is poised to capitalize on fifty years of local health care planning and community collaboration by building an information system that enhances academic-community partnerships. IAIMS efforts here will reach beyond URMC, emphasizing vital linkages between the health sciences center and the larger community. The overarching goal of URMC IAIMS planning is to bring together a rich array of institutional and community resources into an integrated information environment, employing computing and communication technologies to further research, education and practice goals of all participants. The specific aims of IAIMS planning at URMC are to: (1) Express our shared vision in a master plan for the URMC integrated information environment; (2) Develop network-based partnerships with information and health care communities on and off the campus; (3) Educate the URMC and community partners to work effectively in the IAIMS environment; and (4) Design a governance architecture to guide implementation of the plan. The IAIMS planning structure consists of seven Planning/Review Groups, the IAIMS Project office, the IAIMS Advisory Board, and the Committee of Group Chairs. Existing institutional mechanisms for planning and policy development at the Medical Center are responsible for review and approval of the Master Plan. This strategy embeds IAIMS into the life of the institution, providing stability for the implementation cycle to come. Seven planning/review groups undertake intensive examinations of the current and future information environment. Four core groups represent URMC's four central program areas: Education, Basic Research, Clinical Practice/Research, and Administration. Three infrastructure groups (Information Policies, Governance and Funding, Network Infrastructure) address broad issues relating to the long-term success and stability of IAIMS at URMC. Planning group membership reflects the diverse constituencies which create, manage and use URMC information resources, including community partners. The planning groups define preferred scenarios, prepare recommendations and propose priorities that form the basis of the Master Plan and governance architecture. Chairs of the planning committees form the Committee of Group Chairs, which drafts the Master Plan and assists in developing community partnerships. The IAIMS Advisory Board, made up of senior-level faculty and administration, reviews the Master Plan before it is sent to the Medical Center Executive Committee for final approval. The IAIMS Project Office, sited in the Miner Library, coordinates and supports the work of the planning and advisory groups, sponsors educational events, and coordinates joint ventures with community partners.

The correlation between papillomavirus infection and the development of human cancer is well established and the viral genes involved in this process have been identified. It is becoming increasingly evident that the ability of the papillomaviruses to induce cellular proliferation involves complex formation with certain cellular proteins that are important for the regulation of cell growth. A consequence of these interactions appears to be an alteration of the function of these proteins resulting in mitogenesis and transformation. The E5 protein is the major transforming protein expressed by the prototype bovine papillomavirus type 1 (BPV-1). It is unique in that it is highly hydrophobic, localizes predominantly to Golgi and endomembrane compartments, and consists of only 44 amino acids. Many of the human papillomaviruses (HPVs) also encode small, hydrophobic E5 proteins. However, our current knowledge of the activity of these structurally related E5 proteins is only rudimentary. Several recent studies on both the HPV and BPV E5 proteins suggest an involvement of tyrosine kinase growth factor receptors in the induction of cellular mitogenesis. This research grant application proposes to demonstrate that mitogenetic and transforming activity induced by the BPV-1 E5 protein is a direct consequence of the activation of growth factor receptors. In addition, we intend to define the molecular and biochemical mechanisms involved in the process of receptor activation. The molecular cloning of growth factor receptor genes has made it feasible to investigate the ability of specific receptors to couple with intracellular components needed to evoke a functional response in foreign host cells. To investigate the ability of the E5 proteins to induce mitogenic signalling through growth factor receptors, a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin 3 (IL-3) for sustained proliferation in culture will be employed. The strict requirement for IL-3 for the growth of 32D cells can be functionally substituted by the stimulation of signal transduction pathways initiated by the expression of specific growth factor receptors and the addition of the appropriate ligand to the culture medium. This phenomenon will be exploited to identify and characterize those components of signal transduction pathways that are capable of responding to E5 expression to induce mitogenesis and cellular transformation in the absence of ligand. The data generated from these studies, in addition to assisting in the elucidation of the complex mechanism of papillomavirus transformation, may also provide new insights into molecular aspects of growth factor receptor tyrosine kinases, receptor-mediated signal transduction, and the control of cell growth. As a long-term goal of this project, a similar approach will be applied to the structurally homologous HPV E5 proteins. This approach should assist in the identification of the function(s) and cellular targets of these less well-characterized proteins.

Dr. Wheatly and her colleagues will conduct a program of Undergraduate Mentoring in Environmental Biology (UMEB), with three objectives: (1) to provide long-term research experience under the guidance of a faculty mentor for students with physical disabilities and from ethnic/racial minority groups, (2) to increase the entry of these students into graduate programs in environmental biology, and (3) to foster interdisciplinary approaches to environmental biology upon which to build curricular and research initiatives.

Students with physical disabilities are the most underrepresented group in science, while ethnic minorities also remain significantly underrepresented. Undergraduate participants will be recruited from these two groups both at Wright State University (WSU, a leader in disability services) and at Wilberforce University (a historically Black university), whose students can get credit for WSU coursework through an articulation agreement. Researchers at WSU have built a highly visible, extramurally funded program in environmental stress that involves undergraduate students. With appropriate infrastructure and attitude, WSU faculty members will mentor students through didactic coursework and long-term research experience. An extended research opportunity will enable students to experience research success and should result in increasing the representation of both groups in environmental biology.

Application materials from the two targeted groups of students will be reviewed by a selection committee, which will accept 10 students based on demonstrated competence in biology and interest in environmental research. Ten faculty mentors were selected from three departments (Biological Sciences, Psychology, Pharmacology & Toxicology) based on funded research in the broad area of environmental stress that will expose students to diverse conceptual, methodological, and organismal approaches. The selected faculty members have all demonstrated effective mentoring of undergraduates from underrepresented groups, and three of them are members of underrepresented groups. They are all volunteering their time as mentors.

During the summer before students enter the research lab, they will take a required course that will be a survival guide to scientific research. They will then enter a selected lab for a minimum of one year, where they will perform research under the supervision of a faculty member. Expectations and responsibilities for mentors and students are clearly defined and contracted. Enrichment activities (project lab meetings, social events, yearly symposium, electronic publishing) that create a community of scientific peers, as well as continuous monitoring and active counseling in professional development, should help to retain students in the program. Students will demonstrate their success in research through oral/poster presentations and written reports/manuscript submissions. An advisory board will monitor overall success of the project through assessment exercises.

Over the past decades, comparative and ecological physiologists have succeeded in describing numerous marvelous ways in which organisms from a variety of habitats are specialized for life in those habitats. However, the great majority of these investigations have been performed in the laboratory, where animals and their environments are under control and manipulable. An important direction in which comparative and ecological physiology is now proceeding is to explore physiological function under natural conditions and with ecological context in mind.

Toward that end, the PIs have assembled a panel of speakers who are pushing forward the ability to measure and interpret physiological function of free-living animals. These speakers will focus on vertebrate animals and will approach this topic from several directions. First, they will address a variety of physiological systems; presentations will focus on energy, body condition, temperature regulation, digestion and metabolism, osmoregulation, reproduction, stress, and others. Second, they will explore a variety of methodological approaches; presentations will focus on endocrinology, telemetry, stable isotopes, phenotypic engineering, immune testing, evaluation of tissue and body fluid composition, and more. Third, they will discuss a variety of vertebrate types; authors will speak on migratory birds, marine mammals, nectarivores, and others. This symposium will be held in conjunction with the annual meeting of the Society for Integrative and Comparative Biology (Chicago, IL, January 2001).

Together, it is anticipated that these presentations will provide an overview of conceptual and methodological advances in the investigation of physiological systems under natural conditions with a view to answering questions of ecological importance. In the process, they will continue to enhance our appreciation of how animals survive under varied and stressful circumstances. Moreover, they will help to provide insights into ecological questions such as what limits the distributions of animals on Earth, and how this might be affected by patterns of environmental change.

Many birds change during growth or seasonal cycles from eating diets low in protein, such as seeds, to diets high in protein, such as insects. In mammals, changing to a high protein diet induces a number of changes in the kidneys, including enlargement of the organs and changes in the production and composition of the urine. In humans, these changes are important contributors to the progression of renal failure, and there is concern about these consequences also associated with the current popularity of high protein/low carbohydrate dieting. Birds possess many of the same elements to their anatomy and physiology as are suggested to be involved in the mammalian response to eating protein; however, birds lack others of these elements. Thus, birds can serve as a natural model for testing theories about the links between diet and consequences for the kidneys. In the proposed studies, Dr. Goldstein will explore the ways in which diet influences the responsiveness of the kidneys to hormones released in response to feeding. He will do so by raising bobwhite quail on high-vs.-low protein diets, and then exploring organ structure, kidney function, and the biochemical responses of kidney cells and tissues. These studies will help to define the physiology of a bird (the bobwhite quail) that is valuable both as a domestic and a wildlife species. They will also clarify assumptions and hypotheses proposed for mammalian kidneys, and thereby help to elucidate the link between dietary protein and the progression of renal failure.

Although people dream of being able to freeze whole bodies and later return them to life, currently successful biomedical cryopreservation is restricted to relatively small structures like blood or embryos. However, there are animals, including several amphibians and reptiles, that survive northern winters in part by an ability to tolerate being frozen. This research will examine the physiological mechanisms responsible for freeze tolerance in one of those species, Cope's gray treefrog. This frog is unusual among freeze-tolerant vertebrates in that it accumulates glycerol in its body fluids as a cryoprotective agent; glycerol is also commonly used in biomedical preservative solutions. Specific questions to be addressed are how glycerol moves in and out of tissues of the treefrogs as they acclimate to cold and actually freeze, and how the glycerol movement is related to the distribution of water among tissues, which is critical to preventing ice formation inside cells where it would lead to lethal damage. In particular, experiments will investigate the role of aquaporins in accomplishing that glycerol and water movement. Aquaporins are proteins known to form passageways that allow water and, sometimes, other small molecules like glycerol to cross cell membranes, and it is hypothesize that they are critical to freeze tolerance. The objectives are to enhance the understanding of this fascinating natural phenomenon, and to use a natural model of freeze tolerance to elucidate principles that can be applied to biomedical cryopreservation. In the process, this work will provide training to undergraduate students at multiple levels of analysis, from principles of comparative biology to whole animals and molecular biology. This training will encourage integrative thinking and application of diverse methods to the study of animal physiology.

Wright State University (WSU) and Sinclair Community College (SCC) are collaborating to develop a common first-year STEM experience with the following intended outcomes:

1. A 10% increase in first-to-second year retention of STEM majors at both institutions

2. A 10% increase in articulation of STEM majors from SCC to WSU. The result is expected to increase the number of WSU STEM graduates by at least 50 students per year by the close of the project. This model is readily transferable to other community college/university dyads in urban settings with comparable open admission policies.

Prior NSF support of WSU's National Model for Engineering Mathematics Education has shown that the introduction of EGR 101 "Introductory Mathematics for Engineering Applications," coupled with a significant restructuring of the early engineering curriculum, has resulted in a 10% increase in first-to second year retention as well as increased student motivation and confidence in math and engineering.

Based on this early success, this research:

1. Implements EGR 101 and the associated engineering curriculum reforms at SCC.

2. Develops a companion lab-based class for science majors (Scientific Thought and Method), SM 101/ASE 101, for instruction at both WSU and SCC.

3. Provides professional development opportunities for faculty at both institutions.

4. Trains STEM seniors/graduate students to serve as lab/recitation assistants, and peer tutors for any introductory STEM classes.

5. Disseminates the curriculum and associated first-year experience through production of a textbook and through presentations at professional meetings.

The overall goal of this CHAVI application is to elucidate viral and host determinants of HIV-1 transmission, persistence, and partial containment in primary human infection, and to translate these findings into rational vaccine design. Core A will support this effort by providing expertise and service in the areas of host genomics and HIV-1 genetics. The Core is comprised of three sections: 1. The Host Genomics Section (under the leadership of Dr. David Goldstein) will elucidate host genetic differences affecting susceptibility to HIV-1 infection and disease progression in early HIV-1 infection. Specific tasks include to (i) identify, validate, and genotype a robust set of tagging SNPs (tSNPs) for 500 high priority human genes to facilitate indirect genetic association studies, (ii) identify putative functional variants among the candidate genes for direct association studies; (iii) develop uniform experimental genotyping approaches; (ivj optimize sequencing primers for direct association studies in the rhesus monkey; (v) investigate all associations at the genetic and functional level; and (vi) establish a robust data management system for clinical as well as host and viral genetic data. 2. The HIV-1 Molecular Biology and Sequencing Section (under the leadership of Dr. Beatrice Hahn), will generate functional HIV-1 env clones and full-length HIV-1 sequences from acutely and chronically infected individuals to elucidate the genetic, biologic, antigenic, and structural characteristics of sexually transmitted virus and the mechanisms underlying this transmission. Specific tasks include to (i) amplify, clone and sequence functional env genes from acute HIV-1 infections; (ii) examine envelopes from acute and chronic HIV-1 infections for phenotypic differences, and (iii) derive full-length HIV-1 sequences from patients with acute HIV-1 infection. 3. The HIV-1 Computational Biology and Biostatistics Section (under the leadership of Bette Korber), will provide interactive automated HIV-1 sequence entry and analysis tools, and establish and maintain an Acute HIV-1 Sequence Database. Specific tasks include to (i) provide an interactive automated HIV-1 sequence data entry system that enables users to identify sequencing errors and contamination as part of their initial data processing; (ii) provide interactive automated tools for baseline analysis/of CHAVI HIV-1 sequences, (iii) make CHAVI HIV-1 sequences publicly available, (iv) build CHAVI investigator interfaces for data entry of immunological data, (v) analyze HIV-1 sequence datasets for acute infection sequence signatures and (vi) provide statistical support for the CHAVI effort. In Year 02, each of these sections will become an independent core facility (see Strategic Plan), commensurate with the anticipated increase of both host and HIV-1 sequence analysis.

Development of Novel Affinity Reagents: During the past fiscal year, the Office of Science and Technology Partnerships (OSTP) has continued to support an initiative for generating high affinity reagents to serve as tools for molecular interrogation of pathways that become altered during cancer development. Partnerships with two biotechnology companies, Beckton Dickinson Pharmingen and Rockland Immunochemicals, have led to the creation of nearly 200 novel affinity reagents (polyclonal and monoclonal antibodies) against hundreds of key cancer-related targets of interest to the CCR at no cost to CCR investigators. These partnerships represent savings of more than one million dollars to CCR and the NCI. The antibodies that have been developed through these partnerships include: 1) novel candidate proteins for which no commercially available antibodies exist; 2) known proteins for which commercially available antibodies exist but fail to meet investigators' requirements; and 3) proteins with phosphorylation sites that may be of importance in pathways that are altered during cancer development. All of the antibody characterization data generated by participating CCR investigators is made available to the companies involved through a Web site developed in a collaboration between the OSTP and the Advanced Biomedical Computing Center (ABCC). Each company uses this data in their marketing material when the antibodies are released for commercialization. A third collaboration will begin this fiscal year with the company, Epitomics, for production of rabbit monoclonal antibodies. These antibodies are known to be much more stable and have higher affinities than mouse monoclonal or rabbit polyclonal antibodies. The OSTP negotiated a contract with Epitomics to access their antibody production pipeline at a significantly reduced cost ($5,500 versus $20,000 from the catalog price). Similar to the other collaborative programs, CCR investigators will be required to characterize the antibodies and make data available to Epitomics for validation and commercialization. Discovery of Novel Protein-Protein Interactions: The OSTP supported a number of programs for defining novel cancer relevant protein-protein interactions and cell signaling networks. For example, OSTP managed a major screening program to identify and characterize novel binding partners to a large number of cancer-related proteins currently under investigation within the CCR. A CCR-wide partnership with Myriad Genetics was developed to access their automated process for the large-scale identification of protein-protein interactions that is based on the yeast two-hybrid (Y2H) methodology. This large-scale effort between CCR and Myriad generated a host of data (over 1,800 novel protein interactions have been transferred to CCR investigators) that are permitting the delineation of previously uncharacterized signaling pathways and a better understanding of the molecular basis underlying cancer. The data derived from these screens has been made available to CCR investigators through a Web site developed in collaboration between OSTP and the ABCC. Facilitating Access to Genomic Technologies: The OSTP has been evaluating all commercially available platforms for microarray and other high throughput genomic technologies. To make the latest advances easily accessible to CCR researchers, the OSTP has established a number of contracts and pricing arrangements with companies providing genomic profiling technologies and services (Affymetrix, NimbleGen, Agilent, Illumina, GenUs, SABiosciences, etc). These agreements have led to far lower cost and an efficient and easy way for CCR investigators to access these technologies. A subsidy program has been established providing 50% payment to the CCR community for all microarrays that are purchased from these companies or full-service microarray analysis that is requested. This funding mechanism has helped to extend the limited research dollars available to CCR laboratories and has permitted our researchers to conduct experiments that might otherwise be cost prohibitive. Many of the funded projects have led to important publications in top tier journals. Most recently, the OSTP has been coordinating CCR's efforts to access the ultra high-throughput, next-generation sequencing technologies. OSTP was involved in all aspects of acquisition of two Illumina/Solexa genetic analyzers that were installed in two CCR laboratories. The OSTP will be providing investigators with subsidy funds to make this very expensive technology accessible to many CCR labs. The OSTP is also working with SAIC-Frederick, Inc. to create a new sequencing facility to be located at the NCI's Advanced Technology Center (ATC) in Gaithersburg, MD. This facility, which will begin operation during the next fiscal year, will make next-generation sequencing technology broadly available to all NCI researchers. Tools for Bioinformatics and Biostatistics Analysis: In response to CCR's rapidly growing need to manage and analyze large sets of genomic and proteomic data, the OSTP has developed several partnerships with bioinformatics companies. At significant cost savings to NCI, OSTP negotiated a variety of license agreements on behalf of all NCI researchers for universal access to sophisticated tools for bioinformatic and statistical analysis of microarray experiments, arrayCGH studies, proteomic profiling studies, genomic analysis, and genetic screens. In addition, the OSTP has made a number of software applications for the investigation of pathways and biological association networks available to all of NCI. A CCR/NCI Integrated and Collaborative Knowledge Environment: To address the complexities associated with querying across the growing number and ever-expanding public and private databases, the OSTP has established a new program to develop a CCR/NCI integrated and collaborative knowledge environment in collaboration with Sophic Systems Alliance and Biomax. Software developed by Biomax enables the visualization of complex relationships between biological and biomedical data and information. It is designed to institutionalize knowledge by enabling the annotation of information stored in a central repository. The Knowledge Environment was configured to support research on different types of cancer, sources of data, and research strategies. The CCR/NCI laboratories are configuring the Biomax software according to their individual research requirements to determine its benefits to cancer research at the Center. The areas of research include colorectal and liver carcinogenesis, neuroblastoma, radiation oncology, and neuro-oncology. Each lab requires configuration of their software instance that is tailored to the type of research conducted in that lab. The Common CCR Knowledge Environment was configured to provide access to 35 public databases integrated into a single user interface, allowing scientists to query a wide range of data sources. Information derived through genomics, proteomics, pathway analysis, and clinical studies are combined into graphical representations of complex relationships. Future plans for the Common CCR Knowledge Environment include the integration of additional databases, genomic and proteomic analysis tools, and software.

Women are underrepresented in STEM units in Dayton academic institutions, as they are nationwide. Four institutions in the Dayton region with diverse histories, missions and demographics form the LEADER consortium for the purpose of Launching Equity in the Academy across the Dayton Entrepreneurial Region. The institutions include a public doctoral university (Wright State University, host institution), a private Catholic institution (University of Dayton), a minority-serving public institution (Central State University) and a federal graduate institution (Air Force Institute of Technology). All are located in close proximity and collaborate routinely on STEM initiatives. They also share a commitment to regional STEM education, pipeline, and economic development, and recognition that inclusiveness, including directed efforts to recruit and support women in STEM, is a necessary component of that mission. Our ADVANCE collaborative will address these issues through a unique combination of inter-institutional coordination and approaches drawn from social and organizational psychology to improve climate and thereby transform the individual participating institutions.

Intellectual Merit: The underrepresentation of women among academic STEM faculties reflects gender disparities in recruitment, support, and promotion. Underlying the persistence of these problems are features of institutional climate that are rooted in the often nonconscious attitudes and behaviors of individuals. Thus, progress toward gender equity in the STEM academy requires transformation of institutional structures and processes, and transformation of climate. The LEADER consortium will implement models of social/organizational psychology based on gender schemas, persuasion theory, and social contracts, to transform institutional climate in support of STEM women. We will facilitate implementation of strategies proven in prior ADVANCE initiatives to enhance recruitment, retention and advancement of tenure-track STEM women. Implementation of these initiatives within a framework of inter-institutional accountability and administrative architecture (the LEADER Consortium) will catalyze transformation of climate within institutions, thus creating a sustainable women-friendly STEM culture within a region built upon a legacy of STEM innovation.
The specific aims of LEADER are: (a) to conduct a comparative analysis of climate for STEM women across the institutions and thereby identify best practices related to recruitment, retention, and advancement; (b) to initiate gender schema education and a campaign based on persuasion theory that will promote new norms of expectation and thereby facilitate implementation of those best practices; and (c) to implement social contracts across the consortium that promote transparency and accountability for transformation of climate, leading to recruitment, promotion and success of STEM women.
Implementation and Management: Social science research will be undertaken by a social psychologist and a philosopher working in the area of moral psychology and gender theory. Initially climate will be compared across the institutions to inform climate initiatives. At the unit and institutional levels, chairs and faculty equity advisors will implement proposed initiatives with the assistance of a centralized LEADER administrative office. Accountability for achieving benchmarks in recruitment and advancement of women will be centrally monitored using accepted metrics, formative and summative evaluation, and continuous improvement under the direction of the LEADER Council (composed of representatives from each institution) and with external oversight from an Advisory Board.

Broader Impacts: The inter-institutional collaboration and accountability should significantly increase retention and advancement of women in the STEM academy. More broadly, our ADVANCE program is designed to promote equity and that model can be applied to diverse target populations. The consortium includes an HBCU (Central State) and an institution committed to accessibility for the disabled (Wright State); as such, this project should promote significant gains in these two demographic groups within the community of STEM women. Our selection of the acronym "LEADER" recognizes this transferability; advancement of STEM women in the Dayton region today will provide leadership, by example, for efforts toward equity within the academy.

DESCRIPTION (provided by applicant): Large-scale genetics studies on schizophrenia to date have failed to discover a substantial contribution of common genetic variation. However, recent analyses of structural genetic variation have identified and rapidly confirmed a number of rare variants contributing to this disorder. Surprisingly, the same rare genetic contributors have often been found to be risk factors for multiple neuropsychiatric conditions, including schizophrenia, mental retardation, autism and epilepsy. This suggests that these rare variants may confer a "neuropsychiatric vulnerability" and that the ultimate manifestation depends on other genetic or environmental influences. This suggests that patients with multiple co-occurring conditions may be those most likely to carry an elevated burden of rare, high-penetrant risk factors. Additionally, families that have multiple neuropsychiatric conditions associated with the same rare genetic variant could be the most valuable in dissecting the genetic and environmental influences on mental disorders. With this research, we propose to collect 100 chronically mentally ill patients with schizophrenia or schizoaffective disorder and as many of their first, second and third degree relatives as possible, and to perform whole-genome sequencing to identify rare gene variants that predispose to disease with relatively high-penetrance. Using DNA collected from these subjects, we will use next-generation whole genome sequencing technology and a variety of novel bioinformatic tools to select potentially disease-associated gene variants on the basis of predicted function and frequency in healthy control populations. We will then examine the distribution of associated variants in the families, determining which disorders, symptoms or traits they are associated with, and what differences in environment and genetic background are present between affected and unaffected carriers. By focusing on patients with a strong genetic burden and a family history of mental illness, and including the entire genome in our search for genetic susceptibility, we expect to be able to find rare highly penetrant variants that would be undetectable with other approaches. PUBLIC HEALTH RELEVANCE: Schizophrenia is a highly heritable neuropsychiatric condition that affects millions of individuals in the U.S. alone. The proposed study seeks to increase our understanding of the genetic basis of this disease by performing whole-genome sequencing in 100 schizophrenia patients to search for rare, highly penetrant associated genetic variants. Potentially associated genetic markers will be genotyped in affected and unaffected family members to determine their specificity for schizophrenia, and to investigate genetic and environmental modifiers of their effects. This work could elucidate the molecular basis of schizophrenia and potentially indicate novel targets for psychiatric drugs.

DESCRIPTION (provided by applicant): This application is in response to RFA-OD-09-004, "GO" grants program area: ARRA Medical Sequencing Discovery Projects. Despite clear evidence for the importance of genetics in susceptibility to epilepsy, only limited progress has been made in identifying the specific genes that influence risk. Most of this progress has resulted from positional cloning strategies applied to rare families with Mendelian inheritance. In the search for genes that influence risk for complex epilepsies, substantial attention has been directed to the identification of common variants, and several major genome-wide association studies (GWAS) are now underway. However, the few results that have emerged from these studies so far suggest that common variants account for little of the genetic component of the disorder. On the other hand, very recent studies have shown an increased frequency of rare and very large genomic deletions in people with epilepsy, suggesting that rare genetic variation may be important in epilepsy. If this is true, structural variants are unlikely to be the only types of pathogenic variation, and additional rare variants may be identified by sequencing. Thus in the current study, we propose to employ next generation sequencing to identify rare gene variants that contribute to epilepsy and are not represented, either directly or indirectly (through high linkage disequilibrium), on the sequencing platforms used in GWAS. Our strategy for this effort will focus on families containing multiple individuals with non-acquired (idiopathic or cryptogenic) epilepsy. The families to be studied have been previously collected and phenotyped in detail, and contain an average of 3.5 affected individuals with a range of different types of epilepsy. We will select one affected individual from each family and use next generation sequencing approaches to identify most of the genetic variation present in each of the selected individuals. These variants will then be evaluated using bioinformatic criteria to identify those most likely to influence epilepsy. We will then test whether these candidate mutations cosegregate with epilepsy within the families, and whether they are associated with epilepsy in a much larger sample of unrelated patients and controls. For each identified variant with strong evidence for association (in families or unrelated individuals), we will examine whether the effect is specific to one or more clinically defined subgroups (e.g., focal or generalized epilepsy), or whether it appears to raise risk more generally. PUBLIC HEALTH RELEVANCE: Epilepsy is one of the most common neurologic disorders, affecting approximately 4% of individuals at some time in their lives. The discovery of specific genes that influence risk offers a novel opportunity to clarify pathogenic mechanisms, identify susceptible individuals prior to seizure onset, and treat and prevent seizures in people at risk. Despite clear evidence of the importance of genetics in susceptibility to epilepsy, however, only limited progress has been made in identifying the specific genes that influence risk. This study aims to identify new and important genetic contributions to epilepsy by performing very high-throughput genomic sequencing in individuals with epilepsy who have affected relatives. Identification of these genes is extremely important for elucidating pathogenic mechanisms and could point the way to the development of new therapies for epilepsy. Application of the methods proposed in this application may also provide insight into the genetic analysis of other complex human diseases, in other areas of neurology and medicine.

Preservation of biological tissues by freezing has application in fields ranging from reproductive technology to conservation of endangered species. However, while scientists can successfully cryopreserve small living structures like eggs and embryos, application of those strategies to larger structures remains challenging. The natural world can provide insights into overcoming those challenges. Many "cold-blooded" animals encounter sub-freezing temperatures in their natural environment and, among the vertebrates, a few species have evolved physiological and biochemical strategies to survive freezing. This project will study one of those species, Cope?s gray treefrog. Freezing imposes daunting challenges for an animal, including potential physical damage from ice, fluctuating tissue oxygenation as the blood freezes and thaws, and disruption of molecular and cellular structures as liquid water freezes and body fluid concentrations change. A key to surviving these challenges is regulation of the distribution of water and dissolved solutes. Ice formation must be restricted to fluids outside of cells, and so water must be able to leave cells rapidly when freezing begins. In addition, some solutes, such as glycerol are cryoprotective and help to preserve molecular and physiological integrity during freezing. Gray treefrogs present a natural model for unraveling the mechanisms behind these capabilities. During the cooling autumn months they accumulate glycerol. They also regulate the expression of proteins ("aquaporins") that facilitate movement of water and glycerol across cell membranes. This project will evaluate how these molecular mechanisms are regulated, what is the resulting impact on cellular properties, and how this contributes to freeze tolerance of the whole animal. This integrative approach provides an excellent framework for training students from high school through PhD level in methods and ideas of molecular physiological regulation, and it will yield novel insights into the challenge of keeping tissues frozen alive.

DESCRIPTION (provided by applicant): The primary goal of the Epi4K Center Without Walls is to increase understanding of the genetic basis of human epilepsy in order to improve the well-being of patients and family members living with these disorders. This improvement will come in the form of better diagnostics, treatments and cures. To accomplish this goal, Epi4K aims to analyze the genomes of a large number of well-phenotyped epilepsy patients and families collected by investigators from several major research groups. The specific goals of this project (1 of 7 - Administrative Core) are to provide overall leadership to the Center, specifically regarding the Center's charter, universal protocols, plans for data sharing, internal and external communications, and tracking of scientific progress of all cores and projects within the Center.

DESCRIPTION (provided by applicant): The primary goal of the Epi4K Center Without Walls is to increase understanding of the genetic basis of human epilepsy in order to improve the well-being of patients and family members living with these disorders. This improvement will come in the form of better diagnostics, treatments and cures. To accomplish this goal, Epi4K aims to analyze the genomes of a large number of well-phenotyped epilepsy patients and families collected by investigators from several major research groups. The specific goals of this core (3 of 7 - Sequencing, Biostatistics, and Bioinformatics Core) are to 1) sequence and annotate 4,000 genomes, 2) develop computational procedures for calling CNVs in whole exome data, 3) identify and prioritize variants of interest for all three projects, 4) conduct follow up genotypin analyses in a cohort of additional cases and controls, and 5) quickly and efficiently share data among the Epi4K consortium. PUBLIC HEALTH RELEVANCE: Epilepsy is one of the most common human neurological disorders, affecting 3% of the population. Although it is clear that there is a strong genetic component for epilepsy, there are still only a few genes known. The Epi4K project will identify new genes and genetic pathways in epilepsy and will directly benefit individuals with epilepsy and their families through improved diagnostic, prognostic and recurrence risk information. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

DESCRIPTION (provided by applicant): The primary goal of the Epi4K Center Without Walls is to increase understanding of the genetic basis of human epilepsy in order to improve the well-being of patients and family members living with these disorders. This improvement will come in the form of better diagnostics, treatments and cures. To accomplish this goal, Epi4K aims to analyze the genomes of a large number of well-phenotyped epilepsy patients and families collected by investigators from several major research groups. The specific goals of this core (3 of 7 - Sequencing, Biostatistics, and Bioinformatics Core) are to 1) sequence and annotate 4,000 genomes, 2) develop computational procedures for calling CNVs in whole exome data, 3) identify and prioritize variants of interest for all three projects, 4) conduct follow up genotypin analyses in a cohort of additional cases and controls, and 5) quickly and efficiently share data among the Epi4K consortium.

The Genomics Scientific Support Component of CHAVI ID will focus its efforts on understanding the genetic contributions to immunological phenotypes relevant to responses to HIV-1 vaccination, with particular, but not sole, emphasis on the genetics of induction of broad neutralizing antibodies. In support of this work, we will also establish the core areas of competency that will be required for the SRSC to study the genetic and genomic bases and correlates of phenotypes of interest in HVTN and MHRP vaccine trials. Specific Aims Aim 1: To use whole genome sequencing to identify genetic correlates of HlV-1 infected patients who do and do not make broadly neutralizing antibodies during chronic infection. To achieve this aim we will perform whole genome sequencing on 50 broad neutralizing subjects, who exhibit the greatest breadth of neutralizing antibodies as determined by the B Cell Focus group and the Neutralizing Antibody SRSC. Variants of nterest identified in these patients will be followed up in a larger cohort of approximately 600 chronically infected patients who have been characterized for degree of plasma neutralizing activity. Aim 2: To relate host gene variation to restricted VH gene usage and other characteristics of the immunoglobulin repertoire as characterized by deep sequencing of the variable heavy (VH) VDJ region. Aim 3: To use RNA sequencing (RNA-Seq) to characterize the transcriptomes of HIV responsive CD4 positive T cells and Tfh cells in vaccinated rhesus monkeys and humans and to relate transcriptomes of helper CD4 cells to qualitative features of antibody responses to vaccination. To achieve this aim we will build upon our existing experience with RNA-Seq to establish a RNA sequence analysis pipeline that is comparable to our whole genome and exome sequence analysis pipeline. Aim 4: To use whole genome sequencing to study genetic bases of key aspects of the immune responses to vaccination observed in the RV144 trial.

DESCRIPTION (provided by applicant): While the vast majority of individuals are susceptible to HIV infection, a rare subset remains uninfected even after multiple exposures. Research on these persons has led to the identification of a genetic polymorphism (CCR5 32) that, in the homozygous state, provides protection against acquisition. However, this finding accounts for only a minority of persons who remain uninfected after exposure; therefore, other mechanisms must be acting to confer protection. In this application we propose an exploratory analysis of genetic and innate host defense factors that may play a role in protection from HIV infection. With this application we propose to examine mechanisms of protection among cohorts of a) 200 highly- exposed seronegative (HESN) men who have sex with men (MSM), b) 60 low risk MSM controls (LRCs), and c) 60 HIV infected men. In a departure from previous initiatives and a markedly innovative aspect of our proposal, we will not only select subjects with an extremely high self-reported risk of HIV exposure, but will attempt to delineate in exquisite detail those wih definitive evidence of biological exposure by using a multi- layered approach to detect HIV-specific anamnestic responses in the peripheral circulation and in the mucosa. With a robust discrimination of subjects most likely to have been exposed, we will examine intrinsic resistance to HIV replication at mucosal sites and in peripheral blood. We will examine the expression and occupancy of the HIV coreceptor CCR5 by peptide inhibitors at mucosal sites of HIV exposure and will examine the quiescence of mucosal T cells as well as in circulating T cells that we firs reported in HESN hemophiliacs. We will explore in fine detail the role of innate defenses provided by natural killer (NK) and dendritic cell (DC) populations in mucosal and systemic protection from HIV infection., Among subjects who are not CCR5 32 homozygous we will examine the genetic determinants of protection by whole-genome sequencing of blood samples from 250 HESN (50 HESN will be collected in addition to the 200 used for all other studies to increase power of the genetic analyses). Transcriptional profiles will also be performed on rectal mucosal samples from 150 HESN and 60 low risk MSM controls. Using a set of validated virologic assays, impaired infectibility will be localized to specific stages of the viral life cyce to facilitate precise identification of the source of the defect. Finally, these data sets will be interrogated to explore in greater detail the genetic determinants of functional hits and the functional implications of high probability genetic hits in order to harmonize these different search approaches. These novel and comprehensive analyses combining immunologic, virologic and genetic approaches using cohorts at carefully defined risks of HIV exposure will provide critical information on the determinants of mucosal protection from HIV infection.

The CCRIFX is clearly meeting a critical subset of CCR bioinformatics needs, and the staff is continually improving the level of analysis support to the CCR community. It has become increasingly clear that most investigators have vastly underestimated the complexity involved with NGS analysis. The CCRIFX has positioned itself as the central resource for the analysis of NGS data. This is reflected in the fact that the number of publications involving the core's analysis is on the rise. At the end of 2011, the core was involved in one manuscript in development. As of March of this year (2013), the core is actively working as coauthors on at least 6 manuscripts with several more emerging as strong possibilities as research efforts mature. In addition, the core contributed as co-authors on 4 posters and was acknowledged in 2 manuscripts. Investigators have confirmed through post-service evaluations that the CCRIFX is helping to advance research efforts, contribute creativity and innovative thought as well as have a favorable impact on future publications. As expected, the sequencing technologies supported emphasize NGS as well as microarray technologies. A significant majority of requests involve data produced in-house. A substantial number of requests require external consultation with experts outside the core. Both ABCC and CCR bioinformatics personnel have provided critical expertise in regularly scheduled prioritization, status, and science meetings.

DESCRIPTION (provided by applicant): Obsessive-compulsive disorder (OCD) is one of the most common and incapacitating psychiatric disorders. Currently, the cause of OCD is unknown, and while treatments are available, there is no specific cure. Therefore, understanding the molecular and genetic mechanisms leading to OCD will be critical for the development of effective treatments. This is a collaborative proposal to study OCD at the molecular and patient levels. Thus far, human genetic studies in OCD have neither identified a genetic locus with a major effect using linkage studies nor found common variants associated with the condition using GWAS studies. Given the technological advances that permit large-scale sequencing, there is now the opportunity to detect putative rare functional mutations for this genetically complex condition. In this application, we propose to directly test the hypothesis that rare variants influence this disease by using cutting-edge next generation whole-genome sequencing technologies. The efficiency of identifying variants relevant for OCD will be increased by sequencing the 150 cases in large, multiplex families that are most likely to exhibit a rare mutation. Variants will be prioritized using a Bayesian multi-variant liability regression model based upon their frequency in normal controls, their functional annotation, their conservation, their enrichment in cases, and their genomic location with regard to linkage peaks and GWAS signals. The multiplex families in the study will be recontacted and assessed to identify additional relatives who had not passed through the age of risk at the time of the initial family assessment. Identifying additional affecteds will improve the sensitivity of the linkage analysis in each family, this enhancing the prioritization of variants. Subsequently, 8,000 candidate variants will be genotyped in an independent sample of 800 unrelated OCD cases and 750 controls, as well as two relatives in each multiplex family, using a large-scale iSelect chip, to identify potentially causative variants. Additionally, implicated genes will be sequenced using the MiSeq platform to identify cases with different causal variants within the same genes. If successful, these studies will open up the field for neurobiology and human genetic studies of OCD and will provide insight for new strategies to develop more effective treatments for OCD.

DESCRIPTION (provided by applicant): The wealth of information provided by whole-exome and whole-genome sequencing studies can make it difficult to identify variants that cause disease. Family sequencing, in particular the sequencing of trios and quartets with affected children and unaffected parents, allows one to exclude most of the rare variants present in the genome and focus on those few that are de novo or rare and homozygous only in the offspring. There is growing recognition that this is a productive approach in the study of complex diseases. Here, we propose to perform whole exome sequencing in families to identify genetic variants predisposing to obsessive-compulsive disorder (OCD). OCD is among the 10 most disabling medical conditions worldwide and involves persistent, intrusive, senseless thoughts and impulses (obsessions) and repetitive, intentional behaviors (compulsions). The lifetime prevalence of OCD is estimated at 1-3%. Although medication and behavioral therapy, available since the late 1980s, are useful, they control symptoms with only limited success, and the course of the condition remains chronic in most cases; cure is rare. It is therefore critical to understand the pathophysiology of OCD so we can ultimately develop effective treatments. We propose to sequence the exomes of 375 simplex OCD trios and 100 OCD quartets comprising affected sibs and unaffected parents. In the trios, we will identify de novo variants that are annotated as functional. Even if de novo variants are found to explain only a small proportion of OCD cases, their discovery will provide a substantial advance in understanding the pathophysiology of the disorder. Furthermore, we will analyze the distribution of de novo variants found in the trios, comparing this distribution to that expected in the general population and that found in trios from other psychiatric diseases. In the quartets as well as the trios, we will identify causal variants that are homozygous or compound heterozygous in the offspring but heterozygous in the parents. We will identify candidate variants falling into these categories and will prioritize them based upon functional annotation, rarity and overlap of implicated genes between unrelated families. We will then confirm these variants by genotyping them in unaffected family members. Finally, we will identify genotype-phenotype correlations by looking for characteristics that are shared between unrelated cases with causal variants in the same gene and also by looking for genetic similarities within clearly defined clinical subgroups of patients, such as those with tic disorders.

The CCR Collaborative Bioinformatics Resource (CCBR) is an organizational umbrella which provides a mechanism for CCR researchers to obtain many different types of bioinformatics assistance to further their research goals. This entity pulls together many different bioinformatics expertise within NCI, with strengths in a broad range of bioinformatics topics. The CCBR currently is providing support to over 80 Principal Investigators within CCR and are working on 120 projects related to several different general areas, including: 1) Data Mining: Analyzing public microarray data for Merkel cell carcinoma 2) Basic/Bench Research: Study of alternative splicing function of Rbfox1 in knock out and transgenic mice and Disruption of Pol II Elongation with O-GlcNAcylation Inhibitors 3)Translational Research: Genomic characterization of mouse model for GBM and Gene expression analysis of Kras-induced lung cancer mouse model 4) Clinical Research: Germline Exome-Seq analysis of Familial Non-Medullary Thyroid Cancer and Exome-Seq analysis of adrenocortical cancer germ line and tumor DNA

Development of Novel Affinity Reagents. During the past fiscal year, the Office of Science and Technology Resources (OSTR) has continued to support an initiative for generating high affinity reagents to serve as tools for molecular interrogation of pathways that become altered during cancer development. Partnerships with three biotechnology companies (Beckton Dickinson, Pharmingen, Rockland Immunochemicals, and Epitomics) have led to the creation of nearly 200 novel affinity reagents, polyclonal, and monoclonal antibodies against hundreds of key cancer related targets of interest to the CCR at minimal cost to CCR investigators. These partnerships represent savings of more than one million dollars to CCR and the NCI. All of the antibody characterization data generated by participating CCR investigators is made available to the companies involved through a Web site developed in a collaboration between the OSTR and the Advanced Biomedical Computing Center (ABCC). Each company uses this data in their marketing material when the antibodies are released for commercialization. Our partnership with Epitomics is for production of rabbit monoclonal antibodies. These antibodies are known to be much more stable and have higher affinities than mouse monoclonal or rabbit polyclonal antibodies. The OSTR negotiated a contract with the company to access their antibody production pipeline at a significantly reduced cost ($5,900 versus $20,000) from the catalog price. Similar to the other collaborative programs, CCR investigators will be required to characterize the antibodies and make data available to Epitomics for validation and commercialization. The OSTR is supporting a number of protein interaction screens in mammalian cells for CCR investigators through the Laboratory of Protein Characterization at the Frederick National Lab (FNL). The OSTR continues to evaluate all commercially available platforms gene expression profiling and other high throughput genomic technologies. To make the latest advances easily accessible to CCR researchers, the OSTR has established a number of arrangements with companies providing genomic and metabolomic profiling technologies and services (Affymetrix, NanoString, Agilent, Illumina, GenUs, Qiagen, HMT, Metabolon, etc.). These agreements have led to far lower cost and an efficient and easy way for CCR investigators to access these technologies. A subsidy program has been established providing subsidies to support CCR investigator access to advanced genomic, proteomic, metabolomic, and bioinformatic technologies. This funding mechanism has helped to extend the limited research dollars available to CCR laboratories and has permitted our researchers to conduct experiments that might otherwise be cost prohibitive. Many of the funded projects have led to important publications in top tier journals. Most recently, the OSTR has been involved in CCR's efforts to access the next generation sequencing technologies, including the setting up of a new MiSeq Core in building 37, which is available to any CCR investigator. In response to CCR's rapidly growing need to manage and analyze large sets of genomic and proteomic data, the OSTR has developed several partnerships with bioinformatics companies. At significant cost savings to NCI, OSTR negotiated a variety of license agreements on behalf of all NCI researchers for universal access to sophisticated tools for bioinformatic and statistical analysis of microarray experiments, proteomic profiling studies, and genomic analysis including next generation sequencing data. In addition, the OSTR has made a number of software applications for the investigation of pathways and biological association networks available to all of NCI. The OSTR continues to assess CCR's requirements in the area of bioinformatics and biostatistics. We continue to work with the Leidos Information Systems Program to create a CCR dedicated Bioinformatics Program to provide support to CCR investigators. Support for analysis of high throughput genomic and proteomic data is being made available through this new core, which is located on the Bethesda campus.

PI: Varghese, Philip L.
Proposal Number: 1438530

The goal of the proposed research is to develop an improved method to compute flows under conditions where the conventional continuum equations are invalid. If successful, the work would result in solution strategies for a broad range of applications where continuum equations fail. The work proposed will advance the state-of-the-art for hybrid flow simulations that are important in design and simulation of wafer fabrication machines, plasma processing equipment, and micro-sensors.

The continuum approach to fluid mechanics reaches its limitations in rarefied flows and in micro- and nanoflows. A commonly used approach for computing non-continuum flows is the direct simulation Monte Carlo (DSMC) method, which can be thought of as providing a statistical representation of solutions of the Boltzmann equation by modeling the behavior of a large number of the molecules in the flow. This proposal is about a novel scheme to solve the Boltzmann equation numerically that has the potential to alleviate many of the limitations of DSMC, including feasibility for large scales. The proposed work is based on a new idea on how to compute the collisional integral, which appears in the Boltzmann equation and has been the reason for inefficient solutions and approximations to-date, using a projection into a discrete yet conservative velocity space. This approach can be viewed as a variation of DSMC that uses variable-mass fixed-velocity quasi-particles, rather than fixed-mass variable-velocity particles. Preliminary results show good agreement with theoretical predictions. The software developed during this project will be given to other academic and government laboratory users on request. The research center within the Institute for Computational Engineering and Sciences at UT Austin will be utilized for dissemination - it has established high standards for software documentation and code verification and validation.

This award by the Fluid Dynamics Program of the CBET Division is co-funded by CIF 21 Software Reuse Venture Fund Program of the CISE/ACI Division.

DESCRIPTION (provided by applicant): The primary goal of the Epi4K Center Without Walls is to increase understanding of the genetic basis of human epilepsy in order to improve the well-being of patients and family members living with these disorders. This improvement will come in the form of better diagnostics, treatments and cures. To accomplish this goal, Epi4K aims to analyze the genomes of a large number of well-phenotyped epilepsy patients and families collected by investigators from several major research groups. The specific goals of this core (3 of 7 - Sequencing, Biostatistics, and Bioinformatics Core) are to 1) sequence and annotate 4,000 genomes, 2) develop computational procedures for calling CNVs in whole exome data, 3) identify and prioritize variants of interest for all three projects, 4) conduct follow up genotypin analyses in a cohort of additional cases and controls, and 5) quickly and efficiently share data among the Epi4K consortium.

? DESCRIPTION (provided by applicant): The overarching goal of this study is the development of a framework to identify causal regulatory mutations in patients with serious neuropsychiatric presentations through the paired analyses of whole genome sequence and high resolution RNA sequence data from both accessible primary cells (PBMC and buccal cells) and reprogrammed neuronal cells from the same patients. The latter constitutes a particularly exciting opportunity as it will allow us to assay gene expression during different developmental stages of previously inaccessible cell types of much greater relevance to patient phenotype than the circulating cells DNA studies are customarily performed on. Specifically, we will interpret the effects of the regulatory variants in different developmental stages of the reprogrammed neuronal and other cells of interest with a comparison to the PBMC and buccal cells helping to interpret the specificity or generality of the relevant effects in different tissues. These analyses will focus nt only on mapping cis- and trans-acting eQTLs, but will also deploy new variant prioritization schemes that integrate knowledge of regulatory regions of the genome through ENCODE and related efforts as well as population genetic data. While an explicit aim of the work is to identif regulatory variants influencing risk of schizophrenia and autism, we emphasize that this work has primarily the broader goal of the development of appropriate frameworks for the eventual identification of such mutations, which inevitably will require substantially larger sample sizes that currently feasible to facilitate systematic discovery.

? DESCRIPTION (provided by applicant): Despite the recognition that genetic factors play an important role in susceptibility to alcohol dependence (AD), few genes have been identified to date that have been replicated across studies. Because alcohol dependence is a common disorder affecting one in five men and one in twelve women, the prevailing thought has been that common genetic variants (those with minor allele frequency over 3% in the population) would be the appropriate place to look for susceptibility variants. The common disease/common variant hypothesis has provided the impetus for a large number of genome-wide association studies (GWAS) where a large number of common variants have been assessed. While some studies have reported findings meeting genome-wide significance, often the findings for AD cannot be replicated in other studies. Also, across a wide variety of diseases, it is now recognized that only 2-4% of the variance in disease outcome is explained by these common variants. Recent evidence suggests that variants that are rarer may be more penetrant and potentially explain a greater proportion of disease outcome. Members of families in which multiple cases of AD are seen are most likely to carry a burden of rare and highly penetrant risk variants. Using existing DNA collected from members of high density AD pedigrees, we will use next generation exome sequencing and novel bioinformatics tools to identify rare variation in these high risk individuals and examine the distribution of variants in the families in collaboratin with the Center for Human Genetic Variation at Duke. Aim 1 provides a discovery stage and is facilitated by the use of a subset of pedigrees in which DNA is available for parents and affected offspring. Aim 2 provides confirmation of the discovered variation using a larger set of pedigrees in which the existence of the variants and their distribution within the full pedigree will be determined. By focusing on individuals with a strong genetic burden with multigenerational involvement and including the entire exome, we expect to find rare highly penetrant variants that would be undetectable with other approaches. A search for early developmental indicators of AD susceptibility has revealed both clinical (e.g., presence of childhood conduct disorder) and biological endophenotypes (e.g., reduced amplitude of the P300 component of the event-related potential). In Aim 3,we will examine our newly discovered rare variants in a third generation sample of individuals followed from childhood through young adulthood and for whom developmental trajectories of P300 amplitude are available. This sample includes both high-risk offspring from multiplex for AD families and low-risk controls providing an opportunity to find variants associated with P300 trajectories, an endophenotype that is highly associated with AD.

Summary Our project brings together the large NewYork-Presbyterian (NYP) health system with its affiliated Medical Centers ? Weill Cornell Medicine (WCM) and the Columbia University Medical Center (CUMC), as well as the CUMC partner, Harlem Hospital (HH), a community-based public health facility, to achieve the aims of the national Precision Medicine Initiative (PMI). We will leverage our pre-existing Hospital Network Infrastructure to enroll at least 150,000 patients over 5 years from our ethnically diverse New York City population into a cohort named the NYC Precision Medicine Initiative cohort (NYC-PMI). We will collect and pre-process appropriate biospecimens from all participants including blood sample, saliva sample, and a urine sample. We will extract all electronic health record data for our participants, and provide standardized physical exam, survey data and all other phenotypic data as required for the national cohort to ensure harmonization with PMI data standards. Our HPO will also perform real-time epidemiological modeling of the characteristics of the participants enrolled and adjust enrollment to meet the goals of the National Cohort. Finally, we will actively engage community members, leaders, and organizations throughout New York City to ensure that the aims of the National PMI are appreciated by the broader community and that participants understand the initiative, its benefits and the importance of remaining engaged through their lifespan. We will bring into the national study a demographically diverse cohort that will feature a broad range of rare and common clinical disorders for which we provide expert care at our HPO. Thus, we will not only enroll the requisite number of participants and contribute diversity to the national cohort, but we will provide the clinical and research expertise necessary to make effective use of genomic data. We have long-standing experience with community engagement in research, and have long-standing experience collaboratively in cooperative NIH grant activities to ensure that the overall scientific aims of the PMI are achieved.

Over the last year the CCR-SF has sequenced several hundred samples for dozens of different NCI principal investigators, generated Terabases of mappable data, and has completed numerous projects. Sample types that have been successfully sequenced include: whole genomic DNA, exome capture DNA, Chromatin Immuno-Precipitated DNA (ChIP),DNase HS DNA, 4C DNA, mRNA, short hairpin DNA, microRNA and Bisulfite samples. The labor intensive library preparation step is the major workflow bottle neck. To reduce library preparation costs and increase library preparation throughput and the efficiency of laboratory operations, the CCR-SF team has implemented Beckmans SPRIworks Fragment Library System (SPRI-TE). The SPRI-TE has the capability to produce three times the number of libraries prepared by a technician, uses a tightly controlled micro fluidics system to reduce manual liquid transfer variability, and the reagent costs are currently at or below the cost of the Illumina library preparation reagents before optimization. CCR-SF bioinformatics has implemented standard analyses on all applications. These analyses have been incorporated into an automation pipeline providing additional time to bioinformatics staff for optimizing the data quality and automation process. CCR-SF bioinformatics takes all new customers through an Introduction to Illumina session complete with a document for future reference. This course has been given across NCI. The CCR-SF Quality Management team has implemented a robust QC workflow to help identify potential failures as early as possible further conserving resources and increasing overall efficiency.

PROJECT SUMMARY/ABSTRACT The overarching goal of this study is the development of a framework to identify causal regulatory mutations in patients with serious neuropsychiatric presentations through the paired analyses of whole genome sequence and high resolution RNA sequence data from both accessible primary cells (PBMC and buccal cells) and reprogrammed neuronal cells from the same patients. The latter constitutes a particularly exciting opportunity as it will allow us to assay gene expression during different developmental stages of previously inaccessible cell types of much greater relevance to patient phenotype than the circulating cells DNA studies are customarily performed on. Specifically, we will interpret the effects of the regulatory variants in different developmental stages of the reprogrammed neuronal and other cells of interest with a comparison to the PBMC and buccal cells helping to interpret the specificity or generality of the relevant effects in different tissues. These analyses will focus nt only on mapping cis- and trans- acting eQTLs, but will also deploy new variant prioritization schemes that integrate knowledge of regulatory regions of the genome through ENCODE and related efforts as well as population genetic data. While an explicit aim of the work is to identif regulatory variants influencing risk of schizophrenia and autism, we emphasize that this work has primarily the broader goal of the development of appropriate frameworks for the eventual identification of such mutations, which inevitably will require substantially larger sample sizes that currently feasible to facilitate systematic discovery.