The long-term goal of our work is to understand the mechanism of transcriptional regulation by steroid hormone receptors and their dysregulation during disease. We seek to reveal how specific coactivators and corepressors translate the hormonal signals into promoter-dependent transcriptional outputs. Our work centers on the mechanism of gene activation by the glucocorticoid receptor (GR). GR harbors two major transcriptional activation functions (AF1 and AF2) that are differentially utilized in individual promoter and cellular contexts. In contrast to the C-terminal AF2 function, little is known regarding the coregulators that interact with, and mediate the functions of the N-terminal AF1. This region plays key roles in important therapeutic effects of glucocorticoids such as the induction of thymocyte apoptosis. We recently generated specific mutations in AF1 that pinpoint the critical residues responsible for function. We have used them in complementary approaches to discover AF1 coregulators. In a genetic approach, we isolated two extragenic suppressor yeast strains where the function of deficient AF1 mutants is restored. The genes responsible are likely to encode conserved factors intimately involved in AF1 function. Through an independent functional approach, we identified the 150kDa subunit of the DRIP/TRAP/Mediator complex as a protein that interacts with a transcriptionally competent AF1. In Aim 1, we propose to define the nature and structural basis for the interaction of DRIP 150 with GR AF1. We plan to map the minimal regions necessary in each protein and identify key residues through random and targeted mutagenesis coupled with screening in yeast. We will explore the structural basis of the interaction through a biophysical characterization of the complex. In Aim 2, we will ascertain the functional consequences of AF1 binding to DRIP 150 and define the effector regions of DRIP 150 involved in transcriptional activation. Using multiple approaches, we will test the hypothesis that AF1 recruitment of the DRIP complex determines whether GR activation of a given promoter requires participation of AF1. In Aim 3, we propose to identify novel conserved AF1 coregulators by cloning the genes responsible for the extragenic suppression in the yeast strains we have isolated. We will then identify mammalian counterparts and begin to ascertain their role in AF1 function using some of the assays outlined in Aims 1 and 2.


The pathogenesis of asthma results from interactions between the innate (Project 1) and adaptive (Project 3) immune systems. A subset of immune cells, regulatory CD4+ T cells, play an important role in suppression of both innate and adaptive responses. Although there are indications that regulatory T cells participate in asthma, much remains to be learned before therapeutic interventions can be entertained. The goal of the first part of this Project is to better define the role of complement regulator (CD46) activated human T cells, which appear to represent a unique adaptive regulatory T cell subset. In Aim 1, we will determine if complement activation plays an important role in asthma, as well as providing a physiologic in vivo stimulus for these adaptive regulatory T cells. We will ask if genetic deficiency in various complement components (beginning with C3) modulates the Sendai virus-induced acute infection and the chronic asthma phenotype in mice (as described in Project 1). We will also determine whether markers of altered complement activation occur in situ in human patients with asthma. In Aim 2, we will look for the presence of T cells with the CD46 adaptive regulatory T cell phenotype in human patients with asthma. We will also determine if human CD46 adaptive regulatory T cells inhibit the function of pathogenic cells in asthma (Th2 cells and B cells). If so, this would argue for further studies into the use of these adaptive regulatory cells for the treatment of asthma. In the second part of this Project, we will complement the above studies on adaptive regulatory T cells and define the role of natural regulatory T cells in a mouse model of asthma. By moving to this model, we can utilize a newly described marker Foxp3-GFP for natural regulatory T cells to facilitate studies of natural regulatory T cell specificity. In summary, our goal is to further address the role of the complement system in asthma and whether adaptive or natural regulatory T cells participate in controlling the severity of asthma. Both avenues represent attractive therapeutic targets for the treatment of asthma. Asthma is a growing problem in the United States, but current therapies are expensive, chronic, and, unfortunately not curative. By exploring the body's innate immune system and natural regulatory mechanisms that inhibit excessive pathology, our hope is that curative therapies can eventually be generated not requiring global immunosuppression.


M. tuberculosis is the world's leading cause of death from a single infectious agent and the leading cause of death in AIDS patients. The emergence of multidrug resistant tuberculosis poses a major threat to the public health, giving new urgency to research aimed at combating this ancient scourge. Moreover, multidrug resistant strains of M. tuberculosis (MDRTB) are a potential weapon of bioterrorism, and such strains have been classified as NIAID/CDC Category C Bioterrorism Agents. Studies proposed in this grant application build upon advances made in collaborative efforts between the Horwitz laboratory at UCLA and the Griffith laboratory at the U. of Wisconsin over the past two years to develop novel antimicrobials against M. tuberculosis for treatment of drug resistant organisms. During the past several years, the Horwitz and Griffith laboratories have laid the groundwork for the development of a new antimicrobial strategy against M. tuberculosis - targeting M. tuberculosis glutamine synthetase (GS). Thus, Horwitz et al. demonstrated that M. tuberculosis GS is a promising antimicrobial target, and that the high production of this enzyme is correlated with pathogenicity in mycobacteria and with the presence of a poly-L-glutamate/glutamine structure in the cell wall of pathogenic mycobacteria. Horwitz et al. showed further that inhibition of GS with L-methionine-SR-sulfoximine (MSO) inhibits M. tuberculosis growth in cell-free culture, in human macrophages, and in vivo in guinea pigs challenged by aerosol with M. tuberculosis. In combination with ascorbate, MSO is almost as potent as isoniazid, the leading antituberculous drug. Importantly, working in a collaboration during the past year, the Horwitz and Griffith laboratories have identified analogs of MSO that are highly potent against M. tuberculosis in vitro but lack certain drawbacks of MSO. This application has two major goals: 1) Develop novel MSO analogs that are better drug candidates than MSO because they are a) more selective for glutamine synthetase (GS) vs gamma-glutamylcysteine synthetase (gamma -GCS); and/or b) less well taken up into the brain where MSO exerts its major toxicity in sensitive species; and/or c) even more selective for M. tuberculosis GS vs. mammalian GS, and test the toxicity of the analogs in mice. 2) Test the novel MSO analogs for their capacity to inhibit M. tuberculosis growth in broth culture, in human macrophages, and in guinea pigs.


The Core Support Grant is in support of the organizational structure of the Cancer Center of Hawaii. The grant supports leadership personnel and key investigators in the Center's five departments of Basic Sciences, Clinical Sciences, Epidemiology, Data Resources and Cancer Control. The grant also supports shared resources which consists of computer facility, animal facility, tumor cell bank and a carcinogen biohazard laboratory. Epidemiological research is being conducted at the Center in the multi-ethnic population of Hawaii. Case control studies on diet and breast cancer, trace metals in prostate, role of vitamin A in lung cancer and the mechanism of action of nitrosoamines in gastric cancer are just a few of the many projects funded at the Center.


Catecholamine regulation of vasopressin in hypertension. The major aim of this proposal is to examine the role of catecholamine regulation of vasopressin in the supraoptic nucleus on the development and maintenance of hypertension. Secondary aims are the establishment of normal parameters of CA regulation of vasopressin and the identification of afferent nuclei and pathways to the supraoptic nucleus. The methodologies to be used will be specific and regional catecholamine depletions, immunocytochemical analyses, biochemical assays, and modulations of fluid content of the cardiovascular system. These experiments will be carried out on normotensive and hypertensive rats.


The concept of the neurovascular unit states that the pathophysiology of stroke, brain injury, and degeneration cannot be investigated as a purely neuronal phenomenon. Instead, interactions between cells from all neuronal, glial, and vascular compartments must be considered. The first cycle of our P01 program was focused on dissecting acute mechansims of disease in the neurovascular unit. In this renewal, we now propose to look at how the neurovascular unit undergoes repair and recovery. Project 1 (Lo/Xing: Gliovascular regulation of the microglial switch) examines how endothelium and astrocytes differentially regulate the activation of microglia. Microglia can span the range from good to bad. We will test the hypothesis that signaling at the gliovascular interface regulates the microglial switch as the neurovascular unit transitions from initial injury into repair. Project 2 (Arai/Huang: Mechanisms of recovery after white matter injury) examines how oligovascular signaling between brain endothelium and oligodendrocyte precursor cells mediate remodeling in diseased white matter. This allows us to extend the neurovascular unit concept for understanding how white matter recovers and reconnects. Project 3 (Ayata/van Leyen: Targeting ROCK for neurovascular recovery after stroke) will ask how the rho-kinase system may be a central signaling system for specific neuronal and vascular recovery endpoints during stroke recovery. This project is also our attempt at translation since it provides a potential therapeutic target for promoting neurovascular unit repair. Direct collaborations exist between all projects. All studies are supported by three cores. Core A (Boas/Sakadzic) provides powerful tools for in vivo and cellular imaging. Core B (Whalen) supports neuro-behavioral assays for assessing recovery in animal models across all projects. Core C (Lo) is our program coordination core that provides infrastructure for all collaborations We are excited about this next phase of our highly integrated P01 that dissects new mechanisms of cell-cell signaling in endothelial, glial and neuronal systems as the neurovascular unit transitions from initial injury into recovery after stroke, brain injury and degeneration.


Elevation of body temperature to between 41.8 and 42.4 degrees centigrade is now being used both alone and together with chemotherapeutic agents to treat patients with cancer. The mechanisms by which hyperthermia is cytotoxic are poorly understood as are the ways in which elevated temperature affects cell killing by chemotherapeutic agents. We propose to study the interaction between hyperthermia and selected chemotherapeutic drugs utilizing both in vitro and in vivo systems. Our general approach will be to test for synergistic cytotoxicity by measuring in vitro colony survival in chinese hamster ovary cells and by measuring the in vitro colony survival of RT9 astrocytoma cells after in vivo treatments. We then plan to study the mechanism behind the observed increased cytotoxicity by examining such things as altered drug uptake and enzyme inactivation. We will also study the scheduling of the drugs with heat in order to maximize cell kill. These studies should suggest clinically useful combinations of drugs with heat as well as provide basic cellular pharmacologic data. This work is already underway and we have discovered an interesting relationship between methotrexate and adriamycin cytotoxicity when each is combined with heat. In addition we have found that the rate of heating to peak treatment temperature is a critical determinant of the cell kill achieved. Our ultimate aim is to use these studies as starting points in the selection of drugs and hyperthermia schedules for clinical use.


In order to understand the role of the Xenopus forkhead protein (Xfkh3) in the differentiation of presomitic mesoderm, I have tried to overexpress and misexpress this protein by injecting synthetic mRNA in the animal pole region of both blastomeres of 2 cell stage embryos. 0.25, 0.5 and 1 ng of mRNA was injected. At stage 8, animal caps were dissected and cultured until sibling embryos reached tailbud stage in the presence or absence of activin. Although misexpression of the message by itself does not induce any muscle in the animal caps, still, muscle induction by activin is not inhibited. Further analysis of these results is currently under progress. By whole mount in situ hybridization, I have localized the distribution of Xenopus nonmuscle myosin heavy chain B (Bhatia-Dey et al., Proc. Natl. Acad. Sci. USA 90, 2856, 1993) using a probe that comprises part of the rod and the entire 3' UTR. The transcript is expressed ubiquitously in blastula and gastrula stage embryos, however, at stage 21, the transcripts begin to localize in the anterior part of the embryo, especially in the developing eye and fully differentiated anterior somites. At early tailbud stage, the transcripts become more concentrated in differentiated somites and in the eye. No expression is detected in the presomitic mesoderm. In swimming tadpoles, the transcripts are localized in differentiated somites, the eye and the branchial arches. Using a probe from a similar region of Xenopus nonmuscle myosin heavy chain A, no localized transcripts were detected throughout embryogenesis. However, Northern analysis, using the same probe, detected 2 transcripts during development. A 7.5 kb transcript is detected throughout embryogenesis from unfertilized eggs to swimming tadpoles and in a number of adult tissues. Another 8.3 kb transcript is first detected around stage 15/16, peaks between stage 25 to 30, gradually decreases in swimming tadpoles and is no longer detected in stage 47 embryos. Preliminary analysis suggests that the larger transcript arises due to differential polyadenylation.


Project Summary This study aims to prove the concept and feasibility that TendonCure, extracellular vesicles that contain signaling molecules (i.e. exosomes), secreted by mesenchymal stem cells (i.e. tendon stem/progenitor cells [TSPCs] or adipose-derived stem cells [ADSCs]) from individual patients (donors) cultured on a novel scaffold exert therapeutic efficacy on tendinopathy when injected into the diseased tendon of the same donor. Tendinopathy is a common chronic tendon disorder that affects 30-50% of individuals over 60 years old. It is characterized by pain, swelling, loss of function, and impaired performance. There is currently no cure for tendinopathy. Spontaneous repair or treatment typically leads to scar formation, resulting in a weakened tissue with reduced function and mechanical properties that may ultimately rupture with further use. Our previous and preliminary studies show that MSCs grown on the TendonCure scaffold are modulated towards tenocyte differentiation with enhanced tenogenesis-related gene expression. Furthermore, TSPCs cultured with exogenous TendonCure-T or TendonCure-A, TendonCure derived from TSPCs or ADSCs, respectively, exhibited tenogenesis-related gene expression profile changes, with increased expression of tenogenic markers and decreased expression of gene markers for the adipo- and chondrogenic lineages. Importantly, local injection of TendonCure showed therapeutic efficacy in an overuse-induced supraspinatus tendinopathy rat model in vivo. While the results are promising, the suggested efficacy of TendonCure was based on statistically significant results in TendonCure from MSCs of a young to middle-aged group. However, whether TendonCure derived from MSCs of aged individuals exerts such therapeutic effects is unknown. We therefore hypothesize that TendonCure from MSCs of aged individuals exerts therapeutic efficacy on tendinopathy. TendonCure-T and TendonCure-A will be harvested from TSPCs and ADSCs, respectively, which are isolated from aged patients at 65-80 y.o. and cultured on TendonCure scaffolds. Control exosomes will be derived from TSPCs and ADSCs from the same patients as the TendonCure groups and grown in regular 2D culture. Adult nude rats subjected to decline treadmill at two weeks (with expected mild tendinopathy) will be injected weekly in the supraspinatus tendons with TendonCure or control exosomes, or placebo (phosphate buffered saline), or kept for normal cage activity (control). Four weeks after injection, animals will be evaluated for efficacy on pain behaviors, and supraspinatus tendons will be dissected for efficacy evaluation with assays for histology and mechanical properties testing. Upon successful completion of Phase I and II studies, we will carry out clinical trials focusing on common sites of tendinopathy. TendonCure will be marketed as a biologic for treating tendinopathy and other tendon disorders. 1


Oxidative stress is thought to be a major contributor to the development of retinal degenerative disorders such as macular degeneration, retinopathy of prematurity and diabetic retinopathy. In order to understand the mechanism(s) of oxidative damage to the retina, we have investigated the gene expression in the retina of rats exposed to intense visible light. The expression of heme oxygenase-1 (HO-1), a marker for oxidative stress, in the retina is highly increased following light exposure. The expression of HO-1 mRNA in the retina in response to acute intense light was investigated as a function of age in dim cyclic light reared and dark reared rats. The HO-1 mRNA in the retina of treated rats was analyzed by Northern blotting. The intense light exposure markedly increased the HO-1 mRNA expression in the retina irrespective of the rearing environment. However, the response was more pronounced in the dark reared rats. The HO-1 induction increased with age in both dim cyclic light reared and dark reared rats. The increase in HO-1 mRNA is accompanied by apoptotic photoreceptor cell loss. DMTU, an antioxidant, was highly effective in blocking HO-1 induction and photoreceptor cell loss resulting from the intense light exposure. The age or rearing light conditions did not influence the DMTU effect. Thus, age, light history, and antioxidant status are important factors affecting the retinal gene expression in response to stress. Activin, a member of transforming growth factor-beta superfamily, is known to be expressed in the vertebrate retina. We have identified the first invertebrate activin gene from Drosophila. A genomic clone representing 102 F region of Drosophila chromosome 4 was found to encode a putative activin b. The predicted protein sequence showed a multibasic protease site that would generate a mature 113 amino acid C-terminal peptide having more than 60% similarity to vertebrate activin bB. A TGF-b family signature as well as 9 cysteine residues conserved in the vertebrate activins were also present in the mature peptide sequence. The gene was expressed in embryo, larva and adult stages of Drosophila.


Microbial communities associated with the human body, in particular the gastrointestinal tract, play crucial roles in health and disease. The objective of this proposal is to understand how specific patterns in gut microbial succession are related to health and disease, and specifically to neonatal necrotizing enterocolitis (NEC). Although available evidence suggests that intestinal microbes contribute to the pathogenesis of NEC, the details of this relationship remain poorly understood. At present, relatively little is known about the gut colonization process in premature newborns, and about differences between this process in premature infants with and without NEC. We propose complementary high-throughput phylogenetic and metagenomic analyses to study microbial colonization of the premature infant gut during the first three weeks after birth. Our proposed work will elucidate, at high resolution, the population structure of microbial communities that develop during colonization of the premature infant gut and examine the roles of early colonists, gastrointestinal tract-mediated selection, immigration, the effects of mobile elements on genomic variation and microbial survival, and examine how these processes relate to onset of NEC. We will use next-generation sequencing to resolve species- and population-level community succession patterns during the critical initial period of gut colonization in babies that do and do not go on to develop NEC. We will profile community development using high-throughput 16S rRNA tag sequencing of stool samples collected daily during the first three weeks after birth. We will then carry out deep metagenomic sequencing of microbial DNA from half of these fecal time series samples to reconstruct genomes for coexisting bacterial, archaeal, phage, and plasmid populations. This will allow us to track species membership, community structure, metabolic potential, and population-level genetic heterogeneity. We will use these data to test the extent to which initial consortia predict succeeding community diversity [Aim 1], the importance of in situ diversification mediated by phage, insertion elements, and plasmids vs. immigration in determining population structure and metabolic potential [Aim 2], and to define ecological trajectories that correlate with health and disease [Aim 3]. Our preliminary metagenomic data conclusively demonstrate that the proposed approach can be used to reconstruct near-complete genomes of coexisting organisms from premature infant gut fecal samples with sufficient population depth to analyze population heterogeneity and dynamics. Improved understanding of the colonization process in the premature infant gut could translate to improved outcomes for premature babies by suggesting more effective strategies for disease prevention and treatment. More broadly, this research will uncover aspects of ecosystem colonization dynamics that have implications for other aspects of human health and the environment. !


The overall goal of the Duke University - University of North Carolina at Chapel Hill (UNC) Training Program in the Genome Sciences and the Hemoglobinopathies is to train a cohort of both domestic and foreign postdoctoral fellows to be outstanding investigators of the basis for and treatment of the hemoglobinopathies, using genomics and related large-scale and high-throughput approaches in the genome sciences. Whether each trainee's chosen area of focus is in genomics, proteomics or computational biology and whether one's future research is basic, translational or clinical, each fellow will be expected to gain a broad and comprehensive understanding of the genome sciences as applied to ongoing research projects in the hemoglobinopathies. Training towards this goal will be achieved through: advanced coursework;extensive rotations in genomics, proteomics, and computational laboratories;a weekly colloquium involving researchers in the genome sciences;research in laboratories with active programs in hemoglobinopathy research;and co-mentoring by training faculty with expertise both in the genome sciences and in the hemoglobinopathies in order to develop a full appreciation for the multidisciplinary nature of this training program. The training program will be directed jointly by the Director of the Duke Institute for Genome Sciences &Policy and by the Director of the Duke-UNC Comprehensive Sickle Cell Center. Outstanding fellows will be identified from among a strong pool of applicants to existing postdoctoral training opportunities at both Duke and UNC in hematology, transfusion medicine, medical genomics, vascular disease and the genome sciences;from applicants to individual mentors at Duke and UNC;and from applicants identified by an international advisory group consisting of colleagues and collaborators in Tanzania and Thailand. Research opportunities will be provided by a group of more than 25 Duke and UNC faculty members with expertise in the genome sciences and in the hemoglobinopathies. We will develop a group of domestic and foreign colleagues who are dedicated to advance our understanding and treatment of the hemoglobinopathies. This network of investigators will form the basis for an ongoing series of international collaborations to reduce the pain and burden of these disorders.


This research concerns quality of life among younger women (premenopausal) diagnosed with, and treated for, early stage breast cancer. There is evidence that breast cancer leads to more distress among younger than older patients, and many possible reasons have been suggested for this difference. Study 1 (descriptive study) will examine this issue, assessing the levels of concerns expressed by younger breast cancer patients with regard to a range of issues suggested by various sources as important. We will also assess factors that we view as vulnerability/resilience variables (personality qualities of optimism and investment in body image as a source of self-esteem, contextual qualities of social support/social integration), to determine how the latter variables render individuals vulnerable to higher levels of various concerns. Finally, we will assess mediating variables (coping, perceptions of partner reactions to the surgery) and levels of quality of life (affective, social, and psychosexual disturbance) to determine the extent to which the concerns reported are linked to the distress and dysfunction that are the ultimate outcomes of interest, and to examine the role of coping differences as pathways in this chain. We will also collect open-ended data to ensure that we have identified all meaningful sources of concern among these patients. Subjects will be equivalently represented on extent of surgery (mastectomy vs lumpectomy), and 3 periods of time since surgery (3, 6, and 12 months). We will also recruit as close to equal numbers as we can from the 3 ethnic groups represented in large numbers in this population (Black, non-Hispanic White, and Hispanic). Study 2 (in a new sample) will implement and test the effectiveness of an intervention designed to meet the special needs and concerns of younger breast cancer patients. This intervention is based on findings from the literature of breast cancer, on previous intervention studies of breast cancer patients, on the experiences of this research team in intervention research using other populations, and on the results of Study 1. The once-a-week, 10-week intervention will target participants' sense of confidence about remaining cancer free, their coping responses (increasing acceptance and reframing of the experience, and decreasing avoidance coping), their utilization of social support, and their self- image, in a group-therapy format. The control group will be an education/standard of care group, in which subjects learn of the benefits of implementing some of the changes as described above in their orientation, but will not undergo the group experience in solidifying and acting on this information. Treatment efficacy will be assessed at 3, 6, and 12 months post-surgery, in terms of all the quality-of-life outcome variables listed above. This study will also have the benefit of addressing the generality of the treatment effects across ethnic/cultural boundaries.


The purpose of this study was to determine the feasability and utility of adenoviral mediated suicide gene therapy in women with recurrent ovarian cancer. A phase I trial was conducted in women with recurrent intraperitoneal ovarian cancer. Adenovirus encoding Herpes simplex virus-thymidine kinase (AdHSF-TK) was administered intraperitoneally through a percutaneous Tenckhoff catheter; the dose of AdHSV-TK was escalated in cohorts of patients between 1 x 10(9) pfu. Two days later, patients were treated with intravenous ganciclovir (GCV) at a dose of 5 mg/kg BID for a total of 14 days. Patients were monitored at regular intervals for eight weeks post-treatment for evidence of clinical toxicity and response. Serum samples and peritoneal aspirates were obtained at various time points to assess for evidence of gene transfer and immunologic response. Fourteen patients with persistent/recurrent ovarian cancer were treated per study specifications. Most patients had been extensively treated with a variety of chemotherapy regimens. Transient and easily ameliorated vector-associated fever was experienced by 4/14(29%) of treated patients. Other possible vector-associated constitutional (fatigue, insomnia) symptoms, abdominal pain, and gastrointestinal (nausea, diarrhea) symptoms were experienced by 6 of 14 (43%) treated patients. No other vector specific side effects were noted. Six patients experienced catheter-associated complications (infection-5, partial bowel obstruction-1). One patient expired due to a pulmonary embolism three weeks post therapy.


Computed Tomography (CT) has had an enormous impact on medicine since its introduction, and many physicians consider CT (along with MRI) to be the most important recent technological innovation in medicine. Further technical improvements would have important clinical benefit, but the system design in use in current CT systems, even the most advanced clinical scanners, is not able to achieve the combination of capabilities that is needed. We recently proposed a radically different CT system design, Inverse Geometry CT (IGCT) that promises to deliver wide volumetric coverage in a single rapid scan with no "cone-beam" artifacts, high spatial and temporal resolution, improved dose efficiency, and reduced radiation dose to the patient. Our preliminary results provide strong evidence that these goals can be achieved The goal of this proposal is to perform research leading to and including designing and constructing a fullscale prototype IGCT system capable of animal and human scanning, to quantitate its performance, and to perform pilot in-vivo studies. The research involves a collaboration between Stanford University and GE's Global Research (GEGR) Center, building on the pioneering work on IGCT performed at Stanford and the important advances and unique capabilities of the team at GEGR. The groups will collaboratively optimize the system design, perfect calibration and reconstruction algorithms, and perform detailed evaluations. The Stanford group, led by the PI, will be responsible for defining the clinical requirements and performing the animal and human studies. The GEGR group, led by Bruno De Man, will be responsible for detailed system design and construction. While the significance and potential impact of this research are very large, the scope and risk preclude it from being performed by industry alone and public support is required. At the same time, the impact of the requested budget is amplified by existing funding and ongoing research at both Stanford and GEGR, and by a commitment of 1$M from GE Healthcare to fund construction of the gantry based system. We believe that important research and clinical application would be possible with CT systems capable of much wider volumetric coverage in short scan times, requiring lower radiation dose than present systems, and delivering uncompromised image quality and temporal resolution. CT technology currently in use is not up to this task, and that a new approach is needed. We believe, and our preliminary studies show, that our IGCT approach will be able to open this new era in CT scanning. [unreadable] [unreadable] [unreadable]


Methods for the synthesis of N-hydroxy-pyrroles and -imidazoles will be extended and the chemistry of these substances will be explored with a view of preparing new heterocyclic ring systems related to known antineoplastic agents. It is hoped that these will function as pyrimidine and purine antagonists. Substituents will be introduced to confer desirable properties to some of these molecules: alkylating groups, groups that increase lipid solubility, or groups that permit the molecules to cross the blood-brain barrier and thus make them more target-specific. Similar studies will be carried out with derivatives of pyrido(1,2-b)indazoles.


Considerable research suggests that major depression (MD) is common in women with bulimia nervosa (BN). Yet, despite more than two decades of research, the precise relationship between these forms of psychopathology remains unknown. This project aims to examine the depressive symptoms and cognitive correlates of MD, BN, and comorbid MD and BN. It is hypothesized that the depression experienced by individuals with comorbid MD and BN will differ from that experienced by individuals with "pure" MD and that evidence for a depressive subtype of BN (i.e., depressive bulimia) will be illuminated. Approximately 100 women meeting one of four diagnostic criteria (MD, BN, comorbid MD and BN, and asymptomatic control) will be recruited for the study. Proposed methods include diagnostic interviews, self-report questionnaires, and information processing tasks designed to assess: (1) depressive and bulimic symptoms, (2) depressogenic cognitive biases, (3) dysfunctional attitudes regarding needs for achievement and approval, and (4) dysfunctional attitudes and cognitive biases regarding weight, shape, and food. The results of this project are expected to illuminate important symptomatic and cognitive distinctions between MD and depressive bulimia. Such findings will have significant implications for the classification of MD and BN and will inform treatment protocols for both forms of psychopathology.


Ionizing radiation is widely utilized in cancer therapy. While some cancers have been treated successfully, an unacceptable fraction of treatments still fail. The ensuing recurrences and secondary tumors are often more aggressive than the original, possibly because of treatment-induced mutations. These mutations are not well defined due to the varied types of DNA damage ionizing radiation, the randomness of the damage, and its relative infrequency in the genome. Recent advances in nucleic acid chemistry make it possible to synthesize many of the types of damage detected in DNA subjected to ionizing radiation. These modified nucleotides can be incorporated into specific sites in cloned genes by standard molecular biology techniques. The proposed work will utilize this combined approach to place types of DNA damage, characteristic of ionizing radiation, at designated sites in a cloned DNA fragment. We will then determine how such damage affects eukaryotic gene expression and gene integrity. DNA damage will be positioned in synthetic oligonucleotides corresponding to transcription factor binding sites. A gel shift assay will be utilized to determine the binding and stability of transcription complexes containing either normal or damaged DNA elements. Damage will also be incorporated into specific sites in a synthetic DNA template and transcribed in vitro with a nuclear extract. The effect of specific types of DNA damage of gene transcription and fidelity will be determined. Related experiments will also determine if transcription transforms two opposed single-strand breaks into a more lethal double-strand break. Finally, we will measure the relative stability of duplex oligonucleotides containing base damage sites using the competitive mobility shift assay and determine whether a stability correlates with the polymerase by-pass or mutagenicity results obtained from the in vitro transcription studies.


A single injection of cocaine (24 hr prior to injection of carbamazepine) caused a trend (p=0.06) toward increased plasma levels of carbamazepine (single injection). More studies are required to test the effects of chronic cocaine treatment on carbamazepine plasma levels. These data suggest that carbamazepine treatment of humans for cocaine withdrawal may require close monitoring of carbamazepine blood levels to prevent potential toxic effects of this drug. Studies were started to investigate the gamma-aminobutyric acid (GABA-A) receptor system in amygdala-kindled rats that were made contingent tolerant to the anticonvulsant effects of diazepam. A study parallel to this one using contingent tolerance to carbamazepine was reported in last year's project report (Z01 MH 02459-06 BPB). Thus far, only the alpha-4 subunit of the GABA-A receptor has been measured. Results were qualitatively the same as for tolerance to carbamazepine. Kindling increased mRNA for the alpha-4 subunit selectively in the dentate gyrus of the hippocampus, and this increase was significantly less in rats tolerant to diazepam. However, the increase in rats tolerant to diazepam was greater than that observed in carbamazepine-tolerant rats.


Adolescence is a critical neurodevelopmental period that is associated with dramatic increases in rates of substance use. Identifying predictors of substance use and its effects on child and adolescent development is critically important, as substance-related decrements incurred during ongoing maturation could have long- lasting effects on brain functioning and behavioral, health, and psychological outcomes. This Research Project Site application from the University of Michigan and University of Florida is in response to RFA-DA-15-015, as part of the ABCD-USA Consortium (9/13) to prospectively determine the neurodevelopmental and behavioral effects of substance use on children and adolescents. A representative community sample of 975 9-10 year olds will be recruited as part of this application, contributing to the sample of 11,111 to be collected from 11 total sites across the ABCD-USA Consortium. All participants will undergo a comprehensive baseline assessment, including state-of-the-art brain imaging, comprehensive neuropsychological testing, and extensive assessment of substance use patterns and mental health functioning. These comprehensive assessments will occur at 2-year follow-up intervals, with intermediate assessments of functioning and substance use at 6- month intervals. The brain, behavioral, psychological, social, genetic, and environmental data collected during the course of this project will elucidate: 1) the effects of substance use patterns on the adolescent brain; 2) the effects of substance use on behavioral and health outcomes; 3) the bidirectional relationship between psychopathology and substance use patterns; 4) the effects of individual genetic, behavioral, neurobiological, and environmental differences on risk profiles and substance use outcomes; and 5) the gateway interactions between use of different substances. Elements Unique to this Site: This hub's Research Project application leverages site-specific expertise to address two aims focused on the identification of risk and resilience factors for adolescent substance use. Using baseline data categorized into distinct domains (Demographic, Cognitive, Mental Health, Personality, Life Stressors, Family History/Genetics, Environmental, and Brain), we will use cutting-edge, multi-stage analytic methods involving data reduction within each domain (e.g., latent variable analyses), identification of etiologically-distinct subgroups (e.g., community detection), and the construction of integrated multi-modal predictive models (e.g., regularized regression). This approach will delineate subgroups characterized by distinct profiles of risk and resilience. This approach is essential for informing outcomes of substance use during adolescence, which will ultimately inform the development of more efficacious interventions and clarify the toxic effects of exposure on adolescent brain, health, and cognition.


The purpose of this Task Order is to characterize the plasma pharmacokinetics (PK) and bioavailability of small molecule candidate anticancer compounds in mice as outlined below and optionally, provide analytical support for the quantification of such anticancer agents in plasma and/or tumor samples to be provided by NCI. Rapid turn-around is required. There is a base period and options under this task order.


The purpose of this five-year Institutional National Research Service Award is to provide funding for five predoctoral trainees and four postdoctoral trainees per year to prepare them for academic-research careers in the Speech and Hearing Sciences. Because of the multidisciplinary nature of the scientific substrates underlying Communication Sciences and Disorders, the 15 participating faculty for this training grant are drawn from five different departments in three colleges. Each faculty member has agreed to serve as a research preceptor to one or more trainee supported from this grant during a five-year period of time. Faculty members were chosen for participation on this training grant on the basis of the nature of their ongoing research activities, research funding, and experience in supervising doctoral and postdoctoral students in the speech and hearing sciences. Predoctoral and postdoctoral trainee-are selected on the basis of outstanding credentials that reflect a bonafide potential for productive research careers in the Speech and Hearing Sciences. Funding is requested to provide trainees selected for this training grant up to a maximum of three years of support at either the predoctoral or postdoctoral level.


AFC HDPTP Project Summary A disaster, by definition, is an unexpected destructive event that brings harm and damage to a community. While the community members often feel powerless to control the disaster, they can take steps to prepare for their response if it occurs. AFC proposes to use funds from the NIEHS Worker Training Program?s Hazmat Disaster Preparedness Training Program (HDPTP) to provide preparedness training to public safety responders in the southeast US and to Native American tribes throughout the country. The proposed project will continue AFC?s ten-year history with of delivering quality training focused on safety for first responders to a disaster, whether natural or man-made. This project will target public safety (fire, law, EMS, EMA) responders with training that helps them recognize hazards, assess response actions, and bring some order to the chaos of the disaster scene. Disasters often become mass casualty incidents (MCI) and responders may need triage their limited resources to do the most good. Also, unfortunately, these MCI events may be ?man-made? acts of terror in which the perpetrator of violence is still at the scene. AFC proposes to bring Hostile Event Response training that teaches enhanced situational awareness and survival as well as life-saving tactical ?stop-the- bleed? care in an active shooter or improvised explosive device event. AFC projects to bring a total of 70 classes to 1,400 trainees in 11,200 contact hours. It will be split between both the public safety and Native American target populations.


The objective of the project is to determine if cells other than early embryo cells can clone the mammalian blastocyst and participate in embryo development. The mouse will be used as the experimental animal. Cells for transfer will be obtained from late stage embryos, normal adult tissues, and neoplastic tissue. The effect on the cells and blastocysts will be determined by (1) recording microscopic changes in the transferred and blastocyst cells during in vitro development of the blastocyst, (2) following autoradiographically the fate of the cells in the embryo, and (3) placing the recipient blastocysts into foster mothers and studying the offspring for signs of an effect from the transferred cells. BIBLIOGRAPHIC REFERENCE: Brinster, R.L., Can teratocarcinoma cells colonize the mouse embryo? In: Teratomas and Differentiation, (Eds. M. I. Sherman and D. Solter), Academic Press, Inc., New York, pp. 51-58, 1975. Brinster, R.L., Participation of teratocarcinoma cells in mouse embryo development. In: Proceedings of the Meeting on Embryonic & Fetal Antigens. Edited by Dr. J. H. Coggin, Jr., 1976. In Press.


Retrotransposons comprise almost half of the human genome and substantial fractions of other metazoan genomes. Nonetheless, the mechanisms and localization of retrotransposon assembly are poorly understood. Ty3 is a long terminal repeat retrotransposon in budding yeast containing GAG3 and POL3 ORFs encoding structural and catalytic proteins, respectively. Because Ty3 exists in a simple eukaryote that facilitates molecular, genetic, biochemical, and even cytological approaches, it provides a useful model for understanding retrotransposon mechanics. In the current funding period, the assembling Ty3 VLP was characterized by density sedimentation, RNA protection, transmission EM and atomic force microscopy performed on Ty3 VLPs blocked at specific stages and also on mature VLPs. In addition, sixty Ala substitution mutants were analyzed to dissect the functions of Gag3 (capsid, spacer, and nucleocapsid) subdomains. These studies showed that the amino-terminal domain is critical for assembly and nuclear pore association of VLPs and that the nucleocapsid domain is important for targeting Ty3 RNA and Gag3 protein to the P body for assembly. A set of Ty3 RNA variants was developed and used to show that the Ty3 5'and 3'untranslated regions (UTRs) and POL3 contain independent cis-acting P-body targeting activities, but that only the UTR mediates packaging of Ty3 genomic RNA. The finding that the RNA processing body (P body) previously described as a site where nontranslating RNAs are sequestered or degraded, is the site of Ty3 assembly was particularly significant as assembly sites have not been characterized for retrotransposons and how retroviruses preassemble RNA and protein at a perinuclear site is not well understood, but appears to involve packaging signals. In Aim A of the proposed work, we will use mass spectroscopy to identify host proteins which are associated with Ty3 RNA and structural protein. Our collection Ty3 wt and variant RNAs tagged with the MS2- binding site coupled with a novel high affinity TAP-tagged MS2 binding protein and our antibodies against Ty3 proteins will be used to isolate proteins directly involved with Ty3 components. Strains with attenuated functions of identified genes will be tested for defects in Ty3 VLP assembly and proteins identified will be investigated in Aims B-D. A key step in assembly occurs when the mRNA transitions from translation to packaging. Based on the retrovirus model, this is associated with structural rearrangements in the UTRs of the RNA which are facilitated by nucleocapsid binding. In Aim B we will define at higher resolution the packaging site of Ty3 RNA and its interaction with the reverse transcription primer initiator tRNAMet. Previous work in the Darlix laboratory, with whom we collaborated, showed that initiator tRNAMet has a bipartite primer binding site in Ty3 5'and 3'UTRs. Retroviruses have dimeric genomes and we have shown that Ty3 is similar. Darlix proposed a novel model for Ty3 genomic RNA dimerization mediated by two itRNAMet molecules annealing at the 5'ends. We will specifically test whether the PBS, and by implication, the putative dimerization interface, is required for packaging of Ty3 RNA into VLPs. If so, that would suggest that initiator tRNAMet, which has unique roles in translation initiation, and priming Ty3 reverse transcription, is a central player in the key transition from translation to packaging. SELEX and in vitro RNA-protein binding assays will be used to identify important nucleocapsid binding contacts in the packaging site of the Ty3 UTR which could mediate primer annealing and dimerization. In Aim C we will test our hypothesis that the Ty3 UTR structure antagonizes translation initiation and imposes requirements for specialized translation helicases. In addition, we propose that reduced numbers of translating ribosomes downstream of the frameshift site attenuates 60S subunit delivery to the 5'end of the RNA and explains the sensitivity of Ty3 and other retrotransposons to imbalances in translation initiation factors and 60S ribosomal subunits. This hypothesis will be tested using host mutants and variant Ty3 RNAs. We will test whether translation is ultimately repressed by Gag3 binding in cis to the Ty3 UTR or POL3. In Aim D we will characterize the contribution of P bodies to Ty3 assembly. We hypothesize that P-body proteins cooperate with Gag3 to repress Ty3 translation, allow the concentration of assembly factors, and may promote unwinding of the RNA to allow packaging. Nonetheless, isolation of an unusual mutant which does not form large P body clusters suggests that P bodies might repress transposition at a post-assembly stage. Factors identified in genetic and mass spectroscopy screens will be tested for direct interactions with Ty3 components and for their roles in clustering P body proteins and assembling Ty3 viruslike particles. In summary, Ty3 is a well-characterized retrotransposon in a model eukaryote. It offers a unique opportunity to understand the retrotransposon particle assembly process and how P bodies might chaperone this process.


The Environmental Protection Agency calls arsenic the most prevalent environmental toxin and carcinogen in the United States (://www.atsdr.cdc.gov/cercla/07list.html). Arsenic causes cardiovascular and peripheral vascular diseases, neurological disorders, diabetes mellitus and various forms of cancer such as skin and bladder cancer. Arsenic is biomethylated by the liver enzyme As (III) S-adenosylmethionine (SAM) methyltransferase (AS3MT) to mono- and dimethylated species. Because the trivalent products methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) are more toxic than inorganic arsenite, they have been proposed to be associated with arsenic carcinogenesis and other diseases in humans. Individuals with AS3MT polymorphisms produce increased amounts of methylated species. How methylation contributes to disease depends on the mechanism of human AS3MT and differences between wild type and polymorphic enzymes. The uncertainty over the consequences of methylation makes it imperative to understand how this enzyme works. The overall goal of this study is elucidation of the structure and function of hAS3MT and its polymorphic forms.


Approximately 100,000 of the over one million people in the United States infected with HIV are women, 80% of whom are in their childbearing years. Consequently, AIDS has already become one of the leading causes of death in children between the ages of 1 and 4 in the United States. Maternal and infant HIV infection is occurring disproportionately among lower socioeconomic populations and minority women. Poverty, drug use, severe stress, and family disorganization characterize the ecological environment of these infants. As a result, these infants, already at risk for developmental delays because of the impact of the HIV infection, are at even greater risk for delays in their developmental trajectory. The quality of parental caregiving has been found to be a major environmental determinate of a normal child's social, physical, and mental development. Within the context of HIV-infected families, the quality of caregiving is critical. Yet, providing care to a seropositive child creates further strains on the family, which is already compromised by the burden of illness in the biological mother. Inadequate caregiving may also be caused by other lifestyle characteristics of the mother and family. The quality of parenting in these at-risk infants may influence the progress of the child's illness. The field of psychoneurology has demonstrated that stressful caregiving environments may create physiological responses in the child, specifically the immune system, at a time when the child is highly vulnerable to such changes. The overall goal of this longitudinal study of HIV seropositive infants born to HIV-infected women is to identify child, parent, family and community factors that influence the developmental trajectory of these children. Special emphasis will be placed on whether quality of parental caregiving plays a mediating role in developmental outcomes and, with HIV-infected infants, in the progression of the disease. This prospective longitudinal study will be done at the Duke Infectious Diseases Clinic where medical services and supportive care are currently provided in a comprehensive team approach. Infants sero-positive for HIV will be enrolled in the study when the infant first enters the Pediatric Infectious Disease program, generally about 24 months of age, and followed until the child is at least 12 months old, with a subsample followed to 18 and 24 months as feasible within the 3 year time frame. Date will be collected using multiple methods (interview, questionnaires, observation, and assessment) with multiple informants (parents, health care staff, research team) over time. Data collection methods assess selected infant, caregiver, family, and community characteristics, the quality of parental caregiving, and disease progression and developmental outcomes in the child. The ultimate goal is to identify factors which place infants born to HIV seropositive women at developmental or health risk so as to identify appropriate interventions to help parents or other primary parental caregivers cope with HIV and more adequately meet the child's developmental needs.


We recently discovered a thrombogenic mechanism for pregnancy loss in the antiphospholipid antibody syndrome that antiphospholipid antibodies displace the placental anticoagulant protein, annexin-V, from the anionic phospholipid surfaces of trophoblasts and accelerate coagulation reactions at that site. The core hypotheses of this grant are: a) Annexin-V serves a thromboregulatory role at the anatomic site where maternal blood and placental villi interface by shielding anionic phospholipids (which markedly accelerate coagulation enzyme reactions) from participation in coagulation reactions and; b) antiphospholipid antibodies competitively displace annexin-V from that phospholipid surface, disrupting the annexin-V "shield", thereby increasing the exposure of the thrombogenic anionic phospholipid. This process leads to thrombosis in the maternal circulation at the sites of interface with fetal trophoblasts and to ultimately to pregnancy complications and losses and determine their clinical efficacy. Our specific aims are: 1) To determine the mechanism(s) by which antiphospholipid antibodies and cofactors reduce the quantity of annexin-V on trophoblast apical membrane and accelerate coagulation reactions and 2) To develop tests for the reduction of annexin-V binding to phospholipids and the inhibition of annexin-V anticoagulant activity. This innovative project opens a new path toward elucidating the mechanism of pregnancy loss in the antiphospholipid syndrome and is likely to lead to improved diagnosis and treatment.


During the last year, the project has focused on the chemokine system in cancer. Chemokines and chemokine receptors can have roles in cancer by bringing white cells to the cancer and through direct effects on the cancer cells themselves. Although white cells can combat cancer by killing cancer cells, white cell products can also serve to enhance cancer cell growth, which is thought to be a mechanism whereby inflammation can promote cancer. Cancer cells that express chemokine receptors can also receive growth and/or survival signals from chemokines. In analyzing samples from human cancers that typically arise in association with inflammation, we have found that many of these cancers express the chemokine CXCL16. We concentrated on prostate cancer and using immunohistochemistry and immunofluorescence with confocal microscopy, we found that the cancer cells express both CXCL16 and CXCR6, and that high expression was associated with poor prognostic features. We also found expression by tumor-associated lymphocytes, and we hypothesize that both by bringing lymphocytes to the cancer and by directly stimulating cell growth, CXCL16 may be promoting prostate cancer. Another aspect of the project has explored using a second chemokine receptor, CXCR4, which is expressed on many cancers and is thought to be associated with aggressive cancer, in order to image cancers by positron emission tomography (PET). We have synthesized a radiolabeled CXCR4 antagonist, (64)Cu-AMD3100, that could potentially be used to identify CXCR4-expressing cancers and to correlate CXCR4 expression with prognosis and responses to therapy, thereby providing additional clinical information that might guide effective cancer therapy in individual patients.


. Repetitive use of hand tools, in particular those that compress the palm of the hand and are held with a pinch or precision grip may be a large part of the high incidence of carpal tunnel syndrome (CTS) that is overwhelming the industrial work place. This proposal is to conduct preliminary investigations into the relationship of carpal tunnel pressure (CTP) to the design of handles used on squeegees and related hand tools such as pliers and screwdrivers. The purpose of this investigation will be to determine the feasibility of preventing CTS using the squeegee handle as a hand tool model and redesigning the standard shape. In Phase I we will investigate how handle shape and size affects CTP to determine the feasibility of designing an ergonomically sound squeegee handle. If the results of this investigation suggest an ergonomically sound squeegee handle design will prevent CTS then in Phase II we will use the results of our study to design and test prototype squeegee handles and develop the biomechanics to properly use these ergonomic handles. The screen printing industry is actively seeking a solution to the rampant CTS problem.


The research outlined in this proposal deals with several aspects of biologically active compounds produced by microorganisms. The main emphasis is on the biosynthesis of a number of antibiotics. The methods to be used include feeding experiments with precursors labeled with radioactive isotopes, but also to a large extent the use of 13C-labeled compounds in conjunction with 13C Fourier Transform NMR. Antibiotics to be investigated are pyrrolnitrin, chlorothricin, boromycin, granticin, the naphthocyclinones, dihydrophenylalanine, ketomycin and anticapsin. In addition some structural work is proposed which involves the isolation and structure elucidation of some new naphthoquinone-type compounds found in the cultures of the granaticin-producing organism. Also within this project, some pilot studies will be carried out to explore possibilities of biotransformation of antitumor antibiotics. Also in collaboration with some synthetic organic chemists engaged in total synthesis of natural products and analogs we will evaluate their synthetic compounds for biological activity and/or help them establish identity of the synthetic and the natural material.


This application is for the continuation of studies to design and develop inhibitors of O6-alkylguanine-DNA alkyltransferase (AGT) as chemotherapeutic agents. The presence of AGT is a major factor in the resistance of tumor cells to killing by drugs that form adduct at the O6- position of guanine. The aim of these studies is to extend the effectiveness and the range of tumors that can be treated with these drugs. Studies during the first period of support have led to Phase I trials with O6-benzylguanine (BG) but further work is needed to optimize the use of this agent and to develop compounds with better therapeutic properties in order to improve the discrimination between tumors and normal tissue. There are 5 laboratory programs and 3 cores. Dr. A.E. Pegg will serve as the PI and the Leader of Program 1 which will carry out studies on the interaction of possible AGT inhibitors with the control and mutant AGT proteins and design improved inhibitors including compounds able to inactivate BG-resistant AGTs. Dr. M.E. Dolan will head Program 2 in which detailed metabolic studies of novel AGT inhibitors will be carried out. Specific compounds include:metabolism-resistant 8-substituted BG derivatives; compounds with ester linkages that are activated as AGT inhibitors by esterases; and benzyl pyrimidine derivatives which are very potent but rapidly cleared. The use of regional therapy with AGT inhibitors using intraarterial delivery for CNS parenchymal tumors and intrathecal administration for leptomengeal neoplasms and the selection of the most promising compounds for targeting the inhibitor to brain tumors will be studied in Program 3 led by Dr. H.S. Friedman. Program 4 led by Dr. S.C. Schold will focus on the development of 9-substituted BG derivatives including the nucleoside and deoxynucleoside which appear to offer significant advantages over BG in terms of systemic availability, penetration of the blood-brain barrier and localized metabolic activation. Program 5 led by Dr. S.L Gerson will investigate methods for optimizing tumor cytotoxicity after AGT inactivation. He will study the extent of AGT inactivation needed to achieve maximal inactivation, methods to overcome resistance to BG-therapy of colon and breast tumors, and procedures including gene transfer to minimize bone marrow toxicity of the combination of AGT inhibitors and BCNU. Routine evaluation of compounds both for AGT inactivation and for the ability ot sensitize tumor cells in culture to BCNU will be carried out in Core B led by Dr. Pegg. Promising compounds will be examined for in vivo pharmacokinetics, tumor AGT inactivation and sensitization of tumor xenografts in nude mice in Core C led by Dr. Dolan.


= No large-scale study within the United States has examined any aspect of the epidemiology of intracranial meningiomas, a lesion which accounts for up to 25% of all primary intracranial neoplasms. We propose to conduct a population-based case/control study of meningioma within the states of Connecticut, Massachusetts, North Carolina, and Texas as well as the San Francisco Bay area under the mechanism of an integrated R01 (Note that identical applications are submitted for all five sites). Cases will be selected using rapid case ascertainment and will include all male and female individuals diagnosed with a histologically confirmed intra-cranial meningioma between the ages of 20 and 79 years from 7/1/2006 through 6/30/2010 in the above listed regions. Control subjects will be identified via random-digit dial methods and matched to the cases by sex, ethnicity, geographic location, and five-year age category. Study subjects will be administered a telephone questionnaire to collect information on the two primary categories of risk, exposure to ionizing radiation and hormones as well as additional risk factors such as family history of meningioma and other tumors, cell-phone utilization, and head trauma as well as questions on outcome and quality of life. Parraffin-embedded tumor tissue blocks will be obtained for case subjects to allow for 1) a uniform histological review, and 2) immunohistochemical testing for estrogen, progesterone, androgen and MIB-1 receptors. Blood or buccal specimens will be collected from all study subjects for testing of DNA polymorphisms in DNA repair and cell cycle genes. This study represents the first concentrated effort to examine environmental and genetic risk factors for meningioma.


This interagency agreement has the following scope: Clinical research on HIV and AIDS associated complications and co-infections with emphasis on optimizing therapeutic approaches to the treatment of HIV and related co-infections an co-morbidities in resource poor developing countries, including phase I-4 therapeutic clinical trials Clinical research on HIV prevention strategies ranging from novel pre-exposure prevention modalities such as Prevention of Mother to Child Transmission (PMTCT) and between discordant couples, treatment of sexually transmitted diseases, post exposure prophylaxis, and microbicides


The overall goal of this project is to directly test how cholinergic input from the nucleus of the horizontal limb of the diagonal band of Broca (HDB) alters olfactory bulb (OB) glomerular circuits and how this modulation ultimately affects odor coding, perception, and learning. We will use of both wide-field and 2-photon in vivo and in vitro calcium imaging in mice expressing genetically encoded indicators of neuronal activity in defined OB cell types, to test the novel overarching hypothesis that synaptically-released acetylcholine bidirectionally modulates mitral/tufted cell odor responses as a function of the prevailing odor intensity. We further test the hypothesis that this modulation is due to opposing muscarinic and nicotinic receptor actions on inhibitory periglomerular cells that differentially regulate the strength of presynaptic inhibition of olfactory sensory neuron input to the glomeruli. Using imaging and well characterized olfactory-mediated behaviors, we will also investigate how this cholinergic modulation of the glomerular odor representation affects odor perception and learning. Specifically, we test the hypotheses that HDB-evoked acetylcholine release in the OB: (1) enhances olfactory sensitivity, (2) dishabituates odor responses adapted at the peripheral olfactory sensory neuron level and (3) is critical for associative olfactory learning. By investigating receptor type-specific cholinergic modulation in morphologically- and physiologically-distinct neuron types at both the population and single cell levels, the experiments of this proposal will for the first time elucidate how cholinergic modulation of neural odor responses in the OB impacts olfactory perception and learning behaviorally. The overall findings will dramatically advance our understanding of how acetylcholine modulates sensory representation, odor coding, perception and behavior.


The contractor shall serve as the coordinating center for the Epidemiologic Studies of Human Retroviruses in Volunteer Blood Donors program. The objectives are as follows: 1) determine the prevalence of retrovirus seropositivity in first time blood donors: 2) determine the rate of retrovirus seroconversion in repeat blood donors as a measure of incidence of infection: 3) ascertain risk factors for antibody-positive donors: 4) characterize the blood population by geographic location, age, sex, race/ethnicity, and donation history to permit analysis on prevalence, incidence, and risk factors: 5) identify recipients of retrovirus-positive blood units and conduct clinical and laboratory follow-up of these recipients to determine the transmissibility of these agents via transfusion; and 6) establish blood specimen repositories for long-term storage of specimens from study donors and recipients for future testing.


This project investigates the role of mitochondrial integrity during the development of TBI pathology and periods of functional recovery. Treatment paradigms involving cyclosporin A (CsA) are used to test the hypothesis that a triad of injury pathology, including plasmalemmal microporation, cytoskeletal proteolysis and mitochondrial degeneration, can be mediated through direct effects on mitochondrial membrane integrity. We will investigate the effects of blocking the mitochondrial permeability transition pore complex (MPT) with CsA both prior to and following TBI. Given that our original observations show that CsA administration offers protection against axonal injury pathology in TBI, we will now test the hypotheses that somato-dendritic domains within injured neurons also exhibit the same triad of pathologies and that these domains can also be protected with CsA intervention. Our hypotheses will be tested the same triad of pathologies and that these domains can also be protected with CsA intervention. Our hypotheses will be tested in two well known models of TBI, impact acceleration and combined neuroexcitation + deafferentation lesion. Using these models we can directly compare similarities and differences in both diffuse and focal injury paradigms. We will use immunohistochemistry (IHC), electron microscopic permeability tracer methods, assays for mitochondrial oxidative enzyme function and mRNA expression, and behavioral endpoints to measure effects of CsA on TBI pathology. In addition, we will explore the pharmacokinetics of both intrathecal and intravenous administration of CsA, assaying effects of administration time and dose on the above outcome measures, as well as determining the degree of drug permeability through the blood brain barrier. Finally, we will begin to address alternative mechanisms of action for CsA in TBI, first testing its potential influence on calcineurin mediated pathways. To do this we will administer a calcineurin specific inhibitor, FK506, in treatment paradigms shown to efficacious with CsA and determine its effect on structural outcome measures. The value of our proposed studies is twofold. First, they clearly define cellular mechanisms of TBI pathology and recovery associated with metabolic function. Perhaps more importantly, these studies, combined with the related human studies in this program project proposal, will establish a solid knowledge base supporting potential CsA therapeutic efficacy in TBI patients.


Patients with high level of eosinophils in their blood frequently die because of heart disease. Using ultrasound, we are able to detect abnormalities of the heart long before heart symptoms occur. It is hoped that early treatment of this problem will prevent or delay heart abnormalities.


Rodent pests have eaten our food and transmitted diseases to us for millennia. There are an estimated 300 million introduced Norway or brown rats in the U.S. that cause $27 billion of economic losses annually. Historically the strategy has been to kill rat pests through a variety of methods but primarily by using poison baits that are anticoagulant rodenticides. In addition to all of the problems associated with using poison bait, e.g. poisoning nontarget animals/pets, children under the age of 6, and contaminating the environment, poison does not address the problem. A non-lethal strategy that has significant potential to manage rodent pest number is fertility control. But thus far effective control of free-ranging wildlife, such as small, nocturnal rodents, has not been achieved because distribution to dose rats must be via an oral route. The industrial chemical 4-vinylcyclohexene diepoxide (VCD) accelerates the natural process of atresia leading to depletion of rat ovarian follicles causing ovarian failure and permanent sterility. Follicle depletion occurs when VCD is given in repeated intraperitoneal daily doses and on average, complete elimination of primordial/primary follicles and premature ovarian failure occur by day 58 following the onset of dosing and in mice causes infertility. To date VCD induced follicle depletion has been achieved by intraperitoneal administration. However to develop and commercialize a product to cause wild rat infertility it must be given orally in a bait. This Phase I application aims to test the following hypothesis in three specific aims. Oral administration of VCD to rats will lead to complete depletion of ovarian follicle populations, resulting in infertility demonstrating feasibility for development of a fertility control bait for rats. Aim 1. Determine the dose needed to deplete primordial ovarian follicles in rats by oral exposures to VCD. Aim 2. Determine the time course over which VCD completely depletes ovarian follicle populations. Aim 3. Determine the pharmacokinetics of VCD excretion. Results from Phase I experiments will define the dose and duration of VCD exposure necessary to cause complete ovarian follicle depletion. These results will enable us to obtain sufficient information to progress to a Phase II application to develop an oral bait and baiting protocol to manage wildlife rodent populations. Rats are being targeted in these studies, with the understanding that rats are more resistant to VCD than mice. Thus, success with rats, can easily be translated to mice. Furthermore, rats are the primary target pest species based on the enormous economic and significant public health risks they pose both by their contamination and destruction of our food supplies and the dangerous rodenticide control approaches currently being used to manage rat populations. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Management of wild rats is critical to minimize agriculture and property damage and the spread of infectious diseases. Lethal approaches introduce poison into the environment and consequently are being targeted for removal from over the counter availability. The goal of this project is to provide data to support the feasibility of using the industrial chemical 4-vinylcyclohexene diepoxide (VCD) to ultimately develop an environmentally neutral permanent rodent fertility control bait to manage wild rat populations


Major disasters occur with regularity throughout the world, causing widespread destruction and death and leaving stunned survivors in their wake. Relatively little is known of the extent of lasting psychic impairment produced by these events or what can be done to ameliorate it. One such disaster took place at Buffalo Creek, West Virginia, on February 26, 1972, when a dam collapsed, unleashing 150 million gallons of water and coal waste into the valley. A group of approximately 650 survivors are suing the coal company for compensation for physical and mental pain and suffering and the destruction of their former community life. Our research team is in the unique position of having access to all medical and school records, psychiatric interviews and psychological tests, obtained on this group of litigants. We propose to quantify the psychiatric data and the familial life-stress produced by the disaster, making them amenable to statistical analysis and hypothesis testing. These will be supplemented with symptom data on non-litigants and on members of another similar community that has not had such a disaster. A follow-up study is also planned for two successive years after the case is settled, to determine its effect on the community. Independent random samples of 50 litigant and 25 non-litigant families each year will be selected for this purpose.


This SBIR Phase II proposal is a plan to further develop a novel therapeutic approach that will save the lives of patients with sepsis. Despite the increased understanding of the complex pathophysiology of sepsis, severe sepsis still results in significant morbidity and mortality. As such, there is an urgent unmet medical need for an effective novel therapy for septic patients. The global market potential for sepsis treatment is estimated at over $30 billion annually. Thus, successful development of a new anti-sepsis therapy will not only have a positive impact on health care, but also will have significant commercial benefits. A balanced inflammatory response is an essential element of a successful host defense after injury. However, excessive production of proinflammatory cytokines may cause further tissue injury. Macrophages/Kupffer cells play important roles in producing proinflammatory cytokines in sepsis. The nervous system reflexively regulates the inflammatory response in real time. We have demonstrated that the release of the sympathetic neurotransmitter, norepinephrine (NE), from the gut is increased in sepsis, and that NE potentiates endotoxin-induced TNF-( upregulation via the A subtype of (2-adrenoceptors (i.e., (2A-AR) expressed on the surface of Kupffer cells. Pre-treatment with a specific (2A-AR antagonist, 2-[(4,5-dihydro-1H-imidazol-2-yl) methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL- 44408 maleate), downregulates TNF-(, attenuates tissue injury, and improves survival in a rat model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). However, it remained unknown whether the delayed administration of BRL-44408 maleate (which is more clinically relevant) reduces sepsis-induced mortality as well. Accordingly, the primary objective of our completed Phase I project was to determine the effect of delayed administration of BRL-44408 maleate on sepsis-induced inflammation, organ injury, and mortality. We have clearly shown that administration of BRL-44408 maleate at 5 h after CLP (i.e., at the early stage of sepsis) is protective in experimental animals. These results have established the technical merit and feasibility of the proposed Phase II project. We therefore continue to hypothesize that the administration of the small molecule drug candidate BRL-44408 maleate in established sepsis attenuates tissue injury and improves survival. In this Phase II proposal, we will perform detailed toxicological evaluation and pharmacokinetic characterization, and determine the optimal protective dose(s) and time- course of BRL-44408 maleate in sepsis in the rat. In order to advance the technology to clinical trials, the efficacy of BRL-44408 maleate will be tested in a rabbit model of sepsis. These proposed studies should provide important preclinical data that will help us filing an IND application to the FDA to initiate clinical trials in order to obtain commercial utilization of BRL-44408 maleate as a safe and effective therapy for sepsis. PUBLIC HEALTH RELEVANCE: Sepsis is one of the leading causes of death in intensive care units. Over 210,000 people succumb to this overwhelming infection in the United States annually. A recent epidemiologic study estimated that more than 750,000 people develop sepsis each year at a cost of $16.7 billion nationally. Given the intensive and prolonged care necessary to treat patients with sepsis, the economic burden is profound. Thus, there is an urgent unmet medical need for an effective novel therapy for patients with sepsis.


Background Endocrine malignancies (including thyroid, adrenal, parathyroid, and pancreatic neuroendocrine tumors) are among the fastest growing cancer diagnoses in the United States, but it is difficult to distinguish benign from malignant tumors by routine clinical, laboratory, and imaging studies. So, even patients who have seemingly benign endocrine tumors often choose to undergo surgery to get a definitive diagnosis in the hopes of ruling out cancer. Most patients with endocrine cancers have a relatively good prognosis. However, anywhere from 10% to 40% (depending on tumor type) have aggressive disease which often cannot be reliably determined at the time of initial treatment. Prognostic markers which can reliably risk stratify patients with high risk of recurrence and death would help determine which patients should receive aggressive initial treatment and close follow up. Furthermore, prognostic markers may also help identify which patients are likely to respond to standard therapy and which patients do not respond to standard therapy if a distinct molecular phenotype is identified. Summary We have made progress with our pan-genomic (mRNA and microRNA expression, copy number changes, and DNA-methylation) analysis of endocrine neoplasms to identify candidate diagnostic and prognostic markers for endocrine malignancies (thyroid, adrenal, neuroendocrine pancreas), and to understand the dysregulated genes/pathways in endocrine cancers. Our analysis shows key changes in mRNA/protein expression and microRNA expression levels, and DNA-methylation status that serve as excellent diagnostic markers. These studies have been expanded to include the analysis of serum and urine samples to detect altered levels of microRNAs, proteins and DNA-methylation changes found in the tumor samples. In this analysis we found some microRNAs (downregulated in cancer) to be actively secreted in exosomes based on both in vitro and first in human venous sampling studies. The integrated analysis of our genomic data, especially in thyroid cancer and adrenal cancers, have also uncovered possible novel pathways which may have a role in thyroid and adrenal cancer initiation and progression which we are confirming using functional genomics studies.


SHARED RESOURCE CORE CRUMP PRECLINICAL IMAGING TECHNOLOGY CENTER ABSTRACT: The Crump Preclinical Imaging Technology Center will provide comprehensive support for the in vivo imaging studies carried out in the NSBCC projects. Under the direction of Core Director Dr. Michael Phelps, the Imaging Center is comprised of two other faculty, Dr. Arion Hadjioannou and Dr. Jason Lee, and one full-time staff member. All faculty members have extensive experience and recognition in preclinical molecular imaging. The Imaging Center will provide all radio-labeled probes, perform all microPET and CT studies, and data analysis. In addition, they will provide support and training to investigators, including animal preparation and monitoring, quality control and the strictest safety measures. The goal of the Shared Resource Core is to provide advanced molecular imaging support to assess the kinetics of tumor metabolism and therapeutic outcome in response to therapeutic intervention. These experiments will consist of highly-controlled, serially- acquired PET and CT scans for both functional and anatomical assessment, respectively. Since the PET radio- labeled probe used is also widely available in the clinic, the preclinical imaging data acquired by the Shared Resource Core can be translated as biomarkers for evaluation of treatment response in patients.


This is an application to renew a grant to study RNA synthesis in minus-strand RNA viruses. Nonsegmented negative-strand (NNS) RNA viruses include some of the most significant human pathogens that are a major ongoing threat to US public health. To combat those agents, we need a combination of antiviral drugs and vaccines. Our long-term objective is to understand the mechanisms by which the replication machinery of vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, functions. VSV is the ideal choice for such studies because it is the only NNS RNA virus for which robust transcription can be reconstituted in vitro from purified recombinant components. The catalytic core of the RNA synthesis machinery is a 241 kDa large polymerase protein (L) that contains an RNA dependent RNA polymerase (RdRP), a polyribonucleotidyltransferase (PRNTase) that caps the mRNA, and a dual specificity mRNA cap methyltransferase (MTase). During mRNA synthesis, those activities are coordinated so that the nascent mRNA is capped, methylated and polyadenylated. Although L contains all the enzymatic activities for RNA synthesis, it requires a 29 kDa phosphoprotein (P) that bridges interactions between L and the nucleocapsid protein (N) that completely coats the genomic RNA template. In the last grant period, we developed in vitro assays to separately study each of the steps of mRNA cap addition independent of ongoing transcription. Those assays, combined with a powerful reverse genetic system allow mechanistic analysis of lethal mutations in the viral RNA synthesis machinery. We have used those assays to provide a map of where the different enzymatic activities for each step of mRNA cap addition are localized within L. Those studies lead us to the hypothesis that L contains independent functional domains whose activities are coordinated by the assembly of L into the RNA synthesis machine of the L-P complex with the N-RNA template. A major gap to understanding the mechanisms by which the RNA synthesis machinery of NNS RNA viruses function is the absence of structural information for L. During the next funding period, we will use electron microscopy, X-ray crystallography and in vitro assays of polymerase function to provide unique structural and functional insights into the RNA synthesis machinery of VSV. We will: (i) determine the functional organization of the VSV polymerase complex; (ii) determine the three dimensional structure of the VSV polymerase, and (iii) probe the relationship between the mRNA capping and RNA synthesis activities of L. The successful completion of this study will provide a structure of the polymerase of an NNS RNA virus as well as new mechanistic insights into the function of this RNA synthesis machine that may help in the rational design of antiviral therapeutics and candidate vaccines.


The Bypass Angioplasty Revascularization Investigation (BARI) is a randomized clinical trial to compare the relative efficacy of an initial strategy of PTCA vs. CABG in patients with multivessel coronary disease and severe angina or ischemia. Treatment strategies will be compared to respect to clinical outcome and economic impact. The trial was initiated in 1987 and investigators from 14 primary sites and 5 satellites have randomized 1829 patients. Of these, 43% are elderly, 27% are female, and 6% are black. BARI patients are at high risk for cardiac events; 55% presented with a history of myocardial infarction, and 69% with unstable angina or non Q-wave MI. At baseline, triple-vessel disease was present in 41% and 73% had three or more significant lesions (greater than or equal to 50% stenosis). As of July 1993, the first patient randomized will have been followed for 5 years and the last patient will have been followed for 2 years. In addition to the randomized trial, BARI contains a registry comprised of 2013 eligible-not-randomized patients and a sample of 422 patients deemed ineligible based on their angiogram. For both randomized and registry patients, we propose to complete and report 5-year follow-up results and extend follow-up to 10 years. In randomized patients, treatment strategies will be compared with respect to mortality, myocardial infarction, need for repeat procedures and hospitalizations, functional status, radionuclide ejection fraction and quality of life. Periprocedural outcome will also be reported. Similar analyses will be reported for both registry groups. To accomplish 10-year follow-up, we will use the same methods of data collection and management, and continue the same lines of communication between the clinical sites, central laboratories and the NHLBI that have served BARI successfully to this point. The clinical sites will rely on the excellent long-term relationships established with their patients and referring physicians. Ten-year follow-up results will provide the long- term cost of PTCA vs. CABG, not only in terms of dollar expenditures but in terms of quality of life, morbidity and longevity. The results of BARI will have an important impact on the future practice of coronary revascularization and thus affect hundreds of thousands of patients who are expected to undergo these procedures.


Ethnic minorities tend to face more stressors than the majority population and to have excess risk of many health problems. These disparities are strikingly evident for Puerto Ricans living on the mainland, especially for older Puerto Ricans who face psychosocial stressors related to drastically changed social and physical environments. The limited research on Puerto Ricans has provided evidence of much poorer physical health and greater disability', including cardiovascular risk, compared to other Hispanic groups. The effects of psychosocial factors on heart health risk are not well understood for older Puerto Ricans, as research has primarily focused on other Latinos and has been mainly cross-sectional. Theoretically, it is important to understand the mechanisms of mediating factors and the longitudinal causal pathways through which psychosocial factors influence cardiovascular risk. It is therefore critical to identify and explore the interactions between personal, social and environmental factors which contribute to cardiovascular risk for Puerto Ricans. The longterm goal of this work is to contribute to understanding psychological, social and environmental contexts of cardiovascular risk of older Puerto Ricans. The objective of this proposal is to examine in our study population how stressors, personal resources, social networks, and perceptions and characteristics ofthe built environment relate to cardiovascular risk indicators over time. We approach the analysis of these factors using a multi-level approach and drawing on the large scale cohort longitudinal data set with 1350 participants and three points in time. The aims ofthe projects are 1) to assess associations between depressive symptomatology, psychosocial stress and CVD risk over a period of 5 years; 2) to assess the characteristics of social networks and their effects on cardiovascular risk, and 3) to assess the characteristics ofthe local environment and its impact on heart health and cardiovascular risk. This project is innovative in its integration of analj/sis at the individual, social and environmental levels which should yield a richer data set and a more thorough analysis. The work vrill contribute significantiy to gaining a fuller view ofthe context of psychosocial stress and how it influences the cardiovascular health of older Puerto Ricans. We add an interdisciplinaty social science perspective to grounding and contextualizing findings from our proposed work but also those ofthe other projects in this program. Results will allow the development of more effective strategies and programs to reduce health disparities in this high risk population.


Work planned for the coming year will concentrate more heavily on studies of the ultrastructural correlates of ion and fluid transport and macrom lecule secretion in epithelial cells which are possible candidates for the type of cell affected by the genetic defect in Cystic Fibrosis. Attempts at identifying an abnormal cell or cell organelle in patients with CF will entail cytochemical, fine structural, electrophysiologic and certain biochemical analyses of human eccrine sweat glands & possibly specimens of nasal or tracheobronchial epithelium from control & CF subjects. Alterations in these tissues maintained in explant under various experimental manipulations will be assessed along with the effect of so-called factors thought to be present in body fluids of CF patients. Efforts will continue in collaboration with Dr. W.H.J. Douglas to culture epithelial cells from sweat glands of normal and CF cubjects & investigate abnormalities in the cultured cells from the patients again employing fine structural, cytochemical, electrophysiologic, ion flux & biochemical measurements. Investigation of the presence of amiloride channels from passive Na ion transport in the human sweat gland & respiratory epithelium will be initiated. These studies, in collaboration with Dr. E.G. Cragoe of Merck Sharp & Dohme, will include radioautographic demonstration of binding of C14 and H3 labeled derivatives of this diuretic drug to specific epithelial cells. Fluorescence microscopy with various fluorescent derivatives & electron spin resonance measurements with a spin labeled derivative will be utilized for localization of amiloride channels in various epithelial cells. Freeze-fracture studies now in progress will be pursued to characterize further the nature of cell junctions between various cell types in the sweat glands and respiratory epithelium & characterize intramembranous particles in plasma membranes of these cells. Investigations of the pathogenesis of mega inclusions in cells of beige mice with a genetic defect analogous to the Chediak-Higashi syndrome will continue with emphasis on the pathogenesis of inclusions in the proximal nephron, gastric epithelium and culture fibroblasts. Development & application of cytochemical methods for localizing & characterizing complex carbohydrates of immunocytochemical methods for visualization of carbonic anhydrase & other antigens will continue.


Neurocognitive impairments in HIV-infected individuals, collectively known as HIV-Associated Neurocognitive Impairments (or HAND) remains a significant problem in the era of Combined Antiretroviral Therapy (CART). In many HIV-infected individuals there is evidence of accelerated aging, including aberrant processing of amyloid precursor protein (APP). These disruptions seem to result in accumulations of pathogenic forms of amyloid- () in brain and are thus likely to also decrease the formation of soluble APP? (sAPP?), an important neurotrophic peptide. Our preliminary data suggest that accumulations of sphingolipids and complex glycolipids in intracellular compartments accelerates A formation by enhancing the activity of - and ?- secretases (that process APP to A), and by perturbing the intracellular trafficking / clearance of A. Previously we have documented accumulations of multiple sphingolipid species in HIV-infected individuals. These combined findings prompted us to determine if the accumulations of sphingolipid products in endosomes, lysosomes and/or autophagosomes are associated with aberrant APP processing, increased A deposition and decreased sAPP? in the setting of HIV-infection. In this application we propose a comprehensive approach to address this question, using human brain tissues, cellular/molecular approaches, and transgenic model systems to determine if increased brain levels of these lipid metabolites shifts APP processing to a more amyloidogenic (A) and less trophic (sAPP?) pheotype and if interventions that target sphingolipid metabolism can reverse these effects.


Xiphophorus fishes have been used as a vertebrate experimental model for fundamental biomedical research for over 60 years. This R24 proposal outlines both resource and research components designed to maintain and enhance the Xiphophorus Genetic Stock Center (XGSC) that has operated within the United States since the 1930's making it one of the oldest live animal resources centers worldwide and a national scientific treasure. The XGSC houses over 60 pedigreed genetic lines representing 25 Xiphophorus species in over 1,400 aquaria. The XGSC has provided pedigreed fish and materials to researchers in over 30 laboratories representing 11 countries. The traditional strength of the Xiphophorus experimental model involves the non-biased assessment of genetic inheritance patterns associated with complex phenotypes within intact animals. For example, genetic control of tumor susceptibility has been documented in both pure Xiphophorus strains and select interspecies hybrids, for a variety of spontaneous and induced neoplasms including several genetically distinct melanomas. Our preliminary studies represent a first global comparison of the relative abundance of species-specific allele expression in two vertebrate species and Fl hybrids. In this initial study we were able to identify substantial changes in gene expression levels for parental alleles in the interspecies hybrid genetic background. We also found a new class of genetic interactions that lead to substantially suppressed gene expression in Fl hybrids relative to either parent. These initial experiments, employing RNAseq to follow allele specific gene expression in Fl interspecies hybrids, serve as examples of the Xiphophorus model may be used to uncover new knowledge regarding the mechanics of gene interactions, the genetic basis of developmental programs, complex behaviors, and the molecular processes underlying disease initiation and progression. If the goals of this proposal are realized, mechanistic dissection of Xiphophorus interspecies hybrid melanoma models may be approached and the results gleaned will have translational significance to tumorigenesis in humans.