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The Methods and Analysis Core will have three functions: 1. to supply, operate and maintain the specialized equipment used in individual Research Projects for monitoring a variety of overt circadian rhythms in rodents and humans and to implement standard procedures for their computer storage. 2. to provide investigators in the individual Research Projects with standard methods for reporting and analyzing temporal profiles of activity, temperature, sleep stages, hormonal levels, and behavioral variables. In particular, the Core will provide novel methods for statistical analysis that are specially designed for studies with multiple outcome measures, as are all studies proposed in the four individual Projects. 3. to organize and operate a centralized data bank in order to allow for comparisons between standardized recordings of sleep, body temperature, wrist activity and performance tests among subject groups in individual Projects #1 and #2 as well as for comparisons between standardized recordings of activity and other behavioral measures in rodents in individual Projects #3 and #4. The services provided by this Core Facility will be used by all four individual Research Projects. The availability of a centralized pool of monitoring equipment will prevent unnecessary equipment duplication and provide the individual investigators with expert assistance for optimal operation and trouble-shooting of the equipment. The availability of standardized procedures for data reporting and analysis will enable each investigator to extract as much information as possible from the collected data and facilitate the integration of individual projects as well as communication between investigators in the various Research Projects. The operations of a centralized data bank will permit to perform additional comparisons of various study groups across Projects and thus derive more information from the Program Project as a whole than from the sum of each of its components.

Fertility in females requires controlled maturation of the oocyte, supporting granulosa cells (GCs), and thecal cells that comprise the ovarian follicle. Follicle growth is a dynamic process that demands exquisite regulation. Follicles are restrained at the preantral stage until they are stimulated by the pituitary hormone follicle stimulating hormone (FSH). In response to FSH GCs produce steroid and protein hormones and growth factors that regulate the hypothalamic/pituitary axis and uterine receptivity, and promote oocyte maturation and development of the follicle to a preovulatory phenotype. All of the documented responses to FSH are mediated via cAMP and it's predominate intracellular target, cAMP-dependent protein kinase (PKA). Indeed, GCs offer one of the best examples of a cellular model whose responses are orchestrated by PKA. Signaling by PKA is confined to specific locations in cells by virtue of a family of A-kinase anchoring proteins (AKAPs) that localize pools of PKA, their substrates, and interconnected signaling enzymes. This application focuses on the mechanisms by which PKA integrates transcriptional networks to imitate maturation of GCs. PKA accomplishes this integrating function by phosphorylating substrates that directly regulate transcription or by regulating pathways whose targets regulate transcription. The co-activator beta-catenin is emerging as one potential PKA substrate necessary for activation of a subset of FSH target genes. PKA also phosphorylates an unidentified substrate that directs activation of the phosphatidylinositol-3 kinase (PI-3K) pathway fundamental to GC survival, proliferation, and differentiation. Among the many PI-3K pathway targets, the transcriptional factor FOXO1 (forkhead box O factor 1) requires phosphorylation/inactivation to permit induction of at least a subset of FSH target genes. We postulate that PKA phosphorylates both beta-catenin to activate its co- activator activity and a substrate to direct activation of the PI-3K pathway to activate and inactivate a network of FOXO1-regulated target genes. Aims test the following hypotheses: that a specific AKAP targets a pool of PKA to a multi-enzyme complex that directs activation of PI-3K;that induction of FSH target genes like Lhcgr requires activation of beta-catenin;and that the PI-3K pathway target FOXO1 regulates a network of direct target genes that maintain GCs in an immature stage. Understanding how FSH signals to direct follicular maturation can translate into safer and more effective treatments of infertility and early pregnancy loss as well as new approaches for contraceptive drugs. PUBLIC HEALTH RELEVANCE: FSH signaling to mature follicles to the preovulatory phenotype is required for fertility. Understanding how FSH signals to direct follicular maturation can translate into safer and more effective treatments of infertility and early pregnancy loss as well as new approaches for contraceptive drugs.

Fetal alcohol syndrome is a significant preventable health problem world wide. Studies in humans and animals suggest that prenatal ethanol exposure (E) also disrupts the normal development of the immune system, resulting in immune disfunction that may persist well into adult life. Data also indicate that stress can have a profound negative impact on immunocompetence. The proposed research is the first to investigate the interactive effects of prenatal ethanol and postweaning stress on immunocompetence. We have shown that E results in: 1) long-term effects on offspring immunocompetence; 2) alterations in the hypothalamic-pituitary-adrenal (HPA) response to stress, Our preliminary studies demonstrate interactive effects of prenatal ethanol and stress in adulthood on lymphocyte populations. The proposed research will focus on one critical aspen of the immune response, T cell--B cell interactions. The ability of T helper cells to facilitate immunoglobulin production by B cells, and conversely, the ability of B cells to respond to T lymphocyte-derived activation and proliferation signals, will be assessed. In adulthood, offspring will be subjected to a 3 week chronic intermittent stress regimen or will remain undisturbed. Experiments in which mitogen-activated T cell supernatants from both prenatally treated and control animals are cultured with activated B cells from both prenatally treated and control animals will allow us to determine if the potential defect lies on the T cell or the B cell side of this system, or if both cell types are functioning abnormally. Our working hypothesis is that prenatal ethanol will cause abnormal T cell--B cell interactions, via either defective T cell activation or aberrant lymphokine production by activated T cells. Our experimental paradigm will also enable us to determine whether B cells are affected. We also hypothesize that stress will exacerbate the immunosuppressive effects of prenatal ethanol, and based on our previous data, that males will be more affected than females by exposure to prenatal ethanol and stress. This approach will increase our understanding of two fundamentally important issues: the nature of the immunoteratogenic effects of ethanol, and the possible role of stress in exacerbating these effects.

Lyme disease was once restricted to coastal North Atlantic and selected Upper Midwest communities, but the distribution and prevalence of this zoonosis has greatly expanded in the last two decades. The aggressively human biting deer tick vector of the agent of Lyme disease was first recognized to maintain a distinct lineage of Powassan virus (POW) in 1997, but human encephalitis cases attributable to ?deer tick virus? was recognized only in 2006. Severe neurologic disease, a hallmark of classical Powassan fever, remained rare in residents of Lyme-endemic sites until recently. Encephalitis cases attributed to Powassan virus are now being increasingly reported from New England and the upper Midwest. The biological basis for the recent zoonotic emergence of POW requires analysis. The Eurasian tick borne encephalitis virus complex (TBEV) comprises diverse species (including POW), subtypes and geographic isolates that vary in their capacity to cause human disease, and there is a rich literature concerning variation in the genetic, phenotypic and clinical characteristics of Eurasian TBEV that should guide our analysis of the potential public health burden of POW in North America. Our overaraching hypothesis is that Powassan virus, like TBEV, also comprises lineages, genotypes, or populations that differ in capacity to cause human disease. We propose to test this hypothesis using our existing geographic isolates of POW as well as additional isolates that we shall collect for their capacity to cause neurologic disease using a published mouse model for TBEV neurotropism. In addition, it may be that certain lineages, genotypes, or populations of POW are more efficiently transmitted by the human biting deer tick vector of Lyme disease, and this helps to explain the emerging epidemiological situation. Accordingly, we shall also determine whether geographic isolates may differ in their capacity to infect and be transmitted by ticks. Finally, we shall apply the powerful tools of whole genome sequencing to analyze the population structure of Powassan virus as well as identify any genetic correlates of neurotropic capacity. Taken together, these observations may help explain the changing epidemiology of an arbovirus that has been silently enzootic for over 2 decades in Lyme disease endemic sites, but is now apparently an emerging zoonosis. Ultimately, by describing the pathobiological correlates of Powassan genetic diversity, we can better define the potential for tick borne encephalitis to continue to emerge as a public health burden in North America.

The proximal action of parathyroid hormone (PTH appears to be activation of cyclic AMP formation in its target tissues, bone and kidney. Prostaglandin E1, a potent stimulator of bone resorption, also activates cAMP production. In addition, there are a number of other hormonal and pharmacologic agents which affect calcium metabolism, but do not directly activate adenylate cyclase or stimulate cAMP accumulation in bone or kidney. These include thyroid hormone, gonadal steroids, adrenocortical steroids, and benzothiadiazine diuretics. The proposed studies investigate the possibility that these agents modulate the endorgan responses to PTH and PGE1 through an interaction on cAMP formation. BIBLIOGRAPHIC REFERENCES: Marcus, R. Cyclic Nucleotide Phosphodiesterase from Bone: Characterization of the Enzyme and Studies of Inhibition by Thyroid Hormones Endocrinology 96:400, 1975. Marcus, R., C. Lundquist, and I.J. Chopra. In vitro inhibition of cyclic nucleotide phosphodiesterase by thyroid hormones. Abstract #470 Program of the 57th Annual Meeting, The Endocrine Society, New York, June 18-20, 1975.

Reactive hyperemia is a normal physiologic event in response to an abrupt total obstruction of blood flow. In the myocardium this event has been noted for years but only recently has the technique been developed that allows the time related transmural blood flow pattern to this insult to be documented. Two groups of dogs were studied using radioactive microspheres to determine the tramsmural myocardial blood flow during reactive hyperemia. The unrestrictive group, group I, underwent acute occlusion of the circumflex, coronary artery for 15 seconds and transmural flow was determined at 5 seconds, 45 seconds, and 90 seconds after release of the occlusion. The restrictive group, group II, was the same as group I, but in addition had a partial occlusion of the circumflex coronary artery that maintained the distal coronary pressure at approximately 80 mm. Hg mean pressure. Transmural blood flow pattern of group I revealed that both epicardium and endocardium underwent reactive hyperemia of identical magnitude and that the time of peak flow in each was identical. Furthermore, the endocardial/epicardial flow ratio remained normal throughout the reactive hyperemic response. Preliminary data from group II would indicate that the epicardial flow is greater in magnitude and earlier in time than that of the endocardium which is of lesser magnitude and later in time in the reactive hyperemia response.

A hallmark of immunity is its longevity, manifested by the durability of memory B cells (Bmem ) and plasma cells (PC).Through the analysis and isolation of extremely rare antigen-specific Bmem populations, we have scrutinized their functional responsiveness and their transcriptome. Such genetic analyses and functional studies in vivo have opened up new biological leads into the cells and factors that govern the fate of Bmem. The future five years will be devoted to new exciting initiatives in Bmem biology. Over the last funding period, we have: 1) Defined the role of BCR affinity in B cell fate, 2) Developed new strategies to isolate and study Bmem, 3) Resolved the role of BAFF family members in Bmem and PC survival;4) Shown that Bmem can be selectively primed relative to PCs, 5) Identified a lineage commitment factor, Id2, for Bmem, 6) Discounted the contribution of TLR stimulation in Bmem persistence and 7) Identified CCR6 as an important chemokine receptor in Bmem migration and function. Given these findings, the Specific Aims for the next funding period are: 1) To define the role of TLR signaling and bystander T cell help in Bmem survival. The prevailing paradigm is that TLR signaling or bystander T cell help sustains Bmem longevity. Our preliminary data shows that TLR agonists in vivo do not induce Bmem cycling, but instead induce terminal Bmem differentiation. Studies are proposed to comprehensively evaluate TLR and T cell signaling on Bmem fate. 2) Continue studies to understand the role of Id2 as a lineage commitment factor in the differentiation of Bmem. Transcriptional profiling has shown that Bmem over-express Id2. Id2 is a nuclear factor that antagonizes transcription. Studies in ld-2-deficient mice (B-restricted) confirm a selective defect in the Bmem compartment. Studies are proposed to fully evaluate the role of Id2 in post-germinal center B cell differentiation and Bmem in mice where Id2 can be conditionally regulated. 3) Resolve the functional significance that Bmem are selectively responsive to CCR6 stimulation. An hypothesis is presented that selective responsiveness to CCR6 may be central to the longevity of Bmem. Our understanding of Bmem and PC biology is central to the development and improvement of vaccines used to enhance the public health. While vaccines are undoubtedly the most successful form of immunotherapy to date, a broader and more molecular understanding of the factors and cells that govern the durability of immunity is well warranted.

Cancer cells exhibit alterations in parameters of nuclear architecture that control cell fate and compromise control of cell groyvth. Our Program has established new biological paradigms by showing that gene regulatory factors integrate cell signaling at chromatin microenvironments ('subnuclear foci') and support epigenetic mechanisms through association with mitotic chromosomes. In collaboration with other Program Project investigators. Project 1 will now establish new dimensions in gene regulation by defining perturbations in architecturally linked regulatory mechanisms during interphase and mitosis in AML and breast cancer cells. Our central hypothesis is that (i) subnuclear targeting of transcription factors to gene regulatory foci during interphase and (ii) the association of transcription factors wifh their target genes in mitotic chromosomes are fundamental to the retention of biological states of normal and cancer cells. Therefore, we will use IF microscopy, biochemical, genomic and proteomic approaches (i) to characterize modificafions in architectural epigenetics and molecular pathological consequences of expressing the translocation-related t(8;21) AML-ETO fusion protein (Aim 1), (ii) to analyze genes that are transcriptionally and spatially controlled by Runx2 in chromatin micro-environments ('subnuclear foci') during interphase in breast cancer ceils (Aim 2), and (iii) to examine Runx2 mediated architectural epigenetics in breast cancer cells by characterization of Runx2 and cognate gene regulatory factors that associate with mitotic chromosomes (Aim 3). By investigating the functional role of Runx2 in establishing chromatin micro- environments ('subnuclear foci') during interphase and architectural epigenetics in cancer cells during mitosis, we will challenge traditional biochemical views of gene regulation by defining the pathological linkages between modifications in nuclear architecture and gene expression that are fundamental to the molecular etiology of tumorigenesis. RELEVANCE (See instructions): Changes in the overall shape and structure ofthe nucleus are pathological hallmarks of cancer cells that are linked to cellular transformation. This study will use state-of-the-art methods to characterize how targeting of oncogenic transcription factors to specific subnuclear structures and mitotic chromosomes supports gene regulation as components of a novel epigenetic mechanism ('architectural epigenetics').

Certain neuron classes in the cerebral cortex exhibit heightened vulnerability in Alzheimer's Disease, whereas others are relatively resistant to pathology. For example quantitative neuroanatomic analyses suggest that large pyramidal cells that furnish long corticocortical projections and reside in layers III and V may be particularly vulnerable to degeneration and/or neurofibrillary tangle and neuritic plaque formation. The experiments outlined in this project are directed at the development of a detailed molecular/anatomic profile of these pyramidal neurons. In addition, investigation of the cellular pathology of cerebral cortex in other dementing illnesses will be initiated, in order to determine the degree to which other dementias can be traced to the loss of the same subset of cortical cells and connections, regardless of the etiology. A common neuroanatomic denominator for dementia would greatly facilitate attempts to understand the cellular basis of dementing illnesses, and in turn, aid in directing therapeutic and preventative efforts to the appropriate cell class. These studies will involve detailed quantitative neuroanatomic analyses of non-human primate as well as neurologically normal and diseased human cortices. The anatomic methods employed will be chemically-specific histochemical procedures that allow for the correlation between biochemical or molecular data and cellular localization. The biochemical systems to be investigated include, cytoskeletal proteins, calcium-binding proteins, amyloid-beta-protein, and the excitatory neurotransmitter glutamate. The quantitative neuroanatomic analyses rely on computer-assisted microscopy and involve studies of cell distribution as well as morphologic and intracellular characteristics of individual neurons. An important underlying assumption in this proposal is that the selective vulnerability in Alzheimer's Disease is related to the molecular and anatomic heterogeneity of the neuronal constituents of cortex, and that a specific anatomic/molecular profile renders a cell vulnerable. If the elements of the biochemical and anatomic phenotype that are most clearly linked to differential cellular vulnerability in AD can be determined then it will be possible to develop means to protect those neurons that degenerate in AD. The protection of these neurons must be the paramount goal in developing a strategy for the management of AD, since prevention of a neurodegenerative disease is much more likely to be achievable than the development of a cure.

Photodynamic therapy (PDT) with the phthalocyanine photosensitize PC 4 causes rapid generation of reactive oxygen species (ROS), mitochondrial permeability transition (MPT), depolarization and swelling, release of cytochrome c, and activation of both necrosis and caspase-dependent apoptosis in tumor cells. However, PC 4 and a recently synthesized PC 4 derivative PC 181 also localize to endoplasmic reticulum (ER) and lysosomes. We hypothesize that damage to these organelles by PC 4 and PC 181 leads to additional perturbations, such as Ca2+, iron and protease release that ultimately promote MPT-dependent cell killing after PDT. Our goal is to further characterize the role of the MPT in PDT-induced killing of cancer cells and to determine the interactions of damage to ER and lysosomes in promotion of death pathways. In Aim 1, we will characterize changes of cytosolic, mitochondrial and ER Ca2+ after PDT using compartmentally localized fluorescence indicators and Confocal microscopy. Using specific interventions, we will determine whether depletion of ER Ca2+ stores, inhibition of mitochondrial Ca2+ uptake and intramitochondrial chelation act to suppress MPT after PDT and prevent subsequent cell killing. We also determine whether increased cytosolic Ca2+ may activate Ca2+-dependent calpains, to cause Bid processing and translocation to mitochondria with con- sequent mitochondrial dysfunction, MPT, and cell death. In Aim 2, we will investigate the contribution of lysosomal iron and protease release in relation to the MPT and cell death. We will measure cytosolic and mitochondrial chelatable iron using specific fluorescent indicator and determine the effects of compartmentally loaded iron chelators. Similarly, we will assess protease release into the cytosol and investigate the protection conferred by specific cathepsin indicators and knock-out cells. We expect iron and protease release from lysosomes to synergistically promote mitochondrial permeabilization by activating Bid cleavage and translocation to mitochondria. In Aim 3, we will investigate strategies to enhance PDT toxicity in cultured cells and in isolated mitochondria. We will evaluate the ability of salicylate to decrease the threshold of MPT onset and to enhance tumor killing after PDT. The proposed studies will enhance our understanding how to enhance PDT-induced killing of cancer cells and further increase the efficacy of PDT translation to the benefit for cancer patients. [unreadable] [unreadable] [unreadable]

The goals of the proposed work are (1) to establish a role of cortico-cortical feedback in visual decision- making, (2) to examine a potential link between top-down cortico-cortical feedback such as feature-based attention and decision-related activity in visual neurons and (3) to expand the candidate's experimental expertise by optogenetic techniques in behaving primates. Achieving the third objective will complement the candidate's previous training in awake behaving primate visual neurophysiology. Achieving the first and second objective will provide a launching point for the candidate to develop an independent research career, toward a long-term goal of an improved understanding of the role of feedback in perceptual decision-making. These objectives will be accomplished in two phases. In the 2-year mentored phase, the candidate will conduct supervised research in Dr. Callaway's and Dr. Reynolds'laboratory at the Salk Institute to develop and use optogenetic techniques to characterize cortico-cortical feedback connections to V1 (Aim 1) in fixating monkeys. This training environment is uniquely suitable because of Dr. Callaway's expertise in developing genetic techniques for neurobiology combined with Dr. Reynolds'expertise in the study of attention in primates, the immediate availability of monkeys and the intellectual resources available at the Salk Institute, in molecular neurobiology, genetic techniques, viral vectors, neuro-anatomy, visual neuroscience and theoretical neuroscience. In the next 3-year phase, the candidate's independent research will test the hypothesis that decision-related activity in V1, and modulation by feature-based attention in these areas, share a common mechanism, using single unit neurophysiological recordings (Aim 2), and optogenetic techniques (Aim 3). Aim 3 will at the same time allow the candidate to examine the role of cortico-cortical feedback from V2 to V1 in visual decision-making. It represents an innovative approach to study the role of cortical circuits, a merging of established lines of research (attention and perceptual decision-making) and will advance our understanding of the neural circuitry involved in the dynamic processes of cortico-cortical feedback underlying decision making. Impaired control of cortico-cortical feedback appears to be involved in brain disorders such as schizophrenia. Understanding the role of cortico-cortical feedback may therefore inform clinical treatments for patients with schizophrenia. PUBLIC HEALTH RELEVANCE: Relevance to public health The "read-out" of visual cortex serves as a model system for interpretation, decision-making and reasoning. This area therefore sheds light on brain functions central to brain disorders affecting higher mental function, such as Alzheimer's disease, Schizophrenia and neglect. Ultimately, a better understanding of the role of cortico-cortical feedback and of how activity in cortical areas is related to perception, decisions and behavior may also allow the development of more sophisticated neural prostheses.

Drug and alcohol abusing and dependent delinquents represent a large and greatly underserved population that is at high risk of presenting significant deleterious outcomes and long-term costs for themselves, their families and communities, and society (Belenko & Dembo, 2003). As described by recent reviewers (Belenko & Logan, 2003; Cooper, 2002), juvenile drug courts began emerging in the 1980s to address these problems, and 268 juvenile drug courts were in operation by December, 2003 (Huddleston, Freeman-Wilson, & Boone, 2004). Although juvenile drug courts have continued to proliferate in recent years, evaluation of their capacity to reduce offender substance use and criminal activity has lagged. The proposed study builds on our recent research findings supporting the effectiveness of juvenile drug court and the capacity of evidence-based treatments to enhance juvenile drug court outcomes (Henggeler et al., in press) as well as taking advantage of our extensive experience in the transport of evidence-based practices to community treatment settings. The overriding purpose of this Stage 1-B Treatment Development Study is to develop and test a relatively flexible and low cost strategy for enhancing the outcomes of juvenile drug courts by integrating components of evidence-based treatments into existing substance abuse and drug court services. Specific aims include: Aim 1: Adapt existing intervention and training protocols from evidence-based practices (i.e., contingency management for treating adolescent substance abuse; family engagement strategies from evidence-based treatments of juvenile offenders) for integration into juvenile drug court sites. Aim 2: Conduct a study to examine youth (e.g., substance use and criminal behavior) and system level (e.g., feasibility, implementation fidelity, retention and completion rates, consumer satisfaction, cost estimates) effects of implementing the intervention protocols in six juvenile drug courts. Aim 3: Revise intervention and training protocols in preparation for Stage II study if findings are supportive. [unreadable] [unreadable] [unreadable]

The Matrix Biochemistry Section focuses its research on the functions of five major noncollagenous proteins first found associated with the mineralized matrix of bones and teeth but that we later showed are also made by many metabolically active ductal epithelial cells. The five proteins are bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). We have made a strong case for the genetic relatedness of these seemingly different proteins and there is increasing acceptance of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family concept. The genes encoding these proteins are all clustered in a tandem fashion within a short (400,000 base pairs) region of human chromosome 4 and similarly on all other mammals studied to date. After comparing the intron-exon structures and conserved motifs of their respective protein-encoding exons, we proposed that the five genes might be the result of ancient gene duplication and subsequent divergence. The most recent event appears to be a duplication of the DMP1 gene in the common ancestor of mammals and reptiles. Both lines then independently modified a different DMP1 gene to become DSPP-like during the evolution of modern dentin. We and others have shown that all known cases of non syndromic Dentin Dysplasia (DD) and Dentinogenesis Imperfecta (DGI) are the result of either a variety of point mutations at the very beginning of the DSPP gene or deletions later in the gene that result in frameshift mutations within the long repeat domain. In the past we proposed that all known mutations have dominant negative effects (mutations in a single copy of the gene cause the diseases but complete loss of one copy does not) but the mechanisms for this remained unexplored. Recently we have shown that all known mutations (except Y6D) cause the retention of the mutant DSPP proteins in the endoplasmic reticulum of our model system. Furthermore, we have shown that the retained mutant proteins cause the loss in the DSPP protein made from the normal allele. Mutations causing only small amounts of the normal DSPP protein to be secreted out of the cells cause the more severe disease, DGI. Our current research involves a focus on the trafficking of both normal and mutated acidic proteins within the endoplasmic reticulum (ER) as well as how the cells naturally destroy disordered proteins that fail to traffic out of the ER. Searching databases we have found that many secreted, acidic proteins encode similar peptide motifs that we propose are used to interact with a conserved ER cargo receptor. This proposed cargo receptor then traffics negatively charged proteins (as well as a variety of other proteins that have tendencies to either self-aggregate or form complexes with co-translated proteins) out of the ER before they can reach high enough concentrations to form aggregates that cause cells to malfunction.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed research is designed to determine if increased dietary iron and iron stores contribute to the development of alcholic liver disease in African Americans. Our preliminary studies indicate that high dietary iron leads to increased hepatic iron stores in both Africans and African Americans, and that incrreased hepatic iron stores are associated with hepatic dysfunction in Africans who consume alcohol. We postulate that the oxidative environment induced by alcohol in heptocytes and Kupffer cell (hepatic macrophages)leads to diassembly of the ironfur cluster of cytocolic aconitase/IRP 1 and conversion of this enzyme to an apoprotein that binds to RNA stem loops (iron repsonisve elements-IREs)in iron metabolism transcripts. This non-physiologic increase in IRE-binding activity of IRP1 in turn leads to abnormal repression of ferritin synthesis and abnormal increases in transferrin receptor synthesis and potentially toxic cytosolic labile iron concentrations. Based on our cell culture and animal model studies, we further postulate that increased non-heme iron content in Kupffer cells primes these cell for NF-kB activation and proinflammatory gene expression and thereby contributes to the pathogenesis of alcoholic liver disease in African Americans. We aim to test two central hypotheses: i)high dietary iron contributes to an alcohol induced tendency for abnormal iron-loading of cells, and ii)increased hepatic iron contrributes to liver damage in the setting of alcoholic liver disease.

This institutional T32 training application titled: "Clinical Research Training in Oral Diseases for Future Academicians" represents a response to RFA DE-05-008 from the University of North Carolina at Chapel Hill. Our Clinical Research Scholars Program (CRSP) is our School's response to the perceived shortage of dental school faculty who are qualified to conduct research that will advance knowledge in the field. The program primarily focuses on training dentists in clinical research who are also enrolled in advanced clinical education programs to prepare to become academicians who can function as interactive scientists who have the ability to address the expanding opportunities in dental, oral and craniofacial clinical research. As designed, our program embraces the full spectrum of translational clinical research including fundamental mechanisms of human disease, therapeutic intervention, clinical trials, and oral epidemiology. It includes 3 major options: 1) a 2-year postdoctoral fellowship in clinical research, 2) a 3-year PhD program in Oral Epidemiology, and 3) a 1-year Visiting Scientist (mid-career) fellowship. The program integrates Mentorship and curricula primarily from the UNC Schools of Dentistry, Medicine and Public Health. Each student is assigned multiple Mentors from all schools and is expected to write a grant application by the end of their program. The 2-year program is based on our Medical School's K30 curriculum and joint dental and medical school GCRC experiences. The PhD program earns a PhD from the Department of Epidemiology. The Visiting Scholars Program is based on the Robert Wood Johnson Clinical Scholars Program with a second year of Mentorship at their home institution. The CRSP will draw upon the existing interdisciplinary research activities within UNC that currently unites basic scientists with clinicians, clinical investigators and epidemiologists to provide a comprehensive training environment for clinical researchers. [unreadable] [unreadable]

It is the goal of this program to develop efficient, practical (within the context of the complexity of the molecules involved) syntheses of the polyether ionophores. These antibiotics function as agents for ion/amine transport and through their ability to facilitate the transport of biologically relevant ions (Na+, K+, CA2+, etc.) or amines have the capacity to alter significantly cell metabolism. Commercially valuable as coccidiostats and anabolic agents, the polyether antibiotics are also proven cardiotonic agents. Therapeutic utilization of the ionophores as human cardiotonic agents is, however, severely limited by their toxicity and residual character that prevents effective dose regimes. Access to related chemical structures that still retain the ability to chelate metal ions/amines reversibly, but can overcome some of the toxicity and metabolic stability problems associated with the natural products may lead to useful therapeutic agents. It is the purpose of this effort to develop technology that can be generally applied to the synthesis of the characteristic polyether structure and design analogs of these ionophores that through biological testing will help define the requisite structural parameters for activity. Initial targets for the synthetic methodology part of the work are monensin and nigericin and then analogs related to these antibiotics will be prepared by that technology.

Plasmacytoid dendritic cells (pDCs) represent a unique immune cell lineage that plays an essential role in innate immune responses against viruses. pDCs recognize virus-associated nucleic acids and respond by rapidly secreting massive amounts of type I interferons (IFN), hence their other name: type I interferon- producing cells (IPCs). pDCs can also differentiate, upon activation, into conventional dendritic cells that are capable of presenting antigens to the adaptive arm of the immune system. Thus, pDC are critical for the body's control of viral infections. On the other hand, abnormal pDC hyperactivity has been associated with increased type I IFN levels in autoimmune diseases such as psoriasis and systemic lupus erythematosus. Therefore, pDCs do play important roles in both normal and aberrant immune responses and represent potential useful targets in the treatment of some diseases. pDCs possess unique cellular and molecular characteristics and gene expression profiles that enable them to directly recognize viruses, secrete massive amounts of type I IFN, and differentiate into conventional dendritic cells. Over the last few years, these molecular and cellular features of pDCs have been well characterized; however, the genetic basis of pDC lineage commitment, maturation, and function still remains poorly understood. The overall objective of our research project is to study the role of basic helix-loop-helix transcription factors in the development and function pDCs, and elucidate the pathways that these transcription factors control in pDCs. PUBLIC HEALTH RELEVANCE: These studies will give us insights into the genetic network which controls the development and function of pDC, and may pave the way for the development of new approaches in the treatment of viral infections and some autoimmune disease. [unreadable] [unreadable] [unreadable]

This contract is designed to attain extramural support for developing, designing, interpreting, and evaluating clinical trials, epidemiologic and natural history studies. In addition, it will provide for outcomes research involving eye diseases and visual disorders and some preclinical studies. The focus shall be on the design of studies and the collection, analysis, and interpretation of data emanating from these studies, as well as support, and monitoring patient safety and follow-up. Contractor shall also provide analytical and data management support, as described in the work statement, for specified clinical research data bases, cost-effectiveness and economic analyses, quality of life assessment and outcomes research. This will include, but not be limited to, the following areas: analysis of Medicare and other health care databases; evaluation of existing NEI databases such as, centralized NEI Intramural Research database, the Eye Disease Case Control Study, Early Treatment Diabetic Retinopathy Study, Framingham Eye Study, and intramural AIDS and uveitis databases. The 46 active/follow-up trials in 2015 are listed below: For more info: http://clinicalstudies.info.nih.gov/cgi/protinstitute.cgi?NEI.0.html 15-EI-0202: Evaluation of Oral Minocycline in the Treatment of Geographic Atrophy Associated with Age-Related Macular Degeneration * 15-EI-0128: The Genetics of Inherited Eye Disease * 15-EI-0052: Pilot Phase I/II Study of the Evaluation of Interferon Gamma-1b Administered Topically for Macular Edema/Intraretinal Schisis Cysts in Rod-Cone Dystrophy (RCD) and Enhanced S-Cone Syndrome (ESCS) * 15-EI-0041: Patient Expectations When Enrolling in an Early-Phase Clinical Trial * 15-EI-0038: A Phase I/IIa Study of RS1 Ocular Gene Transfer for X-linked Retinoschisis * 15-EI-0020: Adaptive Optics Retinal Imaging * 14-EI-0173: An Investigation of Retinal Findings in Patients with Signs and Symptoms of Alzheimer's Disease Enrolled in 09-M-0198 * 14-EI-0171: The Focal Electro-Oculogram in Macular Disease * 14-EI-0108: Pilot Study to Evaluate Oral Minocycline in the Treatment of Cystoid Macular Edema Associated with Retinitis Pigmentosa * 14-EI-0078: A Phase 2 Multicenter Randomized Clinical Trial of Ciliary Neurotrophic Factor (CNTF) for Macular Telangiectasia Type 2 (MacTel) * 14-EI-0064: Whole Exome and Whole Genome Sequencing for Genotyping of Inherited and Congenital Eye Conditions * 13-EI-0154: Color Vision as an Outcome Measure for Clinical Trials of Inherited Retinal Degenerations * 13-EI-0124: A Pilot Study of Nitisinone in the Treatment of Oculocutaneous Albinism, Type 1B * 13-EI-0072: Microbiome and Ocular Inflammatory Disease * 13-EI-0049: Genetics of Uveal Coloboma * 12-EI-0203: Natural History of ABCA4-Related Retinopathies * 12-EI-0042: NEI Intramural Biorepository for Retinal Diseases * 11-EI-0264: A Pilot Study for the Evaluation of Minocycline as a Microglia Inhibitor in the Treatment of Central Retinal Vein Occlusions * 11-EI-0263: A Pilot Study for the Evaluation of Minocycline as a Microglia Inhibitor in the Treatment of Branch Retinal Vein Occlusions * 11-EI-0245: Generation of Induced Pluripotent Stem (iPS) Cell Lines from Somatic Cells of Participants with Eye Diseases and from Somatic Cells of Matched Controls * 11-EI-0173: The Natural History of Ocular Graft-Versus Host Disease * 11-EI-0147: Longitudinal Investigation of Dark Adaptation in Participants with Age-Related Macular Degeneration * 10-EI-0140: Genotype-Phenotype Study of Patients with Plaquenil -Induced Retinal Toxicity, with Evaluation of the ABCA4 Gene * 10-EI-0093: Immunogenetic Mechanisms in Behcet's Disease * 10-EI-0016: Internal Monitoring of Eye Movement in Schizophrenia * 08-EI-0169: Evaluation and Treatment Protocol for Potential Research Participants with Ocular Diseases * 08-EI-0102: Screening Study for the Evaluation and Diagnosis of Potential Research Participants * 08-EI-0099: Epigenetics, Molecular Genetics, and Biomarkers of Degenerative and Inflammatory Ocular Diseases * 08-EI-0043: Age-Related Eye Disease Study (AREDS) and AREDS2 Follow-Up * 08-EI-0031: Visual Motor Coordination * 06-EI-0236: National Ophthalmic Genotyping and Phenotyping Network, Stage 1 - Creation of DNA Repository for Inherited Ophthalmic Diseases * 06-EI-0068: The Vitreous Proteome and Inflammatory Mediators in Ocular Inflammatory Disease * 06-EI-0059: Clinical and Molecular Studies in Families with Glaucoma and Related Diseases * 06-EI-0058: Clinical and Molecular Studies in Families with Myopia and Related Diseases * 05-EI-0143: Molecular Genetics of Retinal Degenerations * 04-EI-0008: Clinical and Molecular Studies in Families with Corneal Dystrophy or Other Inherited Corneal Diseases * 03-EI-0123: Clinical and Molecular Studies in Families with Congenital or Hereditary Cataracts * 03-EI-0122: Evaluation of Immune Responses to Different Antigens in Non Infectious Ocular Inflammatory Diseases * 03-EI-0033: X-Linked Juvenile Retinoschisis - Clinical and Molecular Studies 13-EI-0206: A Randomized, Controlled, Double-Masked, Clinical Trial of Autologous Serum Eye Drops for Severe Ocular Chronic Graft-versus-Host Disease (GVHD) in Hematopoietic Stem Cell Transplant (HSCT) Patients * 13-EI-0102: Gevokizumab Treatment for Active Scleritis By IL-1 Inhibition (GATSBY) * 12-EI-0167: A Phase I/II Study of the NT-501 Intraocular Implant Releasing Ciliary Neurotrophic Factor (CNTF) in Participants with CNGB3 Achromatopsia * 11-EI-0244: A Pilot Phase I/II Study for the Evaluation of Dextromethorphan as a Microglia Inhibitor in the Treatment of Diabetic Macular Edema (MiDME2) * 07-EI-0213: Laboratory of Immunology/National Eye Institute Repository * 06-EI-0050: Multicenter Uveitis Steroid Treatment (MUST) Trial Protocol * 05-EI-0096: Natural History and Genetic Studies of Usher Syndrome

Incomplete respiratory neuron maturation causes significant morbidity during the perinatal period, yet the mechanisms by which respiratory neuron maturation occurs during this vulnerable time window is not understood. Thus, there is a critical need to identify these basic neural mechanisms of perinatal respiratory control. The objectives of the proposed research are to elucidate developmental processes of respiratory neuron network maturation and to identify brainstem respiratory centers/circuits necessary for perinatal breathing. The central hypothesis is that hindbrain respiratory neuron networks undergo critical developmental maturation during the late embryonic, perinatal, and post-natal periods in mammals, and that developmental abnormalities in neuronal and glial maturation contribute to the pathophysiology of autonomic respiratory neuron dysfunction. The proposed research is inspired by our group's findings of Central Congenital Hypoventilation Syndrome (CCHS), a rare human disorder characterized by an inability to sense CO2 and which is linked to PHOX2B poly-alanine repeat and non-polyalanine repeat (NPARM) mutations. The rationale for the proposed research is that the lack of a basic fundamental understanding of which autonomic neural circuits are required for perinatal breathing represents a barrier to the ultimate implementation of interventions aimed at improving morbidity for premature infants. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the extent to which selective expression of a dominant negative NPARM-PHOX2B mutation regulates perinatal chemosensation-induced respiratory drive, 2) Determine which brainstem circuits are lost in NPARM-CCHS, and 3) Determine the extent to which selected ablation of brainstem astrocyte population promote congenital hypoventilation. Under the first aim, we will test the effects on ventilation control and brainstem anatomy after targeted brainstem expression of a dominant negative NPARM PHOX2B mutation using an already proven conditional transgenic mouse approach. In the second aim, we will combine an innovative transgenic approach to identify which brainstem circuits are lost in congenital hypoventilation. In the third aim, we will determine the extent to which neuronal-glial interaction are necessary for appropriate autonomic respiratory control in the perinatal and post-natal period. The approach is innovative because it uses novel and validated tools, techniques, and reagents from distinct disciplines that allow us to address previously unanswerable questions. The proposed research is significant, because it is expected to vertically advance and expand understanding of which neuronal-glial circuits are required for proper control of autonomic regulation of breathing at birth. The tools and basic knowledge gained from these studies will form the foundation of future studies where interventions to improve autonomic respiratory neuron function in premature babies are designed and validated.

DESCRIPTION: (Adapted from the application) Critical to a better understanding of the pathogenesis of HIV-associated lung disease is an animal lentivirus model that parallels HIV-associated immunodeficiency. Evidence suggests that the feline AIDS-causing lentivirus, Feline Immunodeficiency Virus (FIV) causes serious pulmonary immunodeficiency characterized by increased susceptibility to Toxoplasma gondii pneumonia. The proposed research will employ the use of FIV for examining the defects in alveolar macrophage (AM) function. The application describes experiments designed to test two hypotheses: first, that the constitutive level of activation of AM from HIV/FIV patients is a direct result of viral infection of the AM independent of lymphokine conditioning; secondly, that HIV/FIV infection inhibits an autocrine interferon (IFN)-gamma loop necessary to prime AM for IL-12 production. a. Career Development Plan: The plan consists of two phases. During the first Phase, Dr. Ritchey will complete his Ph.D. training. He entered the Ph.D. program in July of 1993, and has completed the didactic training as well as the preliminary examination. In phase two (years three and four), Dr. Ritchey will remain at North Carolina State University, in a postdoctoral position. b. Research Plan: Feline Immunodeficiency Virus (FIV) will be used to examine the defects in alveolar macrophage (AM) function. Experiments are planned to test two hypotheses: first, that the constitutive level of activation of AM from HIV/FIV patients is a direct result of viral infection of the AM independent of lymphokine conditioning; secondly, that HIV/FIV infection inhibits an autocrine IFN-gamma loop necessary to prime AM for IL-12 production. Studies during the first three years of this program will test these hypotheses by evaluating cytokine expression patterns of AM collected in a temporal fashion following in vivo FIV infection. The last year of this research will correlate the results of the in vitro studies with in vivo challenge of FIV-infected cats with T. gondii.

Scott &White Memorial Hospital provides comprehensive, high quality health care for a 38 county area in Central Texas. Since the early 1900s, Scott &White has demonstrated leadership in providing innovative cancer care emphasized by medical education and research. Continuing with this tradition of quality care for the community, Scott &White seeks to continue its funding for the Community Clinical Oncology Program (CCOP) to support these objectives. The mission of the Scott &White Cancer Institute is "to provide the most comprehensive, highest quality cancer care, focusing on the prevention and treatment of cancer, enhanced by the application of scientific discoveries to ultimately improve the care of those living with cancer at Scott &White and throughout the world." Funding from the NCI since 1997 for our CCOP has enabled the Cancer Institute to evolve into a recognized leader in clinical cancer research and treatment for our service area. The proposed CCOP consists of the Scott &White component and Southwest Regional Cancer Center affiliate. The component and affiliate will work together in promoting innovative cooperative group studies in the community and contributing to quality research through refinement of processes to identify patients for protocol enrollment. The objectives for the CCOP will be to further support the commitment to delivering research options to the community that include cancer control, prevention, and treatment studies. The CCOP will continue to build on the existing strategies to recruit minority participants onto NCI-sponsored protocols by establishing relationships and by providing access to services in underserved areas. The disease-specific teams were developed to expedite patient discussion and treatment plan decisions, will continue to improve processes with a patient-centered approach to treatment. Community-based involvement will be strengthened by increasing participation in cancer-related organizations that support the goal of eradicating life-threatening cancers through education, research, and providing access for underserved areas. A consistent growth in the Central Texas area, increased health plan participation and the expansion of outreach clinics has placed an increasing demand for cancer services at Scott and White.

Preliminary data showed that the relationship between the PAT index and IR signal, calculated as a ratio between temperature before and during the first 100 sec of occlusion, holds promise to simplify and substantially shorten the procedure of endothelial assessment. Thus, peripheral vascular endothelial function testing with both PAT and IR methods correlates with the presence or absence of Sickle Cell Disease. These data suggest that peripheral vascular endothelial function testing with IR imaging is feasible, and this is an important next step in development of this non-invasive imaging technology capable of identifying, differentiating, and locating peripheral vascular beds with endothelial dysfunction.

The project is related to the isolation of proforms of fibrous elastin from lung tissue. A fibrous elastin precursor which is approximately 70,000 daltons in size has been isolated from the lung tissue of the copper-deficient chick. This protein is similar in property to tropoelastin which has been isolated from the aortas of copper-deficient swine and chicks. In addition, a protein, which has an amino acid composition similar to tropoelastin (i.e., eighty-five percent of the total residues as gly, ala, val, pro and over 40 residues/1000 residues lys), has also been isolated with an apparent molecular weight of 100,000 plus daltons. The protein appears to be a proform of tropoelastin. Selective isolation of the "proelastin" is achieved by isoelectro focusing tissue extracts. Studies are currently being conducted to establish more clearly a precursor-product relationship with respect to the putative proform and tropoelastin and their roles in the final formation of fibrous elastin. In addition, dietary copper-deficiency and cadmium intoxication appear to result in severe alterations in lung elastin metabolism.

A method has been developed to measure rapidly and accurately three important material constants of a hydrogel sample. The shear modulus, m, the bulk modulus, k, and the hydraulic permeability, 1/f, can be determined from a single stress relaxation experiment. The material constants are treated as free parameters whose values are optimally estimated X)y minimizing the variance between predicted and empirical force relaxation waveforms. Values of the three constants obtained by this method agree with values obtained by independent free-swelling and permeability measurements.

The goal of this UH2 and UH3 is to study how exosomes can deliver siRNAs across the blood brain barrier to enter neurons and other brain cells. The immediate target is the mutant huntingtin mRNA. Huntington's disease (HD) is caused by an increase in the CAG trinucleotide repeats to ? 36 in series; it necessitates years in a high level nursing facility because of neurodegeneration first in striatum and cortex and then to other brain structures. HD patients have cognitive impairment, depression and aberrant movements. Most HD patient present by 30 to 40 years of age; a few have a juvenile onset. A rational treatment is to decrease expression of mutant huntingtin mRNA; this therapeutic can be accomplished in HD mouse models by siRNA, antisense oligonucleotides (ASO) and adeno-associated virus (AAV) with shRNAmir directed against huntingtin mRNA. However, delivery remains a pitfall to practical implementation of the therapeutics. siRNA and ASO require long-term infusion. In non-human primates, ASO administered to spinal fluid does not reach the striatum and spread of siRNA is limited in brain. Although promising, AAV-shRNA requires several injections into brain areas and the shRNAmir is unregulated. A gap in HD therapeutics can be filled by microvesicles normally extruded by cells, exosomes. Exosomes with rabies virus glycoprotein (RVG) on their surface can be injected into the blood, cross the blood brain barrier, and enter neurons and glia. RVG-exosomes can carry siRNA cargo. Delivered into the blood circulation, the exosomes deposit siRNA in neurons to engage in RNA interference. Our purpose is to develop exosomes as a therapeutic in HD. The UH2 examines the ability of RVG-exosomes carrying siRNA against huntingtin mRNA to cross the blood brain barrier to enter neurons. Localization in brain and RNAi dependent knock down will be studied. Hyper-functional siRNAs will be sought. Because exosomes are made from cytoplasm of cells, exosome mRNA, miRNA, and implaced siRNA will be identified by deep sequencing. Immune reactivity and immune-neutralization will be studied, since exosomes have potential antigens, like RVG, and will need to be administrated often. The UH3 further establishes exosome-based therapeutics, by study of reversal or prevention of neuropathology and aberrant movement in HD mouse models. Dosing of exosomes will be secured. A team of experts in HD pathogenesis, siRNA development, RNA identification and measurement, RNAi mechanisms and exosome production and brain delivery will carry out the studies. Harnessing exosomes for brain delivery is expected to form a viable therapeutic to reduce expression of mutant huntingtin in patients with HD. Patients with other genetically- based neurodegeneration will benefit.

Human plasma high density lipoproteins (HDL) from normolipemic sera were analyzed as a distribution of at least three major components, each with characteristic particle size, hydrated density, and consituent apolipoprotein composition. The plasma levels of two components, HDL2b and HDL2a, were variable among 160 normal individuals while HDL3 levels remained essentially constant. Polydispersity of apolipoprotein composition was demonstratable in all three HDL components from both normal and abetalipoproteinemic individuals. In addition to these three components, the HDL from cord blood plasma of neonates was found to contain additional components which accompany a threefold decreased activity of the plasma enzyme lecithin: cholesterol acyl transferase. This work suggests that HDL2 (HDL2b plus HDL2a) is the major contributor to the inverse correlation of HDL cholesterol with the prevalence of coronary heart disease.

Project Summary Computational methods for mechanistic understanding of inter-sample variability The overall goal of this R21 application is to develop a computational framework that will allow for the prediction of physiological differences between experimental samples. Differences between individual samples can be catalogued and described at the physiological level, in terms of properties such as action potentials, and also at the molecular level, in terms of measurements such as gene expression. Linking variability at one level to variability at another level in a quantitative manner, however, is not straightforward. Here, through an innovative combination of experimental studies, mathematical modeling, and statistical analyses, we will develop methods that allow for molecular-level differences between samples to be translated into quantitative predictions of physiological differences. This Multiple Principal Investigator proposal utilizes the complementary expertise of the two PIs. Dr. Eric Sobie is expert in cardiac physiology, mathematical modeling, and computational approaches for understanding variability~ Dr. Christoph Schaniel is expert in stem cell biology, differentiation of pluirpotent cells into specific cell types, and high-throughput methods. The combined efforts of the two PIs will generate new quantitative data and will yield new computational methods that can be applied broadly to understand variability in different contexts. To achieve the overall project goals, we propose to: 1. Collect measurements of cardiac physiology and expression of relevant ion channels, pumps, and transporters. These measurements will be matched on a sample-by-sample basis. 2. Perform population-based simulations with dynamical mathematical models to develop quantitative and mechanistic predictions regarding how differences between samples in expression of important genes are translated into physiological differences. 3. Use regression-based statistical methods to analyze the experimental and simulation results, and to relate the two sets of predictions to each other. Not only is this exploratory research likely to provide insight into the physiology of cardiac myocytes derived from stem cells, it is also likely to demonstrate a novel computational framework that can be used for quantitative treatments of variability between samples in many biological contexts.

The quest to characterize the human microbiome is a daunting goal, but one that promises to enhance significantly our understanding of health and our management of a wide variety of disease states. In this quest, two features of the human microbiota in particular, pose major challenges: the large proportion and number of as-yet uncultivated species, and the extreme unevenness of the microbial communities, with a resulting large number of potentially important community members that fail to be "seen" in routine surveys. The ability to identify, isolate, and sequence the genome of single bacterial cells would allow us to characterize and understand both rare and uncultivated microbial species, and materially advance our understanding of the human microbiome. In recent work, a microfluidic device has been designed and fabricated, with features that mimic an integrated electrical circuit; this device isolates individual bacterial cells, and allows their genome to be amplified in nanoliter volumes. In this Application, a plan is proposed for optimization and augmentation of this microfluidics device, so that environmental contamination is reduced, rare cell types are more easily captured, larger numbers of cells are screened more quickly, and gene expression is more easily measured from single cells. The long-term objectives of this work are to enhance our understanding of the human microbial communities, and in particular, of novel or poorly-characterized, uncultivated microbial community members. This proposal responds to critical unmet needs posed by the NIH Human Microbiome Project. The following are the Specific Aims of this proposal: Aim 1. To reduce the contribution of environmental DNA to single cell genomic sequence data, and increase the "signal-to-noise" ratio of the sequence data obtained with our cell- sorting, genome amplification microfluidics device. The experimental approach involves the integration of optical (laser) tweezers into the device. Aim 2. To improve the ability to detect and capture rare microbial community members with the microfluidics device. The experimental approach involves the integration of fluorescence in situ hybridization techniques, specific probes, and fluorescence imaging with the microfluidics device. Aim 3. To increase the speed of single cell selection and isolation with the microfluidics device. The experimental approach involves more highly parallel microdevice designs, optimization of laser power and laser optical path, and further automation of cell manipulations. Aim 4. To enhance the capability for gene expression analysis in single bacterial cells. The experimental approach involves the development of on-chip protocols for RNA isolation, reverse transcription, and use of digital PCR to quantify transcript abundance from single cells. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will lead to a better understanding of the microorganisms that live on and in the human body. Because the human indigenous microbial communities help to maintain health and when disturbed, contribute to disease, this research may lead to new tests for diagnosing or predicting disease associated with microbial community disturbance, and new strategies for maintaining or restoring health. [unreadable] [unreadable] [unreadable]

Neuropathologically, Alzheimer's disease (AD) is defined by the presence of plaques composed of the amyloid-beta (Abeta) protein. This alone makes definition of the natural history of amyloid deposition in living subjects an important goal, but this longitudinal information is not obtainable through post-mortem studies. This information will be essential in the assessment of anti-amyloid drugs intended to alter the natural history of amyloid deposition. Our group has developed a novel amyloid-imaging positron emission tomography (PET) radiotracer termed "Pittsburgh compound-B" (PIB) that, in initial proof-of-concept studies, performed well to distinguish AD from control subjects and localized in brain with a regional distribution consistent with that of the post-mortem distribution of plaques. While these results are exciting, certain basic and fundamental characteristics of this PIB PET technology must be further defined before this amyloid imaging technology can reach its potential as diagnostic tool or a surrogate marker of efficacy for anti-amyloid therapies. One of these characteristics is the variation of the quantity and regional distribution of PIB retention over time in individuals. This will be the focus of Project 2 of this Program Project (P01). To address this question, we will perform 12- and 24-month follow-up e studies on 15 control subjects, 15 MCI patients, 15 mild and 15 moderate AD patients who had identical bas line studies in Project 1 of this P01 (or in pilot studies during the year prior to initiation of this P01). All will have been clinically evaluated and diagnosed at the University of Pittsburgh Alzheimer Disease Research Center and further evaluated at each study point with a neuropsychological battery through the P01 Clinical Core. All subjects will be studied longitudinally with fully dynamic PIB PET scans at 12- and 24-months after their baseline evaluation in Project 1. In addition, volumetric MRI and FDG PET will be performed so we can directly compare changes in PIB retention to changes in hippocampal volume and cerebral metabolic rate. Image analysis will be performed in the P01 Imaging, Methodology and Statistics Core. The quantitative PIB PET data will be compared to neuropsychological measures to explore correlations between changes in regional amyloid load and changes in cognition. The association of apolipoprotein-E genotype and education with PIB retention also will be explored. We hypothesize that PIB retention in individual subjects will increase through moderate stages of AD and then plateau and that the rate of increase in PIB retention in individual subjects will be of larger magnitude and be detectable earlier than the rate of decrease in hippocampal volume or cerebral metabolic rate.

The volume and electrolyte composition of saliva are dependent on the coordinated activity of ion transport proteins including multiple Na+-dependent mechanisms. Acinar cells employ a combination of Na+/H+ exchangers and Na+/K+/2Cl- cotransporters to drive fluid secretion and related processes such as cell volume maintenance and intracellular pH regulation. Ductal cells then act on acinar cell secretions to conserve NaCl utilizing Na+/H+ exchangers, Na+ channels and other ion transport proteins. The general functional properties of most Na+ transport proteins are understood. However, it is not clear what contribution individual Na+ transport proteins make to the overall secretion process, or which compensatory mechanisms may arise if the activity of a given transporter is perturbed. Thus, Aim 1 will determine which Na+ transport mechanisms are expressed in mouse salivary glands. The level of expression and the localization of different isoforms will be assessed by northern hybridization blots and immunohistochemistry, respectively. Aim 2 will examine the regulation of the different Na+ transport proteins by secretagogues. It is predicted that functional activity will be modulated for those transporters involved in the formation of saliva during stimulation. The results from the studies proposed in Aims 1 and 2 will provide essential information for predicting the role each transporter plays in the overall fluid secretion process. However, a definitive test of the importance of a given Na+ transport protein can most easily be obtained by studying an animal defective in the expression of that transporter. In Aim 3 the effects of gene disruption will be examined to determine the functional significance of individual Na+ transport proteins. Mice lacking expression of four different Na+ transport proteins are currently available for study (Na+/H+ exchanger isoforms NHE1, NHE2 and NHE3; Na+/K+/2Cl- cotransporter NKCC1). Mice expressing mutations for three additional Na+ transport proteins present in salivary glands are under development (epithelial Na+ channel ENaC; Na+/H+ exchangers NHE4 and NHE5). This multidisciplinary approach to the functional characterization of Na+ transport proteins will define the mechanisms involved in the production of saliva. The results of these studies will provide a foundation for future studies to analyze the in vivo structure/function relationship of a given Na+ transporter, which may ultimately aid in the development of treatments for various forms of salivary gland dysfunction.

The proposed research addresses a fundamental aspect of children's cognitive and social development -- their understanding of the mind. Understanding of the mind plays a critical role in the ability to function as effective social beings. Without an understanding that others have mental states, like beliefs and ideas, people would be unable to communicate and interact sensibly. The proposed work will examine an important yet unexplored aspect of this understanding -- children's knowledge about imagination. Children's understanding of imagination is important to study for several reasons. First, as a component of pretense, it serves as a vehicle for exploration of adult experiences and identities. Second, recent theorizing grants imagination a central role in the emergence of an understanding of human behavior. Finally, for adults and children, imagination serves as a source of much creative thinking and new discoveries. Despite the importance of an understanding of imagination, this question has been neglected in research on children's understanding of mind. Most studies have focused on aspects of children's understanding of epistemic mental states -- those mental states, like beliefs, that originate in direct interaction with physical reality and purport to represent reality truthfully. The conclusions drawn from the research are broader, however, encompassing children's understanding of representational mental states generally. This raises several important questions about children's understanding of fictional mental representations--those mental states, like imagination, that are representational, but do not purport to represent reality accurately. The proposed studies will examine the development of young children's understanding of the fictional mental state of imagination. The proposal addresses questions in three areas: (l) what is children's understanding of the correspondence between imagination and physical reality? (2) are there differences in children's ability to conceive of multiple representations with regard to fictional versus epistemic mental states, and (3) what do children understand about the origin of imagination and other mental states? Answers to these questions will fill a gap in our knowledge of children's understanding of imagination specifically, and will aide in developing more comprehensive theories about children's understanding of the mind more generally.

The goal of this project is to examine the developmental course of alcohol-related problems by focusing on the dynamic associations among family relationships, financial well-being, and drinking behavior from adolescence through young adulthood. To achieve this goal, we propose using four waves of data from the National Longitudinal Study of Adolescent Health (the Add Health Study) in response to PA-08-167. An important predictor of alcohol problems in both adolescence and young adulthood is compromised parent-adolescent relationships. Another predictor is financial stress which may be particularly relevant during times of economic downturn. However, little is known about the dynamics of financial stress and its association with alcohol problems in adolescence and young adulthood. Testing an extension of The Family Stress Model (FSM), this research examines the potential long-term effects of family financial stress and parent-child relationship quality in adolescence on young adult family relationships, financial stress, and alcohol problems. The research will contribute to several areas of basic and practical knowledge relevant to alcohol- related problems, prevention and treatment, and to programs and policies related to reducing financial stress and debt levels during young adulthood. The specific research aims are: Aim 1 (Adolescence and the Transition to Adulthood): To test an extension of the Family Stress Model in which adolescent family financial stress predicts compromised parent- adolescent relationships, which in turn influence alcohol problems in adolescence and into young adulthood; Aim 2 (Young Adulthood): To examine the dynamic associations in young adulthood between relationships with parents, financial stress, and alcohol problems;and Aim 3 (The Full Model): To test an elaborated model of the associations between adolescent family financial stress, relationships with parents, and young adult financial stress and alcohol problems. This combined model posits that family financial stress in adolescence is linked to young adult alcohol problems through its potential lasting influence on relationships with parents and financial stress. In addition to the full model, alternate models will be explored to test alternative pathways of influence: in particular, adolescent and young adult alcohol problems may predict compromised relationships with parents in young adulthood as well as financial stress. Once the suitability of these models has been established relative to alcohol problems, we will extend the analyses to the use of illegal substances. Because alcohol problems and problem trajectories vary by gender and race/ethnicity, analyses of potential gender and ethnic differences, or combinations of gender and ethnicity, will be integrated within each aim. PUBLIC HEALTH RELEVANCE: This research examines the long-term effects of family financial stress and parent-child relationships during adolescence on young adult family relationships, financial stress, and alcohol problems. By using a national study that follows participants from adolescence through young adulthood, we can track developing patterns of alcohol problems and identify factors that contribute to these patterns. This study provides a unique opportunity to extend research in financial stress and alcohol problems in young adulthood, to test multiple models for understanding the mechanisms and directions of influence among these key constructs, and to identify possible points of prevention and intervention.

NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Application The immune system has the potential to eliminate altered neoplastic cells with incredible specificity. A consistent in-frame deletion in the extra-cellular domain of the epidermal growth factor receptor (EGFRvIII) represents a truly tumor-specific target amenable to immunotherapeutic attack. Our multi-institutional Phase II study demonstrated that vaccination with an EGFRvIII-specific peptide in patients with newly-diagnosed glioblastoma multiforme (GBM) induces potent T- and B-cell immunity, produces nearly complete radiographic responses in all patients with residual tumor, and universally eliminates EGFRvIII-expressing cells. Recurrent tumors, however, continue to express wild-type EGFR suggesting that the immune response is specific, but productive intra-molecular cross-priming against other potential tumor-associated antigens is incomplete. We believe that productive extension of such secondary immune responses is hindered by the presence of regulatory T-cells (T ) which are disproportionately represented within the peripheral blood and tumors of Regs patients with GBM. T are characterized by constitutive expression of the high affinity interleukin (IL)-2 Regs receptor (IL-2R1)(CD25) and are uniquely dependent on IL-2R1 signaling for their function and survival. In the context of the existing grant, we conducted a randomized trial demonstrating that an IL-2R1-blocking antibody, daclizumab, significantly reduces TReg levels in patients with GBM with a nadir at 5 weeks without reducing overall CD8+ or CD4+ T-cell counts. Preliminary analysis also suggests that daclizumab enhances EGFRvIII-specific cellular (P=0.01) and humoral (P=0.003) immune responses compared to the saline treated group. The effect of a single dose of daclizumab wanes after 12 weeks consistent with its known half-life, and TRegs recover, however. With this supplement, we seek to extend our results by examining the effects of serial administration of daclizumab in this same patient cohort. We HYPOTHESIZE that serial doses of daclizumab therapy in patients with newly-diagnosed GBM will extend the duration of functional TRegs inhibition and further enhance vaccine-induced immune responses. Consistent with the goals of the American Recovery and Reinvestment Act, this Supplement would accelerate the tempo of our research in this area and allow for job creation and retention. PUBLIC HEALTH RELEVANCE: Treatment for malignant primary brain tumors, which are the most common cause of death among children and account for more deaths in adults than melanoma, currently represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. We have developed a vaccine that eliminates tumor cells containing a tumor-specific protein (EGFRvIII) in patients, but tumor cells that express related normal proteins survive. In this proposal, we will see if prolonged elimination of immunosuppressive "regulatory" T-cells that inhibit immune responses to these related normal proteins will enhance the effectiveness of the vaccine without inducing deleterious autoimmunity. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page

To investigate the physiological function of nonmuscle myosin heavy chain II-B (II-B), we generated a point mutant II-B mouse where amino acid arginine-709 was replaced by cysteine (R709C). Due to the presence of the Neo cassette in the targeted allele intron, both heterozygous (B-R/CN) and homozygous mutant (B-CN/CN) mice showed decreased expression of mutant II-B. The B-R/CN mice were generally healthy and fertile. However, most of the B-CN/CN mice died within the first two days after birth, and the remainder survived for up to 15 days suffering from cardiac and brain abnormalities similar to the B-/B- mice which die prior to birth. The B-CN/CN mice developed an early postnatal ataxia coupled with abnormal development of the cerebellum, which was remarkably reduced in size and had abnormal foliation. Detailed histological examination and neuronal migration assays revealed significant neuronal migration defects in the B-CN/CN mouse. These included an abnormal protrusion of the facial nuclei into the 4th ventricle, premature arrest of the pontine migrational stream, and slowed migration of cerebellar granule cells. This study provides evidence that NMHC II-B is required for migration of certain groups of neuronal cells. Removal of the Neo cassette from B-R/CN mice using Cre-recombinase rescued the decrease in II-B expression (i.e., increased the mutant protein) and generated B-R/C mice, which revealed a novel phenotype. Most of the B-R/C mice (90%) died shortly after birth showing absence of the small intestines from the duodenum through the sigmoid colon and collapsed lung alveoli. The unexpected phenotype was due to the herniation of the liver and intestines during embryonic development and the ingestion of these organs by the mother. Present studies are directed at understanding the role of NMHC II-B in generating this abnormal herniation.

This study will investigate the effect of dietary carbohydrate and aerobic exercise in a 2X2 design on body composition, energy expenditure, insulin secretion and catecholamine excretion in obese subjects receiving a hypo-caloric diet.

Proposed projects include: An extension of studies on the effect of ions, in particular potassium and calcium, on sensitivity of the neuromuscular junction to neuromuscular blocking agents. An examination of the effect of anesthetics on drug-induced membrane electrical noise. Determination of the effect of barbiturates on sensitivity of the neuromuscular junction to competitive neuromuscular blocking agents. Examination of the relationship of physical chemical properties, in particular, pKA and solubility in non-polar solvents, to potency of barbiturates. Determination of the extent of post synaptic effects to "run-down" of end-plate potential trains seen with some "competitive" neuromuscular blocking agents. Refinement of a heuristic model to serve as a basis for determining clinical dosage regimens of competitive neuromuscular blocking agents.

Approximately 20.6 % of the general population in the United States smokes cigarettes and this group may be comprised of smokers with severe nicotine dependence who are resistant to smoking cessation pharmacotherapies and treatments (Hughes, 2011). Varenicline is a first-line medication for smoking cessation that has been shown in meta-analytic reviews to be superior to other smoking cessation treatments (Cahill et al., 2010), however 56% of patients who take varenicline do not achieve smoking abstinence. One strategy to increase quit rates may be to administer a complimentary medication to augment the efficacy of varenicline. The anti-epileptic medication zonisamide is a good candidate for adjunct treatment because it increases dopaminergic tone, normalizes glutamate homeostasis, and potentiates GABA release. Zonisamide also improves sleep and promotes weight loss, two prominent issues not addressed by varenicline. Finally, the PI of this proposal has documented unpleasant changes in the taste of cigarettes and reductions in nicotine withdrawal among patients receiving zonisamide for the treatment of cocaine dependence. The proposed study will explore the efficacy of varenicline + zonisamide for smoking cessation using a controlled, clinical trial. Eligible participants (n=60) will be smokers (>10 cigarettes per day or >1 year) seeking treatment. They will be randomly assigned to receive zonisamide + varenicline or placebo + varenicline under double-blind conditions for a 10-week period. Participants will visit the clinic weekly to receive medication and smoking cessation counseling and also to complete self-report ratings of smoking, nicotine withdrawal and other smoking-related indices. Smoking status will be assessed via urinalysis testing for the nicotine metabolite cotinine (<200ng/ml will be considered abstinent) from specimens obtained at each study visit. Cotinine is a sensitive indicator of smoking status with a longer half-life then carbon monoxide (CO) and is therefore more likely to detect low or intermittent smoking. The study hypothesis is that participants who receive the combination zonisamide + varenicline will achieve greater smoking abstinence when compared to varenicline alone. The primary outcome measure for this study will be the 4-week rate of biochemically-confirmed continuous smoking abstinence during weeks 7-10 of the intervention. Secondary outcomes will include self-reported rates of smoking, subjective effects of cigarettes, mean body weight change from baseline to week 10, self-reported sleep quality, and nicotine withdrawal symptom severity. Results from this study will contribute important information on successful and cost-effective smoking cessation interventions for individuals who do not respond optimally to current smoking cessation medications. This study will advance the science and clinical treatment of smoking cessation, and will provide the prerequisite data to develop a larger scale clinical trial evaluation of the combination zonisamide + varenicline for smoking cessation. PUBLIC HEALTH RELEVANCE: Varenicline (Chantix) is a successful smoking cessation medication, yet 56% of smokers are unable to quit smoking despite using varenicline. Combining varenicline with zonisamide, an antiepileptic medication that has been associated with smoking reduction, may produce higher smoking quit rates compared to varenicline alone. The information collected from this study will be important for understanding whether combination medication treatments (like varenicline & zonisamide) produce more robust smoking cessation outcomes when compared to medications administered independently, and may be used to guide the development of smoking cessation studies for smokers who have not responded optimally to other currently available smoking cessation pharmacotherapies.

Because Crohn's Disease (CD) has an unpredictable course, past research attempted to understand the psychosocial factors that impact disease relapse and exacerbation of symptoms (Searle & Bennett, 2001). However, this literature presents equivocal results, which may result from differing experiment designs. The present study attempts to examine the types of stressors (life events, daily, perceived) that predict relapse and define the within-person temporal relationship between stress and symptom exacerbation. In a sample of 25 CD patients undergoing a biologic treatment, an electronic diary will be used to examine the within-person variations of symptoms and stress within a day and over 8 weeks (time from initial treatment to subsequent treatment). The hypotheses are: 1. Patients who report more stress will relapse sooner compared to those who report less stress. 2. Previous reports of stress will be associated with subsequent reports of symptom exacerbation. Thus, this study will be able to not only identify if stress and disease activity are related, but also the type of stress that most impacts the clinical course of CD. In addition, within-person analyses of the temporal relationship between stress and symptoms may aid in the development of future treatment studies.

PROJECT SUMMARY There is a fundamental gap in how the presence of family companions in medical visits influences indicators of quality of care, such as patient-centered communication (e.g., communication that reflects respect for patients'values and incorporates patient's concerns and preferences into decision-making), specifically for patients with heart failure - a particularly vulnerable population by virtue of its high morbidity and mortality. The long-term goals of this project are to improve the quality and delivery of care and subsequent health outcomes for Heart Failure (HF patients). The overall objective here, which is our next step in pursuit of our long-term goal, is to determine whether family accompaniment influences processes of care within the medical visit and use this information to inform intervention development. The rationale for the proposed research is that once we know how family members influence quality and delivery of care we can fulfill the call to action of policy makers and health care models to "create an integrated, coherent plan for ongoing medical care in partnership with patients and their families." The research strategy is built around the central hypothesis that involvement of family members in clinical care will facilitate patient-provider communication and potentially improve subsequent HF self-management behaviors. The specific aims to achieve our overall objective are: 1) To investigate the impact of family companion's presence on patient centered communication as assessed by the following audio-tape measures: the patient-centeredness ratio (ratio of communication that furthers the patient's agenda to communication that furthers the provider's agenda), patient activation and engagement behaviors, and physician facilitation and patient activation behaviors;2) To assess the impact of family companion's presence on provider communication related to patient education and counseling about HF self-management behaviors (e.g., weighing, diet, symptom management, physical activity, and medication adherence);and 3) To use intervention mapping (IM) to design a family-involved HF intervention based on qualitative data and results of Aims 1 and 2, and pre-test the intervention. The mentored research, formal didactics, and planned presentations/publications will set the stage for independent funding as a clinician-scientist. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE: The proposed research is relevant to public health by seeking to improve the delivery of patient/family centered care for patients with chronic illness by understanding how the patient-physician interaction is influenced by family member accompaniment to medical visits. Thus, the proposed research is relevant to NHLBI's mission to enhance the health of patients living with heart failure so that they can live longer and more fulfilling lives.

This is a competing renewal application that requests continued support for providing pre- and post-doctoral trainees with strong methodological and practical training in quantitative cancer research. The application leverages a unique combination of strengths from the Harvard T.H. Chan School of Public Health, Dana-Farber Cancer Institute (DFCI) and Dana-Farber / Harvard Cancer Center. This training program, now in its 34th year, draws upon a distinguished faculty, consisting of biostatisticians and computational biologists, as well as world renowned experts in cancer treatment and research. Its overarching goal is to provide the trainees with all essential elements of training needed to successfully undertake modern cancer research. The specific goals of this training program are to train students and postdoctoral fellows to be (1) quantitative scientists in cancer research, who are capable of using probability, statistics, computer science and mathematics to increase our knowledge and understanding of cancer; (2) strong team leaders/players as well as excellent communicators in a cancer research environment, who can effectively disseminate their research results and assume active roles in the design, analysis and interpretation of cancer clinical trials, cancer population studies and cancer genomic studies. All predoctoral students supported by this training grant are required to take a concentration in cancer-related courses. During their first and second summer periods in the program, students are required to participate in research activities of the DFCI or DF/HCC, performed under the supervision of faculty mentor/trainers affiliated in the program. Afterwards, many of these students will take up residence at the DFCI or elsewhere at DF/HCC and continue their research in cancer, which eventually evolves into their dissertation projects. All the postdoctoral fellows are closely involved with the practice of quantitative sciences in cancer and are typically in residence at the DFCI. All trainees are required to actively participate in the a working group seminar series on quantitative issues in cancer research, which serves as a primary forum at Harvard to discuss current issues and challenges on this topic, as well as in several of the DF/HCC sponsored symposia and event. This proposal requests 5 years of funding to support 10 pre-doctoral students and 1 post-doctoral fellow annually.

The development of drugs that inhibit the K-Ras oncogene represents one of the greatest unmet needs in the treatment of human cancer. The Ras gene is the most frequently mutated oncogene in cancer, with a greater than 30% cumulative mutation frequency across all cancer types. Cancers with Ras mutations are aggressive and respond poorly to standard therapies. Previous attempts to target K-Ras have failed due to the difficulty of competing with the picomolar nucleotide affinity for the active sit and due to the high similarity of most GTPases. Our scientific co-founder, Professor Kevan Shokat, has discovered a novel, small molecule approach to target the most chemically tractable K-Ras mutant that contains a glycine-12 to cysteine mutation. The G12C mutation is the most common K-Ras mutation in lung cancer. Indeed, 43% of all lung cancers with K-Ras mutations contain the G12C mutation. This mutation positions a chemically reactive sulfhydryl group on the surface of K-Ras. We have carried out a preliminary 500 compound library screen based on mass spectrometry for molecules which bind covalently to K-rasG12C, H-rasG12C and counterscreened against WT K-Ras. 17 hits were identified from the screening library, and the first round optimization of the initial hits led to the discovery of a potent inhibitor JO-01-18. We have now solved the crystal structure of JO-01-148 bound to K-Ras G12C and identified a previously undescribed allosteric pocket on the surface of the protein adjacent to the cysteine moiety. This pocket makes it possible to identify irreversible inhibitors that bind in the pocket and selectively target the cysteine at position 12. Importantly, these small molecules inhibit only mutant K-Ras and not the normal protein. We have now solved more than 10 X-ray crystal structures of irreversible inhibitors bound to this allosteric pocket and synthesized more than 120 compounds. A clear SAR has been established. We are now proposing to further validate our lead G12C compounds in biochemical and cellular assays. The Phase I specific aims are: (1) Develop assays to evaluate K-Ras effector binding; (2) Demonstrate that the G12C irreversible binders can disrupt K-Ras effector binding; and (3) Demonstrate that the G12C irreversible binders can differentially affect tumor cells with G12C mutation compared to cells with wild type K-Ras and other K-Ras mutations. The Phase I milestone is the identification of K-ras G12C inhibitors that suppress proliferation of tumor cells with G12C K-ras mutation five-fold more potently (as measured by IC50 values) relative to tumor cells with other K-Ras mutations or wild type Ras. Collectively, we expect the Phase I results to demonstrate that we can generate a small molecule inhibitor that will specifically inhibit the growth of tumor cells wih K-Ras G12C mutation. If our approach is successful, our Phase II studies will more fully examine the safety, efficacy, and PK/biodistribution of a lead formulation for advancement to an IND application.

The proposed study will investigate modifications of the sympathetic responses of the uterine circulation due to changes in blood gases and temperature in the pregnant sheep. The distal end of the severed right sympathetic chain will be stimulated at varying frequencies as the pregnant uterus is perfused at constant flow with blood whose gas tensions and temperature is altered in the extracorporeal circuit. Alterations in uterine conductance will be expressed as function of frequency of stimulation and a family of curves generated. The decreased responsiveness of the pregnant animal to carotid sinus occlusion will be studied in the pregnant uterus perfused at constant flow to ascertain the role of the uteroplacental circulaton in damping the response. The pressure regulated circuit will be used to perfuse the uterus with mildly hypoxic blood, and after severing of the right sympathetic chain, flow-pressure curves will be recorded in effort to elicit autoregulation.

This project will support the training of six predoctoral students each year in the area of biostatistics, with an emphasis on applications to modern problems in genomic science. The training combines rigorous coursework in statistical methods and theory, additional courses in bioinformatics and genomic science, and an extensive laboratory training experience. For the latter, trainees will begin as supervised statistical consultants for a matched genomics lab, then over the course of a year progress into active collaborators in one or more lab projects. Most students will be supported for the first three years of their graduate programs. The scientific training will be supplemented with training in the responsible conduct of research developed specifically to meet the needs of researchers in this area. The training involves collaboration among biostatistics, genomics, and philosophy faculty members. An active recruiting plan is described for enhancing the diversity of our training and graduate programs, including a summer program bringing faculty and undergraduate students from minority serving institutions to NC State during the summer to initiate collaborative work with training faculty.

We have developed databases and software useful for comparative analysis of protein three-dimensional structure. These tools are distributed freely to biologists and developers of biotechnology software. MMDB (Molecular Modeling DataBase) is the 3D-structure component of the Entrez molecular biology retrieval system. MMDB is an ASN.1 database where all data items describing macromolecular structure are validated and explicitly listed, so that application software need not contain the complex logic required to retrieve this information from text formats such as PDB files. Work has concentrated on addition of accurate taxonomy assignments for macromolecular structures within MMDB, creation of new message and data types for transmission of structure-structure alignment data to local viewers, and on construction of an automated monthly update and indexing system, Pubstruct. CN3D ("see in three dimensions") is a multi-structure visualization program distributed as part to the Entrez client software and in a stand-alone version lauchable via the MIME protocol in World-Wide-Web Entrez. The software differs from other public domain viewers in supporting display of multiple aligned structures from Entrez's "structure neighbor" database, and in supporting simultaneous highlighting/picking of multiple sequence and multiple structure alignments. Other features added this year are on-the-fly alignment of the sequences of homologs, so that an Entrez user may easily map conserved sequence features onto the know 3D structure. These software features are intended to facilitate molecular biologist's identification of important structure-function relationships within protein families. Work this year has concentrated on improvements to CN3D. The software has been modified to use an industry-standard 3D graphics library, OpenGL, which provides much better quality molecular graphics rendering. We have also added core-structure alignment editing and threading tools to the sequence display windows, to support curation of CDD (a Conserved Domain Database). Work is in progress to revise and simplify the data structures underlying CN3D, so that further improvments in graphcis presentation, specific to describing conserved features in protein families, may be added to future versions of CN3D. A new version of Cn3D incorporating these changes was released in June, 2002 and downloaded by over 50,000 users as of October, 2002. This version provides sophisticated alignment editing tools, in addition to greatly improved molecular graphics performance on popular computing platforms. As of October, 2003, over 150,000 copies of CN3D have been downloaded. A new "related structures" link has been added to NCBI BLAST servers, to provide easy-to use mapping to 3D structure whenever possible.

Pulmonary hypertension (PHN) in respiratory distress syndrome (RDS) evolving to bronchopulmonary dysplasia (BPD) results acutely from poor lung inflation and increased blood flow through the patent ductus arteriosus, and chronically from altered reactivity, vascular remodeling, and hypoplasia of the pulmonary vascular bed. The lung develops in relative hypoxia compared to 21% 02 seen at normal term delivery and, especially, to hyperoxia that preterm neonates commonly face. Hypoxia-inducible factors (HIFs) could lower pulmonary artery pressures by modulating surfactant, ductus arteriosus, and pulmonary vascular development. Preliminary data show that HIFs can impact lung expansion at birth, expression of surfactant proteins and lipids, patency of the ductus arteriosus, and angiogenic factors and processes in developing lungs - all of which canmodify pulmonary hypertension. Further data show that HIFs are highly expressed and stable in third trimester fetal primate lungs, while one of them, HIF-1a, declines dramatically after preterm birth. Using cofactors 02, Fe2+, oxoglutarate, and ascorbate, HIF prolyl- hydroxylases (PHDs) specifically regulate HIFstability. We reported that PHDinhibitors (PHDi) profoundly alter stability of HIFs and downstream gene expression (VEGF and its receptors) in lung endothelial and epithelial cells, and fetal lung explants, even in extreme hyperoxia. We hypothesize that PHDi can decrease PHNby restoring the fetal VEGF/eNOS axis and improving perinatal surfactant homeostasis. We propose to examine effects of PHDi delivered antenatally or postnatally, and by differing routes, in ovine models of RDS and persistent PHN in preterm and term ovine models, respectively. Chronically instrumented fetal/neonatal lambs will have pre- and/or postnatal hemodynamic, blood gas, and respiratory physiology measurements. In lung tissue from these animals, we will measure HIFs, HIF- dependent gene products, surfactant proteins and lipids, inflammatory cytokines and cells, and lung morphometry to assess vascular development and remodeling. Results will provide valuable information about pathophysiologic roles of HIFs in perinatal PHN and potential therapeutic uses of PHDi in RDS and PHN.

The subunit composition of electrophoretically isolated human haptoglobin 2-1 polymers was elucidated by 1) determination of the ratio of alpha 2 to alpha 1 polypeptide chains using gel densitometry, 2) analysis of the beta chain composition by enumeration of fractionally saturated hemoglobin-haptoglobin intermediates, and 3) determination of molecular weight of each polymer by sodium dodecyl sulfate (SDS) gel electrophoresis.

font color="#990000";b>Benign and Malignant Thyroid Disease Among Those Exposed as Children to Fallout from the Chornobyl Accident</b;/font;br>The Chernobyl nuclear reactor accident in Ukraine in 1986 contaminated large parts of Belarus, northwestern Ukraine, and bordering Russian provinces. A case-control study in Belarus, with individual dose estimates, demonstrated a statistically significant link between thyroid cancer and environmental radiation dose from fallout related to the Chornobyl accident. With the assistance of the Department of Energy (DOE) and the Nuclear Regulatory Commission (NRC), NCI organized follow-up screening studies of benign and malignant thyroid disease among those exposed as children to fallout from the Chornobyl accident in Belarus and in Ukraine. The thyroid glands of approximately 12,000 subjects in each country have been screened biennially for four cycles in Ukraine and three cycles in Belarus. A prevalence analysis of thyroid cancer based on data from the first screening in Ukraine has been published in JNCI, and a descriptive paper on the pathology of the tumors has appeared in Cancer. A paper on autoimmune thyroiditis has been published in JCEM, a paper on antibody levels among the low-exposed group has appeared in Clinical Endocrinology, and dose-response analysis of follicular adenoma is in the American Journal of Epidemiology. A number of other manuscripts, including one on incident thyroid cancers on the in utero exposed and on hypothyroidism are close to submission. The projects are currently preparing to transition from active screening to other forms of follow-up widening the existing national cancer registries.<br;br><font color="#990000";b>Cancer Mortality Among the Population Exposed to Radioactive Waste Released in to the Techa River Adjacent to the Mayak Nuclear Facility in Russia</b;/font;br>Villagers living along the banks of the Techa River in Russia were exposed to chronic external and internal environmental radiation from radioactive waste released into the river by the Mayak nuclear facility. Cancer mortality is being evaluated in a cohort of approximately 30,000 people who received low to moderate radiation doses from the contaminated river. Current efforts focus on improving data quality, tracing the population for vital status information, and on statistical analyses and manuscript preparation. Associations between solid cancer and leukemia mortality and radiation dose have been observed.<br;br><font color="#990000";b>Thyroid Nodules as a Measure of Radiation Risk Among a Population of 3000 Residents in Kazakhstan Exposed as Children to Fallout from Nuclear Testing at the Semipalatinsk Test Site</b;/font;/br>Thyroid nodule prevalence, determined by ultrasound screening, was used as the main outcome variable for evaluating the effects of environmental radiation fallout exposure in a population of 3000 village residents in Kazakhstan affected by radioactive fallout from nuclear bomb tests at the nearby Semipalatinsk Test Site during 1949-1962. A new dose-reconstruction algorithm, developed jointly by NCI and the Institute of Biophysics in Moscow, was used to estimate individual thyroid doses from external and internal sources of fallout-derived radiation, based mainly upon residential history and estimated consumption of milk from dairy animals grazing on contaminated pasture. Both external and internal dose were found to be significantly and independently associated with nodule prevalence after adjustment for sex and for age at screening. Current efforts, which include obtaining new information through the use of focus group interviews and application of statistical methods that take account of random errors in estimated dose, are focused on sources of uncertainty, especially those involving internal dose from radioactive isotopes of iodine ingested through the pasture-to-milk food chain. Polymorphic variants in 13 genes were studied for effect modification of the radiation dose-response relation and main effects on thyroid nodule risk. A variant in XRCCI was found to interact with radiation dose and thyroid nodule risk.<br;br><font color="#990000";b>Radiation Biodosimetry</b;/font;br>Periodic reviews of the scientific literature are conducted to identify the most current techniques used to estimate doses from past exposure to occupational and environmental radiation. These data are used to inform current epidemiologic studies of irradiated populations (eg. Chernobyl, Techa River, X-ray technologists) where doses may be missing, to estimate dose uncertainty or validate estimated doses.<br;br><font color="#990000";b>Ultraviolet Sunlight Exposure, Ionizing Radiation, and Risk of Skin and Other Cancers</b;/font;br>Exposure to ultraviolet (UV) radiation from sunlight plays a predominant role in the etiology of melanoma and non-melanoma skin cancer. There is accumulating evidence linking melanoma and two major non-melanoma skin cancers (i.e., basal cell carcinoma and squamous cell carcinoma) to different patterns of UV exposure (i.e., total, chronic and acute intermittent), but the nature of the relationship is complex and not fully understood. We have recently completed a study to evaluate the validity of questionnaire-based sun exposure data using histologically assessed sun-related skin damage. Furthermore, it has also been suggested that sunlight exposure may have a protective effect for non-Hodgkin lymphoma (NHL) and some other forms of cancer. We recently completed questionnaire-based collection of lifetime UV sun-related radiation exposure from more than 70,000 U.S. radiologic technologists and have undertaken analyses to assess risks of specific forms of cancer in relation to this exposure. We also plan to evaluate whether skin cancer risks associated with occupational exposures to ionizing radiation might be modified by questionnaire-derived estimates of UV sun-related radiation exposures. <br /;br /><font color="#990000";b>NHANES III: Prospective Vitamin Study</b;/font;br>The NHANES III data set with measured vitamin D levels on about 20,000 people is a unique resource that allows investigation of many issues on the relationship between vitamin D and specific causes of mortality. This cohort includes actual vitamin D measurements and about 2253 deaths, of which 1072 are circulatory disease deaths and 536 are all cancer deaths. We have completed analysis of the relationship between baseline vitamin D and all cancer mortality, with a manuscript published. The Follow-up has been extended- with 60% more deaths. Analyses of cancer outcomes are being under taken in the extended dataset.<br /;br /

The purpose of this study is to assess the role that contextual factors (e.g., community-level health and social resources, community socioeconomic status) play in shaping mental health and recovery in the aftermath of a disaster. The notion that community characteristics are important determinants of post disaster functioning was dramatically brought home in much of the public discourse about Hurricane Katrina that focused extensively on the role that underlying socio-economic position, resource availability, and preparedness may have played in the aftermath of this disaster. The proposed research would be the first study, to our knowledge, to provide a full, rigorous, and theoretically-based test of the various pathways through which community-level factors shape individual risk and community rates of psychopathology and adverse behavioral outcomes after disasters. Our broad aim is to demonstrate the utility of an ecological conceptualization of disaster recovery, focusing on individuals' health outcomes such as the occurrence/severity of posttraumatic stress disorder (PTSD), depressive symptoms, and chronic stress. To achieve these aims we will collect both secondary and primary data to describe (A) community characteristics of Mississippi counties before and after the disasters, and (B) sociodemographics, event exposures, and mental health among 1,000 residents of Mississippi. To characterize pre-disaster community resources in ways that are not subject to recall bias, we will assemble a contextual database from publicly available sources, such as the US Census and the Mississippi Development Authority. We will also assemble objective information about community-level exposure and postdisaster community resources, including estimates of need and amounts of FEMA monies awarded. In order to obtain individual-level information, we will select a random sample of 1,000 adults from three geographic areas formed by levels of damage caused by Hurricane Katrina. Using multiple sampling modalities we will sample persons proportional to their representation in the general population before the hurricane and include both persons who were and those who were not evacuated as a result of the hurricane. Key hypotheses will be tested using multi-level hierarchical models and ecologic analyses. [unreadable] [unreadable] [unreadable] [unreadable]

Our long term objective is to develop a detailed understanding of globin gene activation during erythroid cell differentiation. To model these events we are characterizing globin gene expression during the differentiation of murine erythroleukemia (MEL) cells. We have identified three proteins that bind specifically to the promoter of the alpha-globin gene DNase I footprinting and electrophoretic shift assays were used to monitor the enrichment of each protein through several chromatographic steps. DNA sequence affinity chromatography was then used to purify each protein. We propose to employ these purification protocols to accumulate a sufficient amount of each factor to obtain amino acid sequence data. These data will be used to prepare oligonucleotide probes that will be used to screen MEL cell cDNA libraries. In addition, direct screens of MEL cell cDNA expression libraries will be initiated. For these experiments, double-stranded oligonucleotide comprised of the binding site of each protein will be synthesized, ligated, nick-translated, and used to probe cDNA expression libraries. The identification of clone that encode alpha-globin specific DNA binding proteins will allow us to compare these proteins to other DNA binding factors, to characterize the functional domains of each protein, and to investigate their tissue distribution, synthesis, and turnover. Antisera could also be prepared against synthetic peptides derived from the deduced amino acid sequence of each protein. This analysis will help us to obtain a deeper understanding of how nuclear proteins regulate cell growth and differentiation. In this regard it is of interest to note that MEL cells are transformed erythroid precursors arrested in differentiation at the CFU-e state. We have shown, however, that MEL cell differentiation leads to characteristic changes in the activities of the identified alpha-specific binding proteins. It is not unreasonable to speculate that an understanding of how these factors function and how their activities are modified during differentiation might lead to the development of novel strategies for selectively altering factor activities. Because these factors appear to play important roles in mediating growth and differentiation, these insights could have important implications for approaches aimed at altering the uncontrolled growth and lack of differentiation characteristic of many tumor cells.

Gonorrhea is the second most common bacterial sexually transmitted infection ? the most common is chlamydia, which often coinfects with gonorrhea. About 80 million new cases of gonorrhea occur worldwide annually. Over 555,000 cases were reported in the U.S. in 2017. Serious sequelae of gonorrhea in women include infertility, ectopic pregnancy and chronic pelvic pain. Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea, has become resistant to almost every antibiotic in clinical use. Resistance to ceftriaxone and azithromycin ? the recommended first-line of treatment ? portends an era of untreatable gonorrhea. The CDC has listed Ng as a microorganism with a threat level of ?Urgent.? Development of a safe and effective vaccine against gonorrhea is a public health priority. Our group has shown that a monoclonal antibody (mAb) called 2C7 that targets a widely-expressed Ng lipooligosaccharide (LOS) epitope (the 2C7 epitope) is bactericidal. A peptide mimic (mimitope) of the 2C7 epitope, when configured as a polymer (a ?multiantigen peptide?) elicits bactericidal Abs and attenuates Ng vaginal colonization in mice. Encouraged by the recent retrospective observation that a group B meningococcal vaccine composed of detergent-extracted outer membrane vesicles (MeNZB) designed to curtail a meningococcal epidemic in New Zealand also reduced incident gonococcal disease by 31%, we will incorporate the gonococcal 2C7 epitope into meningococcal outer membrane vesicles to target both meningococci and gonococci. This innovative approach is advantageous because both diseases are highly prevalent in adolescents and young adults. In addition to the economic advantages, a ?pan- Neisseria? vaccine would help address an already crowded vaccination schedule and may mitigate the stigma associated with vaccines against STIs. In Aim 1, we will develop meningococcal native outer membrane vesicles (NOMVs) containing LOS that displays the 2C7 epitope. Meningococcal FH-binding protein (FHbp) will be mutated to abrogate binding of human FH and thereby elicit higher bactericidal Ab against Nm than wild- type FHbp. We will also delete Rmp and H.8, which may elicit blocking Ab against Ng and Nm, respectively. Elicited Abs will be tested for complement-dependent bactericidal activity against homologous and heterologous Ng strains and tested for efficacy versus Ng in transgenic mice we have developed that express the human complement inhibitors, FH and C4BP, to better simulate the complement environment in humans. We will use mice deficient in C9 (the last step in formation of the bacteriolytic pore) or mice depleted of PMNs to elucidate the mechanism of action of the candidate vaccine, which will also define correlates of protection. In Aim 2, we will develop detergent-extracted outer membrane vesicle vaccines (dOMVs) that display the 2C7 mimitope peptide in the framework of the major outer membrane protein, PorA. Immunogenicity and efficacy of this vaccine will be evaluated as described in Aim 1.

PROJECT TITLE: Southwest Tribal NARCH X Administrative Core PROGRAM DIRECTOR/PRINCIPAL INVESTIGATOR: Dr. Kevin English Project Summary/Abstract The Southwest Tribal NARCH X Administrative Core will provide overall leadership to the Center and will coordinate, integrate, and support the various Center components and activities in order to fulfill the overall NARCH mission, which is to implement health research projects prioritized by area tribes, reduce distrust of research among American Indian and Alaska Native (AI/AN) communities, and enhance health research partnerships to reduce AI/AN health disparities, while promoting a cadre of AI/AN scientific and health research professionals. The Southwest Tribal NARCH X Administrative Core will be based at the Albuquerque Area Indian Health Board, Inc. (AAIHB), an intertribal organization, which has extensive experience in the oversight of prior NARCH grants (NARCH I, NARCH III, NARCH V), and currently administers a NARCH VII grant. The Southwest Tribal NARCH X Administrative Core will help fulfill the major aims and goals of all Center components by pursuing the following specific aims: 1) establish a sustainable structure which will facilitate the conduct of Southwest Tribal NARCH X work by providing administrative and technical support as well as by stimulating exchange among all proposed Center components in collaboration with academic and tribal partners; 2) maintain the Southwest Tribal NARCH Community and Scientific Advisory Council (CSAC) to ensure rigorous tribal oversight of all Southwest Tribal NARCH components and activities and foster sustained alignment with tribal priorities and key cultural considerations; and 3) support the continued operation of the Southwest Tribal Institutional Review Board (IRB) to advance the benefits of ethical health research to area tribes and reduce AI/AN community distrust of research and researchers by giving tribes greater control over the research process. Expected outcomes of the Southwest Tribal NARCH X Administrative Core include rigorous tribal oversight of all Southwest Tribal NARCH X components, continued alignment of Southwest Tribal NARCH X components and activities with tribal priorities and important cultural considerations, enhanced protection for individual and community research participants, streamlined IRB review processes, enhanced research alliances among all partners (AAIHB, tribes, and academic partners), and manuscripts and professional presentations highlighting key findings of all Southwest Tribal NARCH X research and innovations.

Carcinoma of the esophagus and carcinoma of the pancreas are among the most lethal of human neoplasms. Efforts to devise successful therapeutic regimens for the primary neoplasm are hampered by lack of an animal model suitable for the study of surgical procedures or radiation therapies appropriate for use in man. Using carcinogens with proven effect in small animals, we have successfully induced invasive bronchogenic carcinoma in dogs. In this application, we propose to extend this approach to the development of carcinoma of the esophagus and carcinoma of the pancreas in dogs. Achievement of a predictable model of esophageal and pancreatic cancer, in which the lesion is reproducible at pre-selected localized sites, will provide 1) opportunities for studying the transition of normal tissues into pre-neoplastic and neoplastic lesions, 2) a preparation for serial diagnostic studies utilizing methods feasible in man but not possible in the small animals usually employed in carcinogenesis research, and 3) a potential for meaningful therapeutic investigations using this large animal model for esophageal and pancreatic carcinoma. As the work progresses and neoplastic lesions are produced, collaborative projects with other members of the City of Hope Cancer Research Center will be developed. These will include basic immunologic and cytogenetic studies of the evolving tumors, and multimodality therapeutic approaches involving radiation and medical oncology.

The mucopolysaccharidoses (MPS) are a group of hereditary diseases characterized by defective metabolism of glycosaminoglycans (GAGs). The disorders are usually associated with severe dysfunction of the nervous system as well as of liver, spleen, heart, bone, and other tissues. Objective of this project is the study of mechanism of pathogenesis of these diseases with emphasis on brain involvement and mental retardation. We are using a comparative approach. For this purpose we study the changes, in GAGs, sphingolipids, and pertinent lysosomal enzymes in tissues of patients with various types of MPS and we make correlation in terms of clinical and ultrastructural findings. Our laboratory contributed significantly in understanding the chemical pathology and in particular the neurochemistry of MPS IH, MPS IS, MPS II, MPS III A and MPS III B. To complement the studies with human subjects, a drug (suramin) induced animal model of MPS has been developed and a canine model, (natural), of MPS I (alpha-L-iduronidase deficiency), has been fully characterized. In an attempt to reverse the progressive deterioration caused by this incurable metabolic disorder, five doges with MPS I were transplanted with marrow from normal or heterozygous littermates. Transplanted bone marrow provides the affected dogs with self-renewing source of cells that produce the enzyme needed to complete the metabolic process. Two of these recipients were killed on post-bone marrow transplantation (BMT) day 628 and 594 at which time both were clinically healthy. Large reduction of stored GAGs, from all tissues including the brain, appears to be consistent consequence of BMT therapy in canine MPS I.

Multiple Sclerosis (MS) is an autoimmune disorder of the central nervous system typified by axonal demyelination and neuronal death. To date, there is no effective treatment to cure the disease, and the available therapies do not alter the outcome of the disease. The development of better therapeutic options requires in-depth understanding of the molecular mechanisms leading to disease development and progression. Our group previously discovered a single nucleotide polymorphism (SNP, rs6897932) within exon 6 of the IL-7 receptor alpha chain (IL7R) that is strongly associated with the risk of developing MS. Our group further showed that the MS-associated allele of the SNP increases skipping of exon 6 both in vitro and in vivo, leading to increased production of a secreted receptor (sIL7R) that is unable to activate the IL-7 signaling pathway. Importantly, sIL7R has been linked to the disease in both human patients and animal models. These results directly implicate splicing of IL7R to the pathogenesis of MS, and posit the trans-acting factors controlling its splicing as candidate MS susceptibility genes. I have uncovered two of the trans-factors controlling IL7R exon 6 splicing: the RNA helicase DDX39B, which activates exon inclusion, and the polypyrimidine tract binding protein (PTBP1), which represses it. Furthermore, we uncovered several SNPs in the DDX39B gene region that are associated with MS, thereby establishing DDX39B is itself a risk factor for MS. None of the associated SNPs are located within the coding sequence of the gene, suggesting that one or more of these SNPs may contribute to the disease association by modifying DDX39B expression. Through bioinformatics and gene expression analyses, I have uncovered mRNA isoforms encoding either the full-length protein or a novel short protein, and several mRNA isoforms that are candidate targets for nonsense-mediated decay. Importantly, I have established two of the SNPs could alter expression levels of these isoforms. The goal of this proposal is to provide functional links connecting DDX39B to the pathogenesis of MS. Specifically, I aim to: 1) elucidate its role in the regulation of IL7R exon 6 splicing; 2) understand the functional roles of the different protein isoforms; and 3) uncover the SNPs responsible for its association with MS. I will combine biochemical and genetic approaches to elucidate the mechanism by which DDX39B activates exon 6 splicing, and to characterize the functional roles of the protein isoforms. I will test the impact of selected SNPs on DDX39B expression by combining in vivo gene expression analysis and functional studies using reporter minigenes. Successful completion of this research will advance our current understanding of the molecular underpinnings of MS, in particular by functionally linking DDX39B to the pathogenesis of MS, and providing a functional characterization of the DDX39B gene. Given that DDX39B has been associated with numerous autoimmune disorders, our results could be relevant to the mechanistic etiology of other autoimmune diseases.

The secretory pathway in the guinea pig exocrine pancreas has been defined in the past in terms of 3H-leucine incorporated into secretory protein (autoradiography and cell fractionation studies). Using a two-dimesional gel technique (isoelectric focusing followed by SDS gradient gel electrophoresis), the secretory pathway will be redefined on a protein specific basis. This will involve studies on (1) cell fractions taken at varying time points during a pulse-chase protocol using a mixture of fifteen 14C amino acids, and (2) radiolabled secretion discharged into incubation medium from pancreatic lobules under varying conditions designed to favor evaluation of (a) discharge, (b) intracellular transport, and (c) the secretory pathway taken as a whole. Using mRNA derived from puromycin-high salt disassembly of bound polysomes from dog pancreas, the primary translation products obtained in vitro will be compared to radiolabeled secretion derived from pancreatic slices by SDS gradient gel electrophoresis and radioautography. Since preliminary studies suggest the presence of pancreatic precursors, attempts will be made using selective immunoprecipitation methods, to isolate several specific precursor-product pairs. Tryptic mapping and sequencing studies will be done on these pairs to determine if the additional peptide sequence is attached to the NH2 terminal end of the precursor molecule, as postulated by the "signal" hypothesis. BIBLIOGRAPIC REFERENCES: Cellular cyclic nucleotides and enzyme secretion in the pancreatic acinar cell. Haymovits, A., and G. Scheele. Proc. Nat. Acad. Sci. USA 73, 156-160 1976. Secretory Proteins of the Exocrine Pancreas. Scheele, G.A., in Cell Biology (P.L. Altman and D.D. Katz, eds.), Federation of American Societies for Experimental Biology, Bethesda, Maryland, 334-336, 1976.

We are conducting studies to determine the function of the Mi-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors in mammalian development. The Mi-Tfe family consists of four related genes- Mitf, Tfe3, Tfeb, and Tfec. The Mitf gene encodes the microphthalmia transcription factor (Mift). Mutations in the human MITF gene are responsible for a common human pigmentation and hearing disorder, Waardenburg syndrome type 2 (WS2). More than 20 independent Mitf mutations have been isolated in the mouse and shown to affect a number of cell types, including melanocytes, osteoclasts, and mast cells. In contrast, Tfeb, Tfe3, and Tfec were isolated by biochemical means and little is known about their function in vivo. In collaborative studies, we showed that the Mitf-Tfe proteins can bind the E-box sequence as homodimers or as heterodimers with other family members. To investigate the function of the Tfe genes in mammalian development, we used embryonic stem (ES) cell knockout technology to make germline null mutations in the three mouse Tfe genes. The effect of each mutation on development was then measured alone or in combination with mutations in other Mitf-Tfe family members. Surprisingly, these studies did not identify any essential functions for Mitf-Tfe heterodimers in development. This is in marked contrast to what has been observed for the Myc/Max/Mad family of bHLH-Zip proteins, where the heterodimers are thought to have essential functions. Future studies will attempt to use chemical mutagenesis and genetic modifier screens to identify other proteins in the Mitf signal transduction pathway.

The long-term objective of this project is to determine in molecular detain the energetic-structure-function relationship in exchangeable apolipoproteins, thereby providing an insight into molecular mechanisms of their action in the pathogenesis of atherosclerosis and other apolipoprotein-related disorders. Exchangeable apolipoproteins are water- soluble protein components of lipoproteins that mediate lipid and cholesterol transport and metabolism and play crucial roles in the pathogenesis of atherosclerosis, coronary heart disease, stroke, and other major human disorders including several forms of systemic and cerebral amyloidosis. Structural adaptability of apolipoproteins to heterogenous lipoprotein complexes and to plasma is absolutely essential for their functions, and has to be understood in detain in order to elucidate molecular mechanisms of apolipoprotein action in normal and in diseased states. The proposed work addresses this long-term goal through studies of the energetics, structure and folding pathway of the smallest human plasma apolipoprotein C-1 (apoC-1, 6 kDa). The ability of apoC-1 to activate lecitin: cholesterol acyltransferase (LCAT) may account for normal plasma levels of cholesterol esters in subjects with deficiency of the major LCAT activator, apoA-1. ApoC-1 delays the clearance of potentially atherogenic triglyceride-rich particles by inhibiting their uptake via the apoE-mediated low-density low-density lipoprotein receptor- related pathway. Thermodynamic and structural analyses of synthetic human apoC-1 and a series of its site-specific mutants targeted towards the predicted amphipathic alpha-helical regions will be carried out by using a combination of far-UV circular dichroism spectroscopy, differential scanning calorimetry, and x-ray diffraction methods. Such analysis will 1) dissect the folding pathway of lipid-free apoC-1 in solution, from partly folded monomeric to fully folded oligomeric or lipid-bound state; 2) determine, at the level of individual amino acids, the critical factors for the stability and cooperatively of the amphipathic alpha-helical structure in apoC-1; 3) determine the conformation apoC-1 in various self- associated status, such as 2D filaments and 3D crystals, to provide models fir a variety of functional apolipoprotein conformations. The results of this analysis will provide the energetic and structural basis for understanding mechanisms of functional apolipoprotein reactions and will help to understand their amyloidogenic properties, thereby leading to identification of rational therapeutic targets in a variety of apolipoprotein-related disorders.

In this application we argue that mechanisms of transition from acute to chronic pain critically depend on brain emotional and motivational learning and memory circuit, namely the properties of the corticostriatal system. This is a radical departure from the classic pain research tradition, which emphasizes mechanisms of nociceptive encoding and representation. We provide human and animal model data consistent with the idea, and propose unraveling underlying mechanisms and translating this information to clinical application by testing a drug treatment strategy for preventing transition to chronic pain We recruit Dr. Surmeier (a world renowned expert on the physiology and reorganization of the striatum) and Dr. Martina (a young scientist with expertise in molecular and electrophysiological studies of the brain), to collaborate with Apkarian on this project, and Drs. Fields and Zubieta act as consultants for the project. In Aim 1 we combine the expertise of the three collaborating labs (Apkarian, Martina, Surmeier) to link large-scale brain imaging outcomes to cellular, molecular, and electrophysiological changes that we predict the corticostriatal undergoes in the transition from peripheral nerve injury to neuropathic pain-like behavior in rodents. In Aim 2 we compare among potential drugs for preventing transition to chronic pain-like behavior in a rat model. In Aim 3 we use the best candidate drug in a human early phase clinical trial, combined with brain imaging, for prevention of transition to chronic pain. The successful completion of the study should dramatically change current notions regarding brain mechanisms of pain chronification, and provide a new concept of treatment strategy for preventing the transition to chronic pain.

Abstract In recent years, the demand for home hospice care has grown rapidly. Family members and friends who act as informal caregivers are essential to the provision of palliative care services;however, this role is not without adverse effects on the caregivers themselves. It is well documented that emotional needs of individuals caring for dying persons in their home are not well attended, and interventions aiming to provide support to informal hospice caregivers are notably lacking. In this context, problem solving therapy (PST) provides an overall coping process that fosters adaptive situational coping and behavioral competence. This reduces and prevents the negative effects of stressful life events with regard to both psychological and physical well-being. We propose a randomized controlled trial to fully evaluate the PST intervention for informal hospice caregivers. Additionally, we aim to evaluate how the modality of the intervention (face to face vs video) impacts its effectiveness. We propose a 4-year randomized trial study in which hospice caregivers will be randomly assigned to a group receiving standard hospice care with the addition of social support visits (attention control group) or a group receiving standard hospice care with the addition of the problem solving intervention delivered face to face (intervention group 1) or a group receiving standard hospice care with the addition of the problem solving intervention delivered via video (intervention group 2). The specific aims include an assessment of the impact of PST on caregiver quality of life, problem solving ability, caregiver anxiety, and caregiver perceptions. Furthermore, we aim to evaluate the cost effectiveness of the PST intervention. PUBLIC HEALTH RELEVANCE: Narrative The proposed research study explores a problem solving intervention that will improve caregivers'quality of life, and cost-effective innovative tools to support its delivery. Thus, the study is highly relevant to public health as it affects the health of a continuously growing segment of our population, namely these who are called to assume the essential role of informal caregiving, and aims to demonstrate the value of a feasible and sustainable problem solving intervention for hospice caregivers.

PROJECT SUMMARY Gastric atrophy and metaplasia are critical epithelial cell changes that promote gastric carcinogenesis. Gastric cancer is a major public health issue world-wide, being the third leading cause of cancer-related death in the world with nearly one million victims every year. Chronic inflammation is a major predisposing risk factor for the development of gastric cancer, demonstrated by the fact that patients with chronic atrophic gastritis have significantly higher risk of cancer than their healthy peers. While the association between chronic inflammation and cancer risk is clear, the exact mechanisms by which the inflammatory process in the stomach leads to carcinogenesis in some patients but not others are not understood. There is a complex cytokine milieu associated with any chronic inflammatory process, and polymorphisms in cytokine genes in human patients have been shown to have a significant effect on the risk of gastric cancer development. Due to the strong association between the prolonged cytokine production during inflammation and increased gastric cancer risk, we are interested in determining the mechanistic role that cytokines serve in regulating the development of gastric atrophy and metaplasia during chronic gastritis. Epstein-Barr Virus-Induced Gene 3 (Ebi3) encodes a protein subunit (EBI3) of the cytokine IL-27, which is well- characterized and has documented effects on CD4+ T cells during inflammation. EBI3 is also a component of the poorly understood immunosuppressive cytokine IL-35 thought to be made by regulatory T cells (Tregs). We use a novel murine model of inflammation-induced gastric atrophy and metaplasia, TxA23. In this model we have discovered that mice deficient in the production of Ebi3 have accelerated development of atrophy and spasmolytic polypeptide expressing metaplasia (SPEM), important preneoplastic epithelial cell lesions. We have also observed that Tregs, which are critical suppressors of gastritis and resulting lesions, exhibit a defective phenotype in Ebi3-/- mice. However, it is unclear whether this defect is due to the extrinsic lack of IL- 27 signaling or the intrinsic inability to express Ebi3, and thereby IL-35. Our hypothesis is that EBI3 expression is critical for suppressing the development of gastritis, gastric atrophy, and gastric metaplasia due in part to the effects of IL-27 acting on Tregs and increasing their suppressive functions. In Aim 1 we will determine whether IL-27 is the critical EBI3 containing cytokine responsible for suppressing disease progression. In Aim 2, we will determine the effect of IL-27 signaling into Tregs during the development of gastric atrophy and metaplasia.

The long term objectives of this research are to identify and characterize the cellular mechanisms underlying ethanol induced changes in neuronal development, and to assess the role of these changes in the etiology of CNS abnormalities associated with Fetal Alcohol Syndrome (FAS). Key neuropathologic features of FAS include altered neuronal morphogenesis and synapse formation in the hippocampus. Recent studies have elucidated some of the molecules and processes responsible for the distinct growth characteristics of axons and dendrites, thus providing the basis for novel experiments to determine the mechanisms underlying ethanol's disruption of neuronal development. The use of primary cultures of embryonic rat hippocampal pyramidal neurons is integral to the objectives of the project. Neurons in these cultures develop axons, dendrites, and synapses in a sequence of events that mimics their development in vivo. Experiments in this proposal are designed to use immunofluorescent cytochemistry coupled with quantitative morphometric analysis, time lapse videomicroscopy, and Fura-2 intracellular free calcium measurements in the following specific aims: (1) Compare the sensitivity of cultured hippocampal neurons exposed to ethanol at different times relative to development of axons, dendrites and synapses, (2) Distinguish whether ethanol induced changes in neuronal development result from direct effects of ethanol on neurons, or indirect effects on neurons mediated by astrocytes, (3) Determine whether ethanol's effects on process outgrowth involve altered regulation of intracellular calcium levels. The results of these studies will establish whether a disruption of process outgrowth and molecular compartmentalization is a key aspect of ethanol's neurodevelopmental toxicity and will provide important insight into the mechanism(s) underlying these actions. This fundamental knowledge can be expected to provide a basis for improving the identification and treatment of affected individuals.

DESCRIPTION (From the Applicant?s Abstract): The studies proposed herein will: (i) determine the roles of membrane FasL (mFasL) and soluble FasL(sFasL) in neutrophil-mediated inflammation within the eye and most importantly, (ii) determine whether sFasL can be used to downregulate neutrophil-mediated inflammation and prevent neutrophil-mediated ocular damage. Preliminary studies demonstrate that within the immune privileged eye, mFasL activates neutrophils and initiates innate immunity, while sFasL inhibits neutrophil activation ad prevents innate immunity. Most compelling though, was the ability of sFasL to block the pro-inflammatory effects of mFasL when both sFasL when both sFasL and mFasL were equally expressed within the eye. The proposed studies will utilize a novel method of expressing DNA directly within the cornea to characterize, in vivo, the inflammatory response induced by the different forms of FasL expressed within the corneal stroma. The ability of both mFasL and sFasL to directly activate neutrophils will be assessed in vitro using stromal cells transfected with the different FasL cDNAs. The final set of experiments will determine, in vivo, whether sFasL blocks neutrophil mediated inflammation in the cornea, and to elucidate, in vitro, the mechanism by which sFasL blocks mFasL induced neutrophil activation. We anticipate that these studies will yield new information as to how FasL regulates inflammation within the eye, and possibly lead to the development of new treatments using the soluble form of FasL to prevent ocular damage secondary to neutrophil mediated inflammation.

The 42 kDa processed fragment of the Plasmodium falciparum Merozoite Surface Protein 1, MSP1-42, is a leading protective human malaria vaccine currently undergoing clinical studies. Protective immunity is antibody-mediated, and directed to the C-terminal, immunodominant 19 kDa region of MSP1-42, or MSP1-19. Available evidence indicates that the vaccine-induced immunity to MSP1-42 or MSP-19 relies on the use of very powerful adjuvants (i.e. CFA) and on repeated hyper-immunizations to elicit high levels of protective antibodies. These are formidable obstacles in the way of field deployment of the malaria vaccine. Recently, we have shown that by refocusing the antibody responses to the N-terminal immuno-silent region of MSP1-42, MSP1-33, biologically active antibodies can be produced when combined with low concentrations of anti-MSP1-42 antibodies. This creates an opportunity to devise new vaccine constructs and/or immunization regimens to efficiently induce protective antibodies under the more favorable logistics of a reduced immunization schedule and the use of readily acceptable adjuvants. Accordingly, Specific Aim 1 will explore strategies to develop bivalent MSP1 vaccines as well as prime/boost immunization regimens, based on MSP1-33 and MSP1-42, in order to achieve a balanced induction of protective antibody responses. The focus will be on the use of less powerful adjuvants and a reduced immunization schedule. Specific Aim 2 will define experimental conditions in which combinations of anti-MSP1-33 and anti-MSP1-42 antibodies will inhibit parasites carrying different allelic and variant forms of MSP-42. This information, together with the down-selected vaccination strategies from Specific Aim 1, will be used to formulate MSP1-33/MSP1-42 vaccines to elicit strain-transcending, parasite inhibitory antibody responses. Significance: This exploratory project provides the critical proof-of- principle on which further development and validation of the dual MSP1-33/MSP1-42 vaccine strategy can be based. The strategy will rapidly lead to a new generation of MSP1-based malaria vaccines deployable with significantly improved efficacy and logistical feasibility, while utilizing the already existing technologies/platforms of demonstrated safety. PUBLIC HEALTH RELEVANCE - PROJECT NARRATIVE: Malaria is a major killer in developing countries and the development of effective vaccines is critical in controlling this deadly disease. This exploratory application seeks to improve the efficacy and the logistical feasibility of deployment of a leading candidate human malaria vaccine, Merozoite Surface Protein 1, MSP1. This is based on new and significant discovery of the immune responses to the MSP1 vaccine, which allows for novel re-design of the vaccine. Success in this project will move this vaccine forward in terms of significantly improving its potential to be effective in large populations in the field.

During mammalian development, the heart is the first organ to develop; the product of exquisite spatial and temporal control over cardiogenic mesodermal cell fate specification. From heart looping to cardiomyocyte cell fate determination, multiple transcription factors play critical roles during the development of the heart. Genetic silencing of Nkx2.5 in mice, the mammalian homologue of D. melanogaster tinman, results in embryonic lethality between 9.0 and 10.0 days post coitus due to abnormal heart morphogenesis. Clinically, missense and nonsense mutation in Nkx2.5 are highly conserved across multiple generations of families with high incidences of congenital heart defects. In Nkx2.5-/- mice, the cardiac specific isoform of the DEAD-box RNA helicase Mov10l1 (Csm) was found to be absent. DEAD-box RNA helicases are involved in virtually all biological processes involving RNA; specifically the nuclear DEAD-box RNA helicases p68 and p72 have been shown to be required for the processing of microRNA primary transcripts. MicroRNAs are critical for normal developmental processes are disregulated under pathological conditions. We demonstrate that Csm physically interacts with argonaute-2, a member of the RNA induced silencing complex, at P-bodies which are known to be sites of mRNA degradation. In addition, morpholino knockdown of Csm in the Danio rerio model system completely abrogates the heart looping process inhibiting cellular proliferation, whereas alpha-MHC promoter FLAG- Csm transgenic mice exhibit a hyperplastic heart. Transgenic animals have a significantly reduced survival rate. FLAG-Csm was found to co-immunoprecipitate a subset of cardiac microRNAs suggesting that it is actively involved in microRNA regulatory pathway. The overall hypothesis is that Csm is critical to both the normal development and function of the heart by facilitating the maturation of mircroRNAs throughout development and into adulthood. To test our hypothesis, we will determine the biochemical function of Csm and its interacting partners. Finally we will determine the biological and physiological effects Csm overexpression, and in conjunction with our biochemical and molecular analyses, determine the functional significance of Csm in the regulation of cardiac gene expression and the maintenance and function of the normal myocardium.

Project Summary Active transport of vesicular cargoes is vital to the targeted delivery of organelles, proteins, and signaling mol- ecules in the complex and crowded cellular environment. Accordingly, defects are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano- chemistry and structural dynamics of isolated motor proteins is essential for integrating the divergent mechani- cal and kinetic properties of cytoskeletal motor families. We have developed a number of powerful new bio- physical tools that reveal the modulation of mechanical and ATPase reaction kinetics under applied mechani- cal force, elucidate the essential rotational conformational transitions of specific domains within the motor pro- teins and produce reliable force, stepping dynamics, and local viscoelastic parameters of the cellular environ- ment. We will apply these unique tools to investigate the divergent biochemical and mechanical properties of myosin-I (Myo1) and dynein isoforms that have that have not yet been approached at the mechanistic detail now possible. Aim 1: Using simultaneous optical trapping and TIRF microscopy, determine stress- and strain-dependence of the binding and dissociation of ATP and ADP that fuel motion and control motor stepping; Aim 1A: in myosins 1b and 1c; Aim 1B: in budding yeast and mammalian cytoplasmic dynein. Aim 2: To determine the rotational motions of motor heads and lever arms that generate force and cargo translocation using single molecule polarized TIRF (polTIRF) microscopy; 2A: in myosins 1b and 1c; 2B: in yeast cytoplasmic dynein. We discovered that Myo1b and Myo1c have markedly different strain-dependent attachment lifetime, even though their unloaded kinetics and protein sequences are quite similar. We will apply our formidable single-molecule mechanical and fluorescence technologies to understand how these closely related motors have optimized their kinetic and structural variations for their different cellular roles. Mammalian and Saccharomyces cerevisiae cytoplasmic dynein have overlapping roles in their respec- tive cells, but their properties are very different. We are in the unique position to compare them using advanced biophysical methods. We will determine their strain-dependent ATPase dynamics and rotational motions. We anticipate that these studies will provide insight into the motors' mechanisms, and will also reveal biochemical and mechanical adaptations to their distinct functions. Aim 3: We will use our unique new technology for measuring and calibrating optical trap signals within live cells to 3A: determine the functional speciali- zations that vary the complement of motors among early and late endosomes, and small and large la- tex bead compartments (LBCs) endocytosed or phagocytosed into live cells. 3B: measure forces and motor dynamics during remodeling of endoplasmic reticulum (ER) by interaction with endosomal vesi- cles. All three aims represent close, essential collaborations with Drs. Ostap, Holzbaur and Shuman. Many cargos have opposing motors bound simultaneously; these motors may operate in teams functioning either cooperatively or competitively. We will focus on the role of oppositely directed motors at critical junctures in cellular organelle trafficking: early and late endosomes and the remodeling of endoplasmic reticulum we have observed when transport vesicles interact with the ER. Together, these studies will provide insights into the roles of the molecular motor families in regulating organelle motility, morphology and remodeling. These stud- ies will lead to a much improved understanding of myosin I and dynein isoforms that operate very differently from the better understood kinesin and myosin motors, leading to a more rigorous understanding of their func- tions in cell biology and disease.

Marfan syndrome is a systemic disorder of connective tissue with autosomal dominant inheritance and a prevalence of at least 1 in 10,000 individuals. Cardinal manifestations involve the ocular, skeletal and cardiovascular systems. While all clinical features impair quality of life, cardiovascular features are uniquely associated with early mortality due to myocardial failure or aortic rupture. Variability in the age of onset, tissue distribution, and severity of manifestations is common both in and among families. Despite major advances in the medical management of Marfan syndrome, the effectiveness of such therapies is compromised by the need for accurate and early diagnosis. It is now known the mutations in the gene on chromosome 15 encoding fibrillin (FBN1), a major glycoprotein component of the extracellular microfibril, can cause Marfan syndrome. Despite this understanding, gaps in knowledge include the molecular basis for clinical variability, the functional significance of various domains and residues of fibrillin, whether MFS truly lacks genetic heterogeneity, and the clinical spectrum that can be associated with fibrillin gene defects. The overall goal of this proposal is to gain insight into the normal and aberrant biology of the fibrillin protein (and of microfibrils) by analyzing the effects of naturally occurring mutations on clinical, cellular, and biochemical phenotypes. This will require comprehensive study of the FBN1 gene in patients with Marfan syndrome and clinically related disorders. Using refined mutation defection techniques and a novel panel of highly informative intragenic microsattelite polymorphisms for haplotype segregation analysis, it will be possible to determine whether FBN1 gene defects are associated with all cases of Marfan syndrome, to what degree allelic and/or genetic heterogeneity account for interfamilial clinical variability, and whether FBN1 gene defects underly divergent phenotypes that appear to manifest primary disruption of the elastic fiber-microfibrillar array system. By similar methods, the genes encoding other constitutive elements of extracellular microfibrils, or proteins known to associate with microfibrils, can be scrutinized as potential disease loci or as the site of sequence variants able to modify the clinical expression of mutant fibrillin alleles. Immunofluorescence and quantitative pulse-chase methods will allow determination of the cellular and protein phenotypes. In addition, much can be learned about the fundamental structural and functional properties of FBN1 domains by the study of wild-type and mutant forms using NMR spectroscopy. Finally, expression studies in cell culture and transgenic mice will allow the examination of the pathobiology of fibrillin in models that mimic the physiologic complexity of the human system. Correlation of mutant genotype to clinical, cellular, and biochemical phenotype will yield insight into the normal functions of specific fibrillin domains and residues, will enhance our understanding of pathogenesis, and should have both diagnostic and prognostic significance.

Latex rubber articles in the form of condoms and gloves are used as barriers against the transmission of infectious diseases. The medical community have identified the use of condoms as an effective means for preventing the transmission of the human immunodeficiency virus (HIV) and other sexually transmitted diseases. Universal precaution procedures for health care workers requires wearing, and changing, latex examination gloves. Formed rubber latex items frequently reduce but do not eliminate completely the possibility of transmission of viruses such as HIV. This proposal suggests a method for coating latex articles with a thin, biologically bland, non-porous polymer to eliminate the possibility of virus transport via micropores in the latex membrane.

The proposed Michigan Regional Comprehensive Metabolomics Resource Core (MRCMRC or MRC2) is a fully integrated program that will provide researchers nation-wide with the expertise and infrastructure to determine the levels of known and unknown metabolites in cells, tissues and biological fluids. In addition, the MRC will provide opportunities for training in the technology of metabolomic analysis, statistical analysis and bioinformatic evaluation of metabolite data as well as approaches to Incorporation of metabolomics into basic, preclinical, translational and clinical research. Incorporated into this service component will be a robust research component directed towards improving the breadth of metabolite detection, the quality and efficiency of metabolomic analysis and importantly, tools to turn spectral data into knowledge to for the researcher The MRC2 will contain an Administrative Core to oversee and integrate operations; an Analytical Core to help design experiments for directed quantitative measure of metabolites or high-throughput evaluation of large numbers of known and unknown metabolites; a Data and Information Technology Core which will perform primary data processing or re-mining of archival data and will serve as a data repository; a Statistics and Bioinformatics Core which will assist researchers in the statistical evaluation of metabolomic data and integration with other 'omics or phenotypic data; and a Promotion and Outreach Core which will organize and provide seminars, symposia, workshops and web-based videos to increase the lay and research communities knowledge and use of metabolomic data. PUBLIC HEALTH RELEVANCE: The RCMRC grant is directed towards providing increasing the technology to measure small molecules (metabolites) found in the body. These metabolites are the building blocks of all cells and participate in all body processes. By providing researchers with ways to measure the metabolites accurately from cells and tissues and in the blood, new insights into the ways In which changes in metabolism can contribute to diseases, such as diabetes and caner will be found. With this knowledge, new ways to prevent and treat a variety of diseases may be possible.

Recently investigators from several countries have reported an increased rate of birth of malformed or maldeveloped infants to alcoholic mothers. These authors suggest that the observed defects are the direct result of maternal alcoholism. The observation of withdrawal symptoms in neonates whose alcoholic mothers had been intoxicated preceding (just before) delivery, raises the additional question whether such children may have an increased sensitivity to alcohol in later life. The present series of studies using a rat model are aimed at investigating the effects of ethanol during the period of gestation on the prenatal development and later behaviors of the offspring. The proposed research has 3 objectives: 1) At maturity, to compare the two groups of offspring for increased sensitivity to alcohol using intragastric self-administration techniques, 2) To compare the offspring of alcohol treated and control mothers with a battery of development and behavioral tests including measures of learning, 3) To isolate the effects of the administration of ethanol from nutritional stress by the isocaloric replacement of ethanol by carbohydrate and by pair feeding of control and experimental animals.

Whether by administration of a vaccine or anti-pathogenesis substances, intervention of schistosomiasis by immunologic means is a major goal of schistosomiasis research efforts today. This application is to renew our program examining the role Schistosoma mansoni irradiated cercariae play as a model for inducing anti-schistosome immunity and anti-pathogenesis, and as candidates for a live vaccine. To determine vaccine effectiveness in situations approximating that to be encountered in the field, immunity will be assessed aginst multiple 'trickle' cercarial challenges. Using mice immunized with irradiated Schistosma japonicum cercariae, the specificity of vaccine induced resistance will be more thoroughly examined by challenge with the homologous or a heterologous schistsome species (S. mansoni). Anti-pathogenic effects of live vaccine administration will be examined in the mouse, along with the possibility that immunologic suppression of protection develops in cases where suboptimal levels of immunity are stimulated. Basic issues related to a recently developed cryopreserved live vaccine will also be addressed. These are: (1) by autoradiographic techniques determine the vaccine's migratory properties in the naive host, and (2) Attempt to enhance the vaccine's efficiency by concurrent administration of immunopotentiators and the cryopreserved vaccine. These experiments should answer pressing questions related to anti-schistosome immune mechanisms, while evaluating more critically the feasibility of irradiated larve as components of a live vaccine.

The proposed work addresses several of the Institute of Medicine's (IOM's) national priorities in clinical effectiveness research: disparities in oral health and health care. Combining extant dental data with other components of the rich VA patient databases, we will be able to examine the effectiveness of fluoride in preventing caries in patients with five of the 14 priority conditions specified by AHRQ: medically compromised veterans with dementia, depression and bipolar disorder, diabetes, HIV-Aids and persons with alcohol dependence. Recent data suggest that dental caries is as much of a problem in adults as children (Dye, et al., 2007). However, significant knowledge gaps remain in our knowledge base regarding prevention of caries in adults (Griffin et al., 2007). Using an extant, rich electronic data base from a health care system providing dental treatment to over 342,000 veterans annually, this proposed retrospective, longitudinal study takes maximum advantage of a unique opportunity to examine the following specific aims in a population of medically complex veterans. Aim 1. Examine the effectiveness of prescription-strength, self-applied fluoride and professionally applied fluoride in the prevention of caries in medically compromised veterans who are at high risk for caries. Veterans will be defined as at risk for caries if they have had two or more restorations in the previous year. Aim 2. While recent evidence suggests that fluoride is effective in preventing caries in adults, and multiple exposures to fluoride reduces caries risk in children (Weintraub, 2006), limited data exist showing that multiple exposures or modalities are more effective than a single modality in adults. Thus, we have a unique opportunity to examine whether multiple exposures to fluoride will be more effective than a single exposure. Aim 3. Over the past year, the Department of Veterans Affairs introduced a quality measure (the fluoride monitor) that examines, on a quarterly basis, the percent of veterans at high risk for caries who receive fluoride treatments. This project will examine the effectiveness of the introduction of this fluoride monitor in reducing the rate of restorations in medically compromised veterans at high risk for caries. PUBLIC HEALTH RELEVANCE: Despite dramatic gains in oral health and access to dental care during the past 50 years, dental decay remains a problem in medically complex older adults. The aging of the 76 million baby boomers, who are retaining more teeth than ever before, makes this a compelling problem. The purpose of this study is to examine the effects of prescription strength, self-applied, brush on fluorides and in-office fluoride treatments in preventing decay in a real-world setting of medically compromised older adults at high risk for dental decay.

The purpose of this project is to describe the ensemble of afferent neural responses to intracochlear electrical stimulation as a function of stimulation variables. Several types of ensemble models will be investigated, including combinations of the various field and neural models from Projects II and III and the addition of stochastic properties to such combined models. Each ensemble model will predict both the spatial and temporal patterns of neural excitation. Conditions will include manipulations in (a) electrode coupling configuration [e.g., monopolar, radial bipolar, longitudinal bipolar], (b) stimulus waveform [e.g., sinusoidal, pulsatile], and (c) the number and geometry of electrode pairs in multichannel electrode arrays. Predictions from models spanning these conditions will test our understanding of the mechanisms underlying psychophysical and behavioral findings with respect to threshold, dynamic range, intensity discrimination, frequency discrimination, channel interactions, and channel discrimination. These predictions will be evaluated directly within this project using single-unit experiments. Such evaluations will, in turn, allow further refinement of the ensemble models and their constituent parts. Iterations of this model/test cycle will periodically refocus or efforts toward key gaps in existing knowledge.

The goal of this project is to develop an Early Vocalization Analyzer (EVA), a computer program to automatically analyze recorded samples of infant vocalizations. This program will be the basis of a screening test, for use in hospitals and clinics, to evaluate which infants are at-risk for later communication or other developmental problems. The major purpose of this Phase-I project is to determine whether the landmark detection theory of Stevens, as implemented for the recognition of adult speech, can be adapted to the analysis of syllabic pre-speech vocalizations produced by typically developing infants. We will also merge this landmark- detection algorithm with a kernel version of EVA, developed by a team at Northeastern University, to produce a proof-of-concept prototype that will count the number of utterances, detect and count syllable-like vocalizations, and classify them according to fundamental frequency and duration. The goal of Phase II is to develop a commercial software program to automatically analyze infant utterances from audio recordings, as a non-invasive research and clinical-screening instrument. PROPOSED COMMERCIAL APPLICATIONS: The product would be useful for hospitals and infant-screening clinics assessing developmental risks, as well as hearing deficits. It could also be used by treatment centers to the efficacy of early intervention. Finally, it would provide a research tool for investigations of the earliest stages of infant speech and of age-specific control of the speech articulators.

Articular cartilage is the remarkably durable tissue that covers the articulating bone surfaces in our joints and permits pain-free movement by greatly reducing friction between bones and distributing stress. Unfortunately, when cartilage is damaged due to injury or disease, it has a limited capacity to heal and can lead to premature arthritis. Arthritis is a major health issue that is predicted to increase as our population ages. As such, there is considerable interest in the development of techniques to repair or reconstruct damaged articular cartilage. The implications of a successful method for cartilage repair would be great in terms of the number of patients affected and their quality of life. Importantly, in this era of escalating health care costs, successful cartilage repair would decrease the long-term costs of health care related to joint replacement and multiple revisions thereof. Our long-term objective is to develop a tissue engineering approach for cartilage repair using periosteum. Periosteal grafts have the potential to contribute to each of the three requirements for tissue engineering (scaffold, cells, growth factors), and have been used successfully in biological resurfacing for the repair of damaged articular cartilage. The principle obstacle to overcome for the use of periosteal tissue grafts for tissue engineering is the declining chondrogenic potential of periosteum with increasing age of the patient. We hypothesize that subperiosteal injection of specific growth factors can be used as a pretreatlnent to "activate" periosteum for articular cartilage tissue engineering. In essence, the engineered tissue will be prepared in vivo for transplantation. In this proposal, we plan to examine the potential for subperiosteal injection of known chondrogenic growth factors to enhance (1) the number of chondrogenic precursor cells in periosteum in vivo (Aim 1), (2) the in vitro chondrogenic potential of periosteum (Aim 2) and (3) the potential of periosteum to repair articular cartilage defects (Aim 3). Beyond articular cartilage repair, these studies have the potential to impact the use of periosteum for other applications such as tissue engineering of bone. In addition, by eliminating the need for cell culture expansion facilities and expertise, the cost of this approach should be considerably less thereby making cartilage repair more globally accessible.

Liver fibrosis is the excessive accumulation of extracellular matrix as a wound healing response to chronic injuries from a broad range of liver diseases, irrespective of the underlying etiology. Advanced liver fibrosis may lead to cirrhosis, liver failure, and neoplasia. Extracellular matrix deposition is crucial to wound healing by promoting tissue formation and integrity, but commonly becomes uncontrolled in chronic injuries and leads to fibrosis. Ductular reaction is part of the injury response in the liver occurring at te interphase of the parenchymal and portal compartments that promotes bile duct regeneration. Although prominent in response to biliary and cholestatic damages, ductular reaction is observed in virtually all chronic human liver diseases. Recent studies have revealed that the matricellular protein CCN1 induces cholangiocyte proliferation and ductular reaction in response to cholestatic injuries through integrin ?v?5-mediated activation of NF?B, leading to Jag1 expression and Jag1/Notch signaling. Furthermore, CCN1 is able to restrict and resolve liver fibrosis induced by either hepatotoxin (carbon tetrachloride) or cholestasis (bile duct ligation) b inducing cellular senescence in myofibroblasts derived from hepatic stellate cells and portal fibroblasts. CCN1 also appears to regulate myofibroblast activation and resolution of inflammation. Based on these observations, we hypothesize that CCN1 serves multiple functions in different stages of hepatobiliary injury repair through binding distinct integrins in different cell types. We will scrutinize this hypothesis in three specific aims: (1) to conduct genetic and functional analyses of the heretofore unexplored CCN1-?v?5-NF?B axis in ductular reaction; (2) to examine how CCN1 induces the reactive cholangiocyte phenotype and differentiation of hepatic progenitor cells in the ductular reaction niche; and (3) to investigate how CCN1 regulates the initiation and termination of fibrogenesis in injury repair. These studies will elucidate the diverse roles of CCN1 as a critical regulator of injury repair in chronic liver diseases, and may lead to new therapeutic targets and treatment strategies for the prevention and treatment of liver fibrosis.

We propose to investigate the impact of the built environment characteristics on body size, physical activity and diet in residents of New York City (NYC). We will use data from three large human health studies. The first data set will be from 1000 Black and Hispanic children enrolled in prospective cohort study of Head Start families. From all of the children we will have complete demographic data, and mother's reports on the height and weight and physical activity of the child. From 500 of the children data will be available on skin fold thickness, body mass index (BMI) and 6 days of physical activity monitoring. Data will be available at baseline and from two annual follow-ups of the cohort. The second data set will be from 18,000 adult New Yorkers from whom we will have demographic data and objectively measured BMI. The third will be from 2,400 healthy Black and Caucasian. From these women we will have demographic data, questionniare data on diet and physical activity and objective measures of BMI, waist and hip circumference and body fat by bio-impedence. From all of these subjects we will have a home address which we will geocode into a CIS model of New York City. Multi-level modeling approaches will be used to determine whether neighborhoods characterisitcs (for example; land use, street design, availability and quality of parks and retail food stores, zoning, public transport, farmers markets) predict body size, physical activity and diet. Because of the very large immigrant population in NYC we will conduct qualitative research on how acculturation to the built environment of NYC impacts body size. Prior work suggests that acculturation to main stream American norms leads to weight increases in immigrants and their children. We do not see this effect in our data and propose that NYC is not "main stream America". We propose to conduct qualitative research on Hispanic mothers and children in the Head Start study and investigate how they relate to the built environment of NYC. Our goal is to develop a new measure of acculturation that includes adaption to the built environment of NYC and will be useful in studies of obesity. The quantitative research will also be used to develop new GIS based measures of aspects of the City that are important to the mothers. This research on the phsyical environment and adaption to it, will identify strategies for building and organizing communites that increase physical activity, improve diets and reduce obesity.

Funds are requested to support six (6) full-time predoctoral trainees per year. The program seeks to develop researchers able to contribute to analysis of social phenomena and problems and an understanding of underlying processes. A foundation of "core" courses in the major subdisciplines of our field, regular research meetings and special topic seminars, and an intensive research apprenticeship are employed to enhance trainees' skills in conceptual analysis, theory development, and research. The overlapping interests and shared commitment of our faculty naturally lead our students to emphasize work that speaks to important social problems and their amelioration. Ongoing work involves the role of basic cognitive, motivational, and affective processes in intergroup conflict in the under-mining of minority students' self-confidence and aspirations, and various social and health problems such as interpersonal violence, debilitating shyness, and depression. Three themes-the role of self and self-reflective processes, the significance of emotion and emotional regulation, and the need for a broader cultural perspective-have played a particularly important role in guiding our efforts. Approximately 85 students apply for admission to the Stanford social program each year, of whom 3 or 4 especially promising candidates enter the program and plan a course of study suited to meet the Department's formal requirements and their own interests and professional aspirations. As they progress, students take increasing responsibility for originating and pursuing their research. Students are encouraged to work with multiple advisors, and to cross traditional disciplinary borders. Numerous interdisciplinary programs at Stanford offer additional research and educational opportunities. Most of our graduates take academic positions, but significant number elect positions in applied settings concerned with health care, education, or new communication technologies.

A role for vitamin D in the pathogenesis and treatment of Mycobacterium leprae (mLEP) has been presumed for many years. Pafients suffering from the progressive, bacilli-abundant lepromatous form (L-lep) ofthe disease are more likely to be vitamin D-deficient and benefit clinically from ultraviolet B (sunlight) irradiafion or dietary supplementafion vitamin D. Such pafients are also at risk for developing dysregulated over- producfion of the acfive vitamin D metabolite 1,25-dihydroxyvitamin D (1,25D) from circulafing 25- hydroxyvitamin D (25D) by disease-activated macrophages. On the other hand, intracellular 1,25D synthesis and acfion at the level of the vitamin D receptor (VDR) is crucial for mounfing an anfimicrobial response to mLEP. Therefore, the mechanism(s) that govern vitamin D metabolism and acfion in leprosy is a key component to the disease. Our recent studies In vitro clearly demonstrate the differenfial expression of the funcfional elements ofthe vitamin D system, including CYP27B1-hydroxylase and the VDR in Type I and II interferon-driven, T-lep and L-lep granulomas, respecfively. Taken together, these clinical and laboratory findings lead us to theorize that mLEP Infection and the respective T-lep or L-lep downstream interferon- directed pathways, will differentially Impact the synthesis, metabolism and function of active vitamin D metabolites in the macrophage and other elicited, VDR-expressing inflammatory cells in the infectious microenvironment ofthe host with leprosy. To test this hypothesis, we will undertake three conceptually novel, mechanisfic aims. First, the vitamin D system components (CYP2R1, vitamin D hydroxylase; CYP27B1; CYP24A1, 24-hydroxylase; and VDR) will be quantitafively mapped in T-lep and L-lep granulomas at the single cell level. Second, the orchestrated effects of T-lep or L-lep immune response secretomes, and associated downstream interferon responses, on the metabolism and immunoacfion of vitamin D in human inflammatory cells will be characterized using innovafive molecular tools developed in Projects 1 and 3. Third, using recently-conceived RNA sequencing technologies, the funcfional consequences ofthe human host vitamin D deficient state, and its rescue In vitro and in vivo, on immune responses to and killing of mLEP will be probed. When analyzed in concert with the experimental results of the other CORT projects, it is anficipated that this work will set the stage for the pracfice of manipulating human vitamin D balance in promofion ofthe innate and adapfive immune response in leprosy specifically and in granuloma-forming diseases in general.

Inductive mechanisms play a major role in the development of the central nervous system as well as of other tissues in the organism. In particular, given the importance of the visual system in vertebrates, induction of eye development is one of the most extensively studied and serves at a paradigm for the inductive process. However, little is known about the induction of the neuroretina. The proposed research will utilize a combination of molecular, biochemical and histological approaches to identify factors that induce formation of the retina. We will undertake two parallel strategies. Firstly, we will investigate the effect of candidate retinal inducers identified in other systems. In addition, we will also perform an expression screen of Xenopus cDNA library for transcripts that induce formation of the retina. We believe that the combination of these two approaches will successfully identify genes that play a role in retinal development. These experiments will also further the understanding of general inductive mechanisms and their regulation. In addition, further comprehension of the inductive process in the eye will advance the prospects for regeneration and repair of ophtamological damage.

Tests whether bisphosphonate (daily oral alendronate) therapy maintains femoral and vertebral bone mass in women over 65 years old.

The Cullin-RING ubiquitin E3 ligase (CRLs) super-family is responsible for much of the signal-dependent protein turnover in eukaryotes. CRL specificity derives from the identity of the substrate adaptor, which often interacts with the substrate in a modification-dependent manner. With more than 200 distinct CRL substrate adaptor proteins for the 7 human cullins, the CRL system controls many facets of biology that impinge on disease and aging. However, our understanding of CRL-substrate relationships is largely limited to a few well- studied adaptors, with many CRL adaptors remaining unstudied. We have taken a multi-pronged approach that uses Global Protein Stability (GPS) profiling, quantitative diGLY capture proteomics, and substrate capture by interaction proteomics using specific CRL substrate adaptors to identify more than 600 high priority candidate CRL substrates. Nevertheless, thus far only a portion of the human proteome has been sampled for substrates and that vast majority of candidate CRL substrates have yet to be paired with the appropriate CRL substrate adaptor protein (such as an F-box protein). To address these major limitations, we propose the following aims: 1) AIM 1 will provide a more comprehensive database of candidate CRL substrates in human cells through further development and screening of a v3.0 GPS system, thereby accessing components of the proteome that are not accessible through the current v2.0 system. Improvements will include new C-terminally tagged GPS libraries, bar-coding, as well as Next-gen sequencing of sorted libraries, thereby avoiding limitations of microarray hybridization methods currently employed. 2) AIM 2 will utilize focused high throughput screening and quantitative diGLY proteomics to specifically link high priority candidate substrates to specific cullins and will use both GPS and RNAi screening, as well as interaction proteomics, to link CRL1 substrates with specific F-box protein adaptors, followed by extensive validation. This analysis will provide the first large-scale analysis of the CRL1-F-box system in mammals. 3) AIM 3 will produce a web-based database for dissemination and analysis of CRL-substrate relationships by the community. In addition, selected high priority candidate substrates and the corresponding F-box adaptor proteins will be analyzed through biochemical and cell biological methods to place these proteins into regulatory and physiological pathways with an initial focus on a BACH1, a signal-dependent transcriptional repressor of NRF2-dependent genes and a novel substrate of SCFFBXL17 discovered via our proteomics platform. Together, this work will substantially improve our understanding of the CRL-substrate landscape and will set the stage for in-depth studies that further define signals that dynamically control the proteome via CRLs.

Abstract Marijuana is the most commonly used illicit drug in the United States, and prevalence rates have increased over the past several years after a period of relative stability. Currently there is no approved medication to treat cannabis use disorder, and relapse rates following behavioral interventions remain unacceptably high. There is an urgent need for pharmacological and psychosocial interventions, but optimal treatment approaches cannot be developed or implemented without understanding mechanisms that underlie and maintain marijuana use. Exposures to drug-related stimuli and stress have been found to increase craving (humans) and drug-maintained responding (mostly in laboratory animals). Given that continued use and relapse likely result from multiple factors, exposure to drug-related cues during periods of stress may increase the likelihood and intensity of drug seeking behavior. Few clinical studies have focused on the interaction of stress and cues on craving and drug-seeking; thus our understanding of the separate and combined effects of stress and cue exposure on drug seeking is currently limited. In a double-blind, placebo controlled cross-over study, 14 males and females with mild-to-moderate cannabis use disorder will receive the pharmacologic stressor, yohimbine (0, 20, and 40 mg), prior to marijuana and neutral cue exposure. Our primary outcome measure is marijuana-seeking behavior assessed using a progressive ratio marijuana choice task. Participants will be given 11 choice opportunities on the choice task to work for either puffs from a marijuana cigarette (6.0% THC; max 11 puffs) or money alternative (max $22). Specific aims are to determine whether: (1) presentation of polysensory marijuana cues increases marijuana craving and choice to self-administer marijuana; (2) placebo-controlled administration of a pharmacological stressor (yohimbine: 0-mg, 20-mg, and 40-mg, PO) alters marijuana craving and choice to self-administer marijuana; (3) marijuana-related cues and yohimbine have additive effects on marijuana craving and choice to self-administer marijuana; and, (4) craving elicited by marijuana-related cues, yohimbine, or both in combination mediates (within subject) marijuana puff-maintained responding. Our overarching hypothesis is that a sustained pharmacological stressor will potentiate cue-induced marijuana craving and seeking. The objective is to understand cue- and stress-induced cannabis abuse to effectively target these risk factors for intervention. To the best of our knowledge, this is the first study to investigate subjective, physiologic, and endocrine responses to a pharmacologic stressor combined with drug-related cues in chronic cannabis users, and to investigate their additive effects on marijuana craving and seeking. Results from this project will advance our understanding of mechanisms that underlie drug seeking behavior and relapse, as well as provide a test for compounds that reduce stress or craving and thus may prove beneficial for treatment of cannabis and other drug use disorders.

In this merit extension proposal, we are requesting funds to extend the critical findings of S1P release and signaling in the vasculature. The first major aim of the research is to understand how S1P metabolic enzymes are regulated in the vascular endothelial cells to impact local (autocrine) as well as systemic S1P signaling. Secondly, we will fully explore the basic mechanisms and physiological impact of the S1P signaling system as a sensor of biomechanical shear stress on the endothelial cells. First, we hypothesize that Sphk1 in the endothelial cell is primarily involved in local/autocrine signaling mode important for endothelial cell homeostatic and angiogenic reactions. We will derive an endothelial cell-specific inducible knockout of Sphk1 and examine angiogenesis, endothelial cell patterning, pathologic angiogenesis, vascular permeability and inflammation. To examine the effect of systemic S1P, which is derived primarily by red blood cells, we will examine endothelial cell functions in the red blood cell-specific Sphk1 knockout. Localized S1P signaling will be defined at the morphologic and ultrastructural level by immunolocalization studies in vivo as well as in primary cells. Second, we will explore the novel hypothesis that S1P metabolism and signaling constitutes a novel shear sensor/ signaling module in the endothelial cells. Mechanisms of how SIP is formed and released will be explored in detail in cultured vascular endothelial cells. In particular, we will focus on mechanisms that down regulate SIP lyase enzyme.\Interestingly, laminar shear-induced downstream signaling mechanisms are critically dependent on S1P1 receptor signaling, suggesting that this receptor is a sensor of physiological shear stress. We will explore this mechanism in detail using cultured endothelial cells. We anticipate that these experiments will allow us to better understand the role of sphingolipid metabolism and signaling in vascular endothelial cells. Since SIP receptor modulators have entered the therapeutic era, this knowledge is likely to have important clinical utility.

Over the past eleven years, we have worked as a Program Project team consisting of four investigators with diverse expertise in the areas of epithelial cell biology, structural biology and membrane trafficking to study, in several closely interrelated projects, the cell and molecular biology and diseases of mammalian urothelium. Our research focuses on, as a central and unifying theme, a group of integral membrane proteins called uroplakins that represent major differentiation markers of mammalian urothelium. During the last granting period (2004-2009), our team has demonstrated that abrogation of uroplakins in transgenic mice resulted in compromised urothelial barrier function and overactive bladder; that defects in Rab27 and Vps33a lead to a depletion of fusiform vesicles and an accumulation of multivesicular bodies, respectively, thus establishing their involvement in uroplakin trafficking; that FimH can induce transmembrane conformational changes in the uroplakin receptor complex thus providing a novel mechanism for the bacterium-induced host cell changes; and that distinctive molecular alterations in genetically engineered mice underlie the two pathways of urothelial tumorigenesis. Our team has therefore functioned well in pursuing biologically important problems related to urothelial growth, differentiation and diseases; in having synergetic interactions and extensive collaborations; in effectively sharing resources; and in having made significant progress advancing the urothelial biology field. During the next five-year grant period, we will continue to work as a team to ask the following questions: What are the roles of uroplakins in the stabilization, enlargement and repair of the urothelial apical surface (Project 1)? What are the roles of molecular machineries including MAL and Rab27b in regulating uroplakin trafficking (Project 2)? What is the structure of uroplakins and how does the uroplakin complex anchor into an underlying cytoskeleton (Project 3)? And what are the roles of individual uroplakins and their subdomains in the uroplakin receptor complex in mediating the bacterial binding-induced signals in host umbrella cells (Project 4)? Results from this highly collaborative and synergetic team effort will lead to a better understanding of urothelial function, and have implications on a number of important urological problems including bladder outlet obstruction and urinary tract infection.

The epidermal growth factor receptor tyrosine kinase (EGFR) is one of the most commonly activated oncoproteins in lung adenocarcinoma, glioblastoma and other cancers. The identification of cancer-associated EGFR mutants, and their predictive power to select patients for treatment with EGFR inhibitors including erlotinib and gefitinib, has led to a major advance in cancer treatment. The collaborative efforts of the Eck and Meyerson laboratories for the past seven years have focused on understanding the structure-function relationship of cancer-derived EGFR mutants with the overarching goal of improved targeted therapies for cancers bearing these mutations. Refinement of our understanding of the mechanisms of action of mutant EGFR, and the spectrum of response of specific mutants to EGFR inhibitors, will permit more efficient development and application of EGFR-directed therapies. In this renewal, we will undertake the following Specific Aims: Aim 1) Characterize the response of novel EGFR mutants, identified by genomic studies, to low- molecular weight ATP-competitive enzymatic inhibitors and to antibody therapies, Aim 2) Perform structural and functional studies of the exon 20 insertion mutants of EGFR, which are resistant to gefitinib, erlotinib and cetuximab, to ascertain potential therapeutic approaches, and Aim 3) Analyze the substrate specificity of wild- type and cancer-derived EGFR mutants through peptide library array experiments, mass spectrometry-based phosphoproteomics, and crystal structure determination. Execution of these aims will help define the pathogenesis of EGFR-driven tumors and will thus provide a critically important mechanistic foundation for clinical trias targeting EGFR in a variety of cancer types using known agents. Additionally, our efforts will structural and mechanistic foundation for development of next-generation EGFR inhibitors that can be effective in patients whose tumors carry EGFR mutants that are resistant to currently available inhibitors.

The purpose of this stud yis to estimate the effectiveness of AR-623 inducing a complete remission in first relapse acute promyocytic leukemia (APL) patients who have not received oral all-trans-retinoic acid (ATRA) within one year.

Myocardial infarction (MI) has emerged as a major health problem during the past two decades. In the infarcted myocardium, loss of contractile myocardium and blood vessels is induced by MI. Cardiac repair at the site of myocyte loss preserves structural integrity and is integral to the heart's recovery. Angiogenesis, the growth of new blood vessels, is critical for cardiac repair following infarction. Impaired angiogenesis in the infarcted heart can lead to rupture and immature/weakened scar tissue. Stimulation of angiogenesis is beneficial in the treatment of coronary artery disease and cardiac repair. Angiogenesis is a tightly regulated process and numerous inducers of angiogenesis have been identified. There is growing recognition and experimental evidence that reactive oxygen species (ROS) plays an important role in stimulating cardiac angiogenesis. However, its underlying molecular mechanisms remain to be elucidated. Oxidative stress is developed in the infarcted heart, particularly in the infarcted myocardium during the early stage of MI, and is temporally and anatomically coincident with the formation of new vessels. The overall objective of this proposal is to explore the underlying mechanisms by which ROS promotes angiogenesis in the infarcted heart. Angiogenesis occurs through degradation of basement membranes/extracellular matrix (ECM) followed by endothelial cell proliferation and migration, tube formation and vessel maturation. Using an experimental MI model created by coronary artery ligation in Sprague-Dawley (SD) rats with pharmacological intervention; inducible nitric oxide synthase (iNOS) gene knockout mice; and cultured endothelial cells, we intend to fulfill the following specific aims: Specific Aim 1: To explore whether ROS promote angiogenesis by regulating endothelial cell migration and adhesion in the infarcted myocardium. Our hypotheses are: 1) ROS regulate the balance of matrix metalloproteinase (MMPs) and tissue inhibitors of MMPs (TIMPs), enhancing degradation of basement membrane and other ECM components, thus allowing endothelial cells to detach and migrate into the infarcted myocardium; and 2) ROS stimulate the expression of angiogenic integrins, thereby activating the integrin signaling pathway and promoting endothelial cell adhesion and migration. Specific Aim 2: To determine whether ROS elevate angiogenesis through triggering of endothelial cell proliferation and tube formation in the infarcted heart. Our hypothesis is that ROS activate the expressions of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), the major angiogenic mediators, and their signaling pathways in the infarcted myocardium, which, in turn, initiate endothelial cell proliferation and tube formation. Specific Aim 3: To examine whether ROS regulate angiogenesis via promoting vessel maturation and survival in the infarcted myocardium. Our hypothesis is that ROS activate the angiopoietin (Ang) signaling pathway in the infarcted myocardium, thus stimulating vessel maturation and stabilization. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to investigate the cellular and molecular mechanisms regulating myocardial repair/remodeling following infarction (heart attack), particularly the role of reactive oxygen species on angiogenesis (new vessel formation) in the infarcted myocardium.

Complex persistent pain conditions (CPPCs) such as headache conditions, fibromyalgia, temporomandibular disorders, irritable bowel syndrome, and vulvar vestibulitis are high prevalent and shared or comorbid chronic pain conditions. There are two features of CPPCs that are fundamental to the aims and goals of this proposal: 1) the etiology of CPPCs is multifactorial and 2) the clinical manifestations of CPPCs are diverse. In this Program Project, we expect to identify a mosaic of risk factors for each of five CPPCs: fibromyalgia (FM), episodic migraine (EM), vulvar vestibulitis (VVS), irritable bowel syndrome (IBS), and temporomandibular joint disorders (TMD). Furthermore, we expect to characterize clusters of patients within each CPPC that vary significantly according to manifestations of their condition in addition to its painful characteristics (e.g., fatigue, dysfunction, sleep loss). Importantly, we expect some clusters of patients to be more alike across CPPCs than within any single CPPC, consistent with our view that there is some overlap in the manifestations of CPPCs. A unifying hypothesis integrating this Program is that multiple genetic factors, when coupled with environmental exposures (e.g. injury, infections, physical and psychological stress), increase the susceptibility to highly prevalent CPPCs by enhancing pain sensitivity and/or increasing psychological distress. To address the aims and goals of the subprojects and cores described in this application, a group of accomplished pain clinicians, pain researchers, psychophysiologists, molecular and cellular geneticists, biostatisticians and epidemiologists have been brought together to form this Program. Studies proposed in this Program Project application seek to identify the psychological and physiological risk factors, clusters, and associated genetic polymorphisms, that influence pain amplification and psychological profiles in enrollees who have established CPPDs. Additionally, the proposed studies seek to characterize the biological pathways through which these genetic variations causally influence CPPCs.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hypertension is a chronic condition associated with increased risk of mortality and morbidity from stroke, coronary heart disease, congestive heart failure, and chronic renal disease. Difficulty in controlling hypertension is due, in part, to the complexity of the pathophysiology of hypertension. In addition to the involvement of multiple pathways and feedback mechanisms, hypertension is often associated with other pathological conditions such as renal dysfunction, which is tightly linked to the renin-angiotensin system (RAS). Angiotensin (AngII), an important regulator of RAS, plays a critical role in regulation of blood pressure and volume homeostasis. These effects are mediated mainly via Ca2+ mobilization. Investigators reported the involvement of sphingosine kinase1 (SK1) in the mobilization of intracellular Ca2+. However, the exact mechanism is poorly understood. Here, we tested whether SK1 plays a role in AngII-stimulated Ca2+ release and regulating vascular tone. Our early observation using FLIPR to measure the intracellular Ca2+ levels indicated a characteristic pattern of AngII induced increase in intracellular Ca2+ level, an immediate peak followed by a sustained Ca2+ release. Additionally, selective inhibition of EGFR by AG1478 or the SK1 inhibitor, (DMS) inhibited the second phase. Also, AngII and EGF increased endogenous SK1 activity and enhanced production of S1P. Deletion of SK1 or DMS treatment abrogated EGF-induced intracellular Ca2+ elevation. Moreover, perfusion of the arteriole of the isolated rabbit glomerular structure with either AG1478 or DMS blocked the sustained elevation of intracellular Ca2+ and pressor response of Ang II. In vivo experiments performed on wild type and SK1-/- mice revealed that deletion of SK1 reverses the effect of AngII on GFR. Based on our data we propose to elucidate the role of EGFR transactivation and SK1/S1P in AngII-dependent intracellular Ca2+ mobilization, vascular tone, and renal hemodynamics. This study will enable us to gain important insight into the pathophysiology of hypertension and may provide novel therapeutic interventions.

Abstract The mucopolysaccharidoses (MPS) are a family of genetic, lysosomal storage disorders characterized by deficiencies in enzymes that degrade glycosaminoglycans (GAGs). Patients with MPS suffer from crippling skeletal abnormalities that are unresponsive to current treatments. MPS VII presents with a particularly severe skeletal phenotype, where patients exhibit progressive kyphoscoliotic deformity and spinal cord compression resulting in chronic pain and paralysis. MPS VII is caused by deficient beta-glucuronidase activity, leading to accumulation of multiple GAG types. The molecular mechanisms linking this GAG accumulation to cellular dysfunction and skeletal disease are poorly understood, impeding development of effective therapies. Our laboratory uses a clinically-relevant, naturally-occurring canine model of MPS VII that closely mimics the progression of skeletal disease that occurs in human patients. In previous work we demonstrated that MPS VII dogs have cartilaginous lesions in the vertebrae that compromise the stability of the intervertebral joint. These lesions are caused by failed conversion of cartilage to bone during postnatal growth. In preliminary studies, we have identified the precise developmental window when abnormal ossification first manifests in MPS VII dogs and that this can be traced to a failure of resident chondrocytes to progress through hypertrophic maturation. We have also shown that there is abnormal GAG accumulation in MPS VII epiphyseal cartilage from an early age, and, using whole transcriptome sequencing, that there is dysregulation of the Wnt/?-catenin signaling pathway at this crucial juncture in the disease progression. Wnt growth factors are critical regulators of chondrocyte differentiation, and GAGs are known to be important regulators of Wnt distribution and activity, suggesting a link between GAG accumulation and dysregulation of this pathway. The objectives of this proposal are to investigate mechanisms of failed bone formation in MPS VII and establish improved treatment paradigms using a clinically-relevant canine model. Our central hypothesis is that abnormal accumulation of GAGs in MPS VII epiphyseal cartilage disrupts the signaling pathways necessary to initiate and sustain chondrocyte hypertrophic differentiation. Further, we hypothesize that to effectively treat bone disease in MPS VII, it will be necessary to both normalize GAG turnover and activate requisite osteogenic signaling pathways in epiphyseal cartilage. In Aim 1 we will define temporal and spatial relationships between GAG accumulation and epiphyseal chondrocyte differentiation potential at key stages of bone disease progression in MPS VII dogs, from birth to skeletal maturity. In Aim 2 we will establish the critical role of Wnt/?-catenin signaling dysregulation in delayed epiphyseal bone formation in MPS VII dogs. In Aim 3 we will establish if therapeutic targeting of Wnt/?-catenin signaling, alone and in combination with enzyme replacement therapy, can enhance bone formation in MPS VII dogs.

Based on successful pursuit of overall goals we will extend in vivo DTH and in vitro cell-mediated immunity tests of patients with breast disease, persons with other diseases and healthy individuals with T antigen. Further development of our quantitative assay to measure alterations in anti-T IgM, IgA and IgG serum levels to establish a sensitive, specific in vitro immunoassay to screen, diagnose and monitor breast cancer accurately and economically. Monitoring is an important aspect as we have now greater than 100 patients under continuous surveillance for up to 6 years. Clear histopathologic definition of the antibody, which clearly delineates benign from malignant breast lesions histochemically.

Our previous work demonstrated that the KSHV-encoded K8.1A virion glycoprotein is essential for infection of B cells (MC116 human B cell line, human tonsillar B cells). This requirement stands in marked contrast to other non-B cell types, for which our results confirm published findings that K8.1A is dispensable for infection of other cell types (the 293 human embryonic kidney epithelium cell line, the African green monkey Vero cell line, human microvascular endothelial cells. In FY2016 we extended this work by demonstrating that another anti-K8.1A monoclonal antibody inhibits KSHV infection selectively for B cells. Thus we have identified a specific virus-encoded tropism determinant for the major cell type harboring KSHV in infected persons, and associated with two KSHV-associated lymphoproliferative diseases. We have extended studies on the effects of producer cell type on KSHV tropism, by expanding the number and types of cell lines constitutively infected with the KSHV.219 reporter virus (GFP linked to a constitutive cellular promoter; RFP linked to the KSHV lytic phase PAN promoter). In these inducible cells, the KSHV lytic phase can be induced by the lytic switch protein RTA (replication and transcription activator), linked to the tet-operator and thereby inducible with doxycycline. We now have inducible variants of the unusually KSHV-susceptible human B cell line MC116, the telomerase-immortalized human microvascular endothelium cell line TIME, the 293 cell line and the Vero cell line. We have characterized in detail the effects of specific activation with doxycycline versus nonspecific activation with BUdR, by measuring both RFP (fluorescence microscopy, flow cytometry) and the production of infectious KSHV (titration on fresh Vero cells). These inducible cell lines will be most valuable in our study of the impact of producer cell type and mode of activation on the target cell tropism of KSHV virions. We have also found a second mode for KSHV infection B cells, namely antibody-dependent entry. This mechanism is K8.1A independent, and in fact was first observed when the anti-K8.1A mAbs were found to greatly stimulate infection of Raji cells, which are otherwise refractory to KSHV. Preliminary data suggest that the antibody-dependent infection pathway is mediated by Fc receptors on the B cell surface. In collaboration with Dr. Robert Yarchoan, NCI, we are analyzing the sera from infected people with different KSHV-mediated syndromes. Preliminary data suggest that sera from MCD and PEL subjects have antibodies that greatly stimulate KSHV infection of Raji cells, whereas sera from KS subjects have lower levels of KSHV-stimulating antibodies. As yet, we do not know the relationships between the stimulating activities and the total levels of anti-KSHV antibodies or antibodies against any specific KSHV-encoded proteins.

The development of movement depends on the maturation of the spinal cord motor circuits. Embryonic motor output is characterized first by spontaneous activity resulting in fetal jerk movements that later become more rhythmic and show limb alternations. In newborns motor development then undergoes a period of reflex and postural maturation leading into coordinated weigh-bearing locomotion. All these changes occur as a consequence of different maturation phases of the spinal synaptic networks and more specifically of local interneurons that ultimately control motor neuron firing patterns and motor output. Our long term objective is to understand the development of these local spinal circuits. Advances in this field were hampered in the past by the diversity and complexity of spinal interneurons. Fortunately, the recent development of mouse models expressing genetically-encoded reporters to identify interneuronal lineages now permits their study through development and allowed new understanding of the principles that govern spinal interneuron development. Our work has been focusing on one lineage of embryonic interneurons, named V1, that provide inhibitory control to ipsilateral motoneurons. Previously, we showed that this group diversifies into different types of adult interneurons, including Renshaw cells and Ia inhibitory interneurons (IaINs) that provide respectively recurrent inhibition to the same motoneurons and reciprocal inhibition between motoneurons with antagonistic actions around single joints. These two interneurons thus perform critical, but different roles in motor control. A basic question is then what mechanisms diversify single embryological groups of interneurons into distinct functional classes in adult. Previous analyses on Renshaw cells provided some important insights, including that this specification might occur early in embryological development and that individual cell types acquire different synaptology and functions through the different phases of spinal cord development. In this proposal we aim to investigate the development of the large group of V1 interneurons, including IaINs, that are interposed in proprioceptive reflexes. The underlying hypotheses are that IaINs are a group of V1-interneurons specified by their early birth (simultaneously with Renshaw cells) to acquire in the embryo transient motor axon inputs and functions that are quite distinct from those in adult. We also hypothesize that later they shed these embryonic inputs and develop connectivity that allows them to mediate reciprocal inhibition. Therefore we propose three aims to find out their birth dates (aim1), their major synaptic inputs and outputs in embryo (aim2) and the maturation of the reciprocal inhibitory circuit postnatally (aim 3). A corollary of this work is that congenital deficits that result in arrested spinal network development do not necessary imply a network with immature adult connectivity, but more likely networks of different connectivity appropriate to an earlier developmental point. Therefore it is important to understand the nature of this earlier connectivity to better appreciate the diversity of neurological motor deficits expressed in newborns. PUBLIC HEALTH RELEVENCE The work proposed will study spinal cord development focusing on the neuronal networks that mediate the maturation of coordinate movements and locomotion. The study will use powerful new mouse genetic methodologies to analyze the cellular elements that organize this network during development. The research is thus guided towards understanding normal and abnormal motor development in newborn and infants [unreadable] [unreadable] [unreadable]

Personal health records (PHRs) integrated with electronic medical records (EMRs) are an essential tool to promote patient-centered care and ultimately improve health outcomes. Although adoption and use of integrated PHRs is increasing, it typically occurs within large integrated healthcare systems that have business models supporting their use. Whether such systems can have broad uptake in typical primary care practices remains unknown. In a previous AHRQ-funded R18, we created a PHR for prevention that is integrated with a clinician's EMR, which we term an "interactive preventive healthcare record" (IPHR). The IPHR also incorporates clinical decision support software, a reminder system, tailored educational materials, and decision aids into one actionable package for both patients and clinicians. In a study that tested IPHR feasibility and effect, the IPHR enhanced clinician-patient communication and promoted the delivery of recommended preventive services. Although these findings are encouraging, the study design permitted only small subsets of the study practices'patients to be included and patients were introduced to the IPHR with only a low- intensity invitational letter. The next logical step is to conduct a feasibility trial to assess whether practices can, under usual practice conditions, sustainably and more intensively integrate the IPHR into care. We propose to test the question in eight practices in the Virginia Ambulatory Care Outcomes Research Network (ACORN) that participated in the first study. All adult patients presenting for care will be considered eligible for IPHR use. Guided by organizational change theory, we will engage practices to create a shared vision on how to integrate the IPHR into care delivery using practice champions, learning collaboratives, and a patient-centered communications strategy. From the IPHR and EMR databases, we will apply the RE-AIM model to measure Reach, Effectiveness, Implementation, and Maintenance. We define Reach as the proportion and characteristics of patients seen for an office visit who establish an IPHR account and receive prevention recommendations, Effectiveness as change in documented preventive service delivery after patients use the IPHR, Implementation as clinician response to IPHR-identified patient needs, practice-level Maintenance as the practice's ability to sustain Reach 6 to 12 months after initial PHR fielding, and patient-level Maintenance as whether patients continue to use the IPHR after 6 months. We will compare the Reach in practices promoting the IPHR to patients using the integrated approach to the historical control rate of the less intensive invitational letter;the eight study sites will provide 80% power, using a one-sided 0.05-level test to detect a 10% incremental increase. We will conduct key informant interviews and record and analyze learning collaboratives to understand how practices integrated the IPHR and mediators and moderators to use. Findings from this study will assist in the design of a future practice-level randomized controlled trial, and inform practices, policymakers, and payers about how to integrate a PHR in typical primary care practices. Patient-centered health information technology systems, such as integrated personal health records, have great potential to improve the quality of care by providing centralized medical information, patient education and activation, enhanced patient and clinician communication, decision support, and reminder systems. However, these systems cannot improve health if they are not used by patients and clinicians. We seek to learn whether primary care practices can encourage patients to use such a system by integrating it into routine care and to determine how the intervention influences healthcare delivery

. National Ovarian Cancer Prevention and Detection Study: The National Prospective Cohort Study of Risk-Reducing Surgery and Ovarian Screening among Women at Increased Genetic Risk of Ovarian Cancer (also known as "GOG 0199") is the cornerstone of CGB"s intervention studies research portfolio. Developed and chaired by Dr. Greene, it is a non-randomized natural history study of risk-reducing salpingo-oophorectomy versus a novel ovarian cancer screening strategy (the ROCA algorithm) [NCI Protocol #02-C-0268]. This study opened in July 2003, and closed to new patient enrollment in November 2006, after having accrued 2605 high-risk women (1029 surgery arm; 1576 screening arm). Follow-up will be completed in November 2011. This complex, multi-institution international study exemplifies the type of project that would be difficult to mount through extramural funding mechanisms. Accomplishments to date include successfully completing accrual to this study, completing the research-based BRCA1/2 mutation testing for the 1,500 mutation-unknown study participants (including both full sequencing and testing for large deletions, rearrangements), nearly completing the central pathology review of 1037 RRSO samples, publishing a report detailing the rationale and design of the study, along with baseline characteristics of the women in each cohort, developing a model of the factors which influence the choice of RRSO versus screening, contributing 1576 screening subjects to a pooled analysis (with the Cancer Genetics Network) of determinants of baseline CA-125 levels (Skates et al., unpublished data), and negotiating a collaboration between GOG and CIMBA under which DNA and clinical data from 199"s 1400 mutation carriers will be contributed to pooled multiple candidate gene association studies of breast cancer risk, and two genome-wide association studies (GWAS), one for each BRCA gene. Breast Imaging Pilot Study in Women from BRCA Mutation-Positive Families (NCI Protocol 01-C-0009): is the second major intervention studies programmatic component. It has reached its accrual goal of 200 women from BRCA1/2 mutation-positive families, 170 of whom are known mutation carriers. Prospective follow-up is ongoing, and scheduled to end in January 2010; 25% of subjects have completed the study. To date, breast cancer and ovarian cancer have developed in 14 and 1 participant, respectively. Analyses of baseline mammographic density and MRI volume in carriers versus non-carriers (the latter including 100 low-risk women from NCI/CCR"s Breast Cancer Susceptibility Study) are well underway. These include a collaboration with Dr. Funmi Olopade and colleagues at the University of Chicago, in which our digitized mammographic images are being used to evaluate, and possibly validate, various novel imaging characteristics that may prove to be better predictors of breast cancer risk than standard mammographic density. We have published the first formal method for enumerating cells in breast duct lavage (BDL) fluid, described the results of BDL in the largest cohort of BRCA mutation carriers yet reported, and quantified the tolerability of the BDL procedure. DNA samples from mutation-positive DNA from BI participants has also been added to CGB"s collection of samples being utilized in our genetic modifiers collaboration with CIMBA. Preliminary data document that fentogram quantities of estrogen metabolites are detectable in both NAF and BDL fluid, with levels in the former much higher than the latter. A Phase II Randomized Trial of Zoledronic Acid versus Standard Care to Prevent Post-RRSO Osteopenia/Osteoporosis (GOG-215): was designed as a companion study to GOG-199, targeting one of the major complications of RRSO-related premature menopause, i.e., significant bone loss, increased risk of osteoporosis and fracture. Participants are enrolled in GOG-215 just prior to or immediately following RRSO; all participants receive oral calcium/vitamin D supplementation, proscription of tobacco exposure, and exercise recommendations. Half are also randomly allocated to receive 3 zoledronic acid infusions, one every 6 months. Bone mineral density and serologic markers of bone metabolism are the primary study endpoints. GOG-199 participants who cross-over from screening to surgery remain eligible for this study. The entry criteria have been relaxed to include all premenopausal women undergoing RRSO. This study is currently open at 50 sites nationwide, and has enrolled 18 participants. Dr. Larissa Korde is Co-PI of the national protocol, and also the local PI of this study which has been opened at the NIH Clinical Center [NCI Protocol #06-C-0204]. A Pilot Study of a 3-month Intervention for Increasing Physical Activity in Sedentary Women at Risk of Breast Cancer: is also being run by Dr. Korde. She designed and initiated this project while still an oncology fellow in the Medical Oncology Branch/CCR, and has continued to run the study since joining CGB. It asks whether a physician recommendation for increasing physical activity along with the use of a pedometer will be effective in increasing physical activity in a sedentary population of breast cancer survivors and women at increased genetic risk of breast cancer [NCI Protocol #04-C-0276]. Changes in mammographic density, total body fat and selected hormones will be explored as surrogate biomarkers. This study has closed to accrual, and a preliminary report has been submitted for publication. Inherited Bone Marrow Failure Syndromes: Our study of persons from families with one of a variety of inherited bone marrow failure syndromes (e.g., Fanconi's anemia) continues steady accrual [NCI Protocol #02-C-0052]. Squamous cell carcinomas of the oral cavity, esophagus, labia and cervix are among the non-hematologic malignancies which occur excessively in patients with these syndromes. Participants in this study are undergoing intensive evaluation in search of both clinical and molecular abnormalities which might represent cancer precursors, particularly with regard to cancers of the head/neck and female genitals. The role of the human papilloma virus (HPV) in the etiology of these cancers is being explored. If suitable clinical or molecular endpoints can be identified, consideration will be given to the development of an intervention program which would target persons from these high-risk families. An HPV vaccine trial among persons with FA is presently under consideration.

Noninsulin dependent diabetes mellitus is associated with increased risk for coronary artery peripheral vascular and cerebrovascular disease (CVD). This could be attributed to metabolic derangements typical of diabetes, disorders of platelet function and coagulation mechanisms, and hypertension. However, the major part of excess risk of CVD in NIDDM cannot be attributed to diabetes per se, because the risk precedes NIDDM, and is uncorrelated with the degree of hyperglycemia. Because insulin resistance is a major cause of NIDDM and appears to precede overt diabetes, it is hypothesized that insulin resistance, per se, and/or the hyperinsulinemia which characterizes resistance without full blown diabetes may be the factor(s) which are responsible for CVD in individuals at risk for NIDDM. This application is for a Central Laboratory Facility, and is responsive to an NIH Request for Cooperative Agreement (RFA NIH-91-HL-03-P). It is submitted in cooperation with a Field Center Application also submitted from the University of Southern California (M. Saad, P.I.). In this application, we propose to make a series of measurements which will establish the degree of insulin resistance as well as cardiovascular risk factors in a group of 1600 subjects to be tested in specific field centers. In this application we propose the formation of two cores: an Assay Core, to carry out a series of biochemical measurements, and a Kinetic Analysis Core, to calculate insulin sensitivity (SI) and glucose effectiveness (SG) from the assay results. This laboratory conceived, validated and developed the minimal model approach which is proposed to be used to assess insulin sensitivity (i.e., resistance). Assays will be performed in 4 laboratories at USC: Bergman (insulin, glucose, C-peptide), Meiselman (blood and plasma viscosity, red blood cell aggregation, zeta sedimentation ratio, and hematological indices), Francis (Fibrinogen, factor VIIc, plasminogen activator inhibitor a, prothrombin factor F1.2, and fibrin D-dimer fragment). In addition, lipoprotein risk factors for CVD will be measured off-site by Dr. Barbara Howard and Dr. Linda Curtiss. Additionally, we propose a Kinetic Analysis Core, which will calculate SI and SG from our assay results. Minimal model calculations will additionally be compared to the same parameters calculated by different software. Results of these determinations will be transferred electronically to a Coordinating Center for analysis. It is believed that this combined Assay/Kinetic Analysis facility will provide an efficient, accurate and precise assessment of insulin resistance in different subject groups, and as such will yield an accurate assessment of the importance of insulin resistance and hyperinsulinemia in the development of cardiovascular disease.

This proposal is based on a fundamentally new approach to the isolation of bacterial virulence genes involved in pathogenesis, termed IVET (in vivo expression technology), that allows for the positive selection of bacterial genes that are specifically induced in host tissues. This methodology will be used to characterize the genetic and environmental factors that regulate the expression of in vivo induced genes (ivi) in the human pathogen, Salmonella typhimurium. Such studies will provide insights into the molecular mechanisms governing the regulation of pathogenicity, which is fundamental to the understanding of infectious disease. Genetic analysis of genes that answer the IVET selection is facilitated because the genes are transcriptionally fused to the reporter gene, lacZ, allowing the recovery of regulatory mutants that constitutively express ivi genes in vitro (normally repressing conditions). Our efforts will be focused on those regulatory factors that coordinately regulate ivi genes, thus defining the regulatory components that ensure a multifaceted bacterial response to a multifaceted host challenge. Trans-acting regulatory mutations will be used to characterize the positive and negative ivi regulatory elements that activate ivi transcription in vivo and repress ivi transcription in vitro. These mutants will be screened for those that confer virulence defects in an animal model, suggesting that they, or some of the products they regulate, play an important role in pathogenesis. Cis-acting mutations will be used to define the ivi promoter region with an emphasis on establishing a consensus site for coordinate ivi gene expression. Defining the nature of coordinate regulation provides a means to under stand the sophisticated signal transduction mechanisms that enable bacteria to grow under such different environmental conditions (e.g., laboratory media vs. host tissues). Additionally, the IVET selection will be extended to screening for ivi fusions whose survival is limited to a particular host tissue and thus may contain tissue-specific ivi genes, whose restricted expression will be examined by genetic analysis. These studies have practical applications for both vaccine and antimicrobial development.

Project Summary The goal of this R21 EBRG project is to develop new optical methods to map high intensity focused ultrasound (HIFU) pressure ?elds. The methods would enable simple, fast, and low-cost in situ HIFU beam measurements, which are needed for quality assurance and safety in the clinic, and to accelerate the pace of research and development of new HIFU applications and technologies. An ideal beam mapping instrument would be low cost, capable of rapidly measuring relevant acoustic parameters of clinical HIFU systems in situ between treatments, and usable by nontechnical experts. Needle hydrophones are currently the gold standard tool for mapping HIFU pressure ?elds, but are poorly suited to the measurement task since they sample only one spatial location at a time, and most can only measure sub- therapeutic pressures. They can be translated in a water tank by a motion stage to produce spatially-resolved pressure maps, but this is a slow and cumbersome measurement that can take several hours to complete. The techniques proposed in this application could meet this clinical need and also provide a fast, ?exible, and spatially- resolved beam mapping instrument that would be invaluable for HIFU research since it would enable rapid val- idation and experimental designs that are currently infeasible, such as mapping pressure ?elds across multiple experimental variables. Standard optical schlieren imaging has a long history in 2D and 3D ultrasound pressure ?eld mapping but has conventionally been applied using sophisticated and expensive high-speed optical setups with limited ?eld-of-view, limited portability and high cost. The methods and devices proposed in this project are instead based on a newer schlieren technique called background oriented schlieren (BOS) imaging, and in their simplest form can be implemented using just a water tank, a tablet PC and a webcam. In essence, BOS trades the sophisticated optical setup for more sophisticated computation, which is a much cheaper commodity. The central innovation in this project is to use BOS imaging to quantitatively map continuous-wave HIFU pressure ?elds in 2D and 3D. The ?rst Aim is to develop portable hardware for BOS imaging and tomography, that can be used with a wide variety of HIFU systems in situ. The second Aim is to develop the mathematical theory underlying the BOS image formation process for HIFU beam mapping, which is different from conventional BOS imaging since the underlying refractive index ?eld is not static. The third Aim is to develop acquisition and reconstruction methods that produce quantitative spatially-resolved pressure ?eld maps, and validate those maps against simulations and optical hydrophone measurements of state-of-the-art HIFU systems. By developing and disseminating BOS hardware, theory, and methods for quantitative 2D and 3D HIFU beam mapping, this development project will lead to fast, simple and robust devices that can be widely adopted and even shipped with each clinical HIFU system for regular quality assurance and exposimetry measurements.

Here we will identify nonhuman primate (NHP) neuron types and build an extensive toolbox of vectors for circuit- based neuroscience studies. NHPs share substantial neuroanatomical, genetic, and behavioral homology with humans, and therefore they are indispensable for investigating the neural circuit basis of cognition and devising therapies to treat neurological and psychiatric disorders. Despite the importance of NHPs, we lack the tools to analyze and manipulate complex circuits in the primate brain. This lack severely limits the use of genetically- coded neuroscience tools to examine circuit specific functions and hinders development of targeted gene therapeutics. Current methods for achieving transgenesis in small model species, such as the creation of genetically modified strains, are prohibitively expensive in NHP and not applicable to human disease. AAVs are the leading alternative to germline modification and selective breeding. AAVs infect adult neurons, confer stable transgene expression, and have proven safe in gene therapy clinical trials. AAVs do not have natural cell-types specific properties, but when altered or combined with cell type specific regulatory sequences (enhancers/promoters) they have been able to achieve cell type-specific transgenesis. This has made possible, for example, our previous optogenetic investigation of midbrain dopamine neurons for learning and decision making. However, before AAV-mediated gene delivery can be generalized to circuits across the brain and for multiple behavioral functions, we must create currently lacking vectors and promoters that permit efficient and specific gene delivery to all required cell types. Here, we will combine single-cell RNA-Seq (scRNA-Seq) with high-throughput screening of engineered adeno-associated viruses (AAVs) to create a complete toolbox of viral vectors and promoters enabling minimally invasive monitoring and manipulation of neurons in NHP brain. We have devised a transdisciplinary approach to classify individual neurons according to their gene expression profile and simultaneously screen for adeno-associated virus (AAV) vectors (capsids and regulatory sequences) capable of specific and efficient transgene delivery to classified neurons. First, we will synthesize massive libraries of mutated AAV vectors and synthetic promoters, in which each variant is paired with a unique DNA barcode. We will then scRNA-Seq to capture the transcriptome for each cell and quantify the AAV and promoter- specific barcodes in every cell?s expression profile. Preliminary experiments in Rhesus monkeys have fully validated and demonstrated the promise of this innovative approach. The outcomes of our Specific Aims will include (1) an inventory of cell types in the retina, prefrontal cortex, primary motor cortex, and striatum, (2) cell type-specific AAVs and promoters targeting all defined cell types, (3) AAVs with broad tropisms, (4) a publicly available dataset of transcription profiles for millions of NHP brain cells, (5) an updated and comprehensive Rhesus macaque reference genome, and (6) anatomical, physiological, and functional validation of cell type- specific circuits tools and their function in the NHP brain.

Temporal lobe epilepsy (TLE), the most common type of epilepsy, is characterized by destruction of specific hippocampal neurons. Recent evidence indicates that this neuronal damage is the result of excessive stimulation by excitotoxic amino acids (e.g., glutamic acid). Two stages of excitotoxic neuronal damage have been identified: and early phase, occurring in minutes, associated with osmotic imbalance, cellular swelling, and lysis; and a delayed phase, characterized by elevated intracellular calcium. One major homeostatic element which counteracts events underlying both phases of excitotoxicity is the sodium pump, Na,KATPase. Three different mammalian genes encode this family of structurally similar heterodimeric isoenzymes which are responsible for ionic and osmotic equilibrium. Although decreased Na,K-ATPase activity has been recognized in TLE and animal models of epilepsy, few studies have examined the distribution, regulation, or role of the sodium pump isoforms in epileptic hippocampal neuronal death. Our preliminary studies indicate a heterogeneous distribution of each isoform in rat hippocampal neurons. In TLE, we have discovered an inverse relationship between the level of one Na, K-ATPase isoform, and the susceptibility to neuronal death. This proposal outlines studies to explore the hypothesis that the activity of the sodium pump in individual hippocampal neurons is the major determinant of their susceptibility to excitotoxic damage. Using in-situ mRNA hybridization, monoclonal immunohistochemistry, in-vitro autoradiography, and a cytochemical assay, the amount of each isoform in the principal neurons of surgically obtained human hippocampus will be correlated with their susceptibility to epilepsy associated damage. Similar studies will be carried out in rats with seizures induced by graded electrical stimulation. Because the hippocampal somatostatin interneuron appears to be an early excitotoxic target, and thus of possible significance in the pathogenesis of TLE, we will particularly focus on the characteristics of its sodium pump. Finally, using hippocampal neurons in-vitro, we will regulate the activity of the NaKATPase isoforms and determine their role in glutamate-induced cell death. These studies will shed light on the complexity of action of the sodium pump, which is fundamentally critical to normal neuronal function. They will also provide new information about the larger issue of excitotoxic neuronal death, and possibly its prevention, which has now been implicated in the pathophysiological of many human neurodegenerative states, including stroke, Huntington's disease, hypoglycemic brain damage, and Parkinson's disease.