Pain as a complication of spinal cord injury (SCI) has been identified as a primary determinant of quality of life. Although pain is a frequent complication of SCI, few studies have directly examined the effectiveness of various pain management interventions in this population. The primary goal of the proposed project is to improve the management of chronic pain in persons with SCI. Although all types of pain can occur in SCI, dysesthetic/deafferentation pain is the most common and disabling. The tricyclic antidepressant medications have been found to be most effective pharmacologic treatment for relieving deafferentation pain. Current research in chronic pain has demonstrated the importance of psychosocial factors, including cognition and emotions. Consequently, behavioral interventions have been successful in improving function and reducing disability and handicap. Multidisciplinary approaches utilizing combinations of treatment modalities, such as pharmacological and psychological interventions, are now recognized as the standard of care in chronic pain management. The proposed project will examine the efficacy of two primary interventions for managing chronic pain, nortriptyline or cognitive-behavioral treatment, and their combination in reducing SCI-related pain, disability, and handicap. In addition to comparing the relative effectiveness of medical and behavioral interventions, this project will examine whether individual patient characteristics, such as depression, predict responses to these treatments and whether treatment gains are maintained at 6-month follow- up. Persons with persistent pain for 6 months or longer following SCI will be enrolled in this randomized controlled study which incorporates a 2 group (nortriptyline versus placebo) by 2 group (cognitive-behavioral versus self-care eduction) design. After completing a baseline assessment, each patient will undergo 10 weeks of active treatment that will include weekly psychological intervention and daily use of medication. Following completion of the active phase of treatment, patients will enter a 6-month maintenance and follow-up period. Outcome measures will assess pain, physical and psychosocial function, use of medication for pain, and use of the health care system. Evaluation of a treatment approach combining commonly used psychological and pharmacological approaches promises to promote the development of truly effective programs. Findings derived from this project will lead to further studies that determine which treatments, or combinations of treatments, are effective for specific subgroups of SCI patients and other chronic pain populations. These results will advance the design of individual treatment programs and further improve treatment outcomes.


DESCRIPTION: The goal of this Phase II proposal is to produce and test a videotape about treatment options for prostate cancer that presents medically rigorous information in nontechnical language using documentaries of real patients. The videotape will be designed as a decision-aid to promote informed decision-making about treatment as well as to increase patient satisfaction with their decisions. During Phase I, a script for one segment of the video and a very good seven-minute video excerpt were developed based on feedback from men treated for prostate cancer, their partners, urologists and nurses. PROPOSED COMMERCIAL APPLICATION: Not available.


With the development of modern immunocytochemical and intracellular electrophysiological techniques, the complexity of local circuitry in hippocampus has become apparent. Although the hippocampus is a relatively simple cortical structure, it is thought to participate in complex information processing tasks. The diversity of interneuron types, their transmitters, and their local circuitry must be key elements in providing the system with the means of modulating the activity of primary projection cells. In the present application, we continue our study of those interneurons and local circuitries. Further, we propose to initiate a series of studies examining the effects of various "traumatic" treatments on the interneurons of hippocampus, and the consequences of damaging specific vulnerable cell populations. The studies will be carried out on parallel, but closely interacting, tracks. Morphological characterization, by light and electron n=microscopic/immunocytochemical techniques, will describe the locations, arborization patterns, and putative neurotransmitter content of specific interneuron populations in the CA1, CA3, and dentate regions of rat hippocampus. These studies will also provide guidance for in vitro slice electrophysiological investigations with will attempt to characterize interneurons on the basis of their electrophysiological properties and their synaptic inter-actions with principal cell types (using simultaneous intracellular recordings of cell pairs). Interneurons will be intracellularly labeled and examined, in turn, at the morphological level. The same morphological and electrophysiological techniques will be used to assess the effects of various treatments on hippocampal interneurons and interneuron-dependent circuitry. Ischemia, anoxia, and stimulation treatments will be delivered to intact animals, which will be examined morphologically, and from which hippocampal slices will be obtained from electrophysiological investigation. Parallel treatments will be carried out in slices while recording from suspected vulnerable interneurons. Procedures will be developed to "protect" vulnerable interneurons from effects of these treatments. These studies will provide a descriptive basis for understanding interneurons and interneuron local circuitry in hippocampus, and will test a number of specific hypotheses regarding interneuron action in hippocampus.


The purpose of this project is to determine the role played by tropomyosin in the organization of the actin cytoskeleton in normal and transformed rat kidney cells in culture. The normal cells contain five isoforms of subunit tropmyosin: two of 30 kilodalton apparent molecular weight (as determined on two-dimensional polyacrylamide gels, one of 35\kilodalton, one of 37\kilodalton, and one of 40\kilodalton. Transformed cells contain the 30\kilodalton isoforms but have only trace amounts of the 40\kilodalton isoform and are lacking the 35 and 37\kilodalton isoforms. Experiments are being conducted on the effects of microinjecting complete mixtures of isoforms of tropomyosin purified from normal cells into transformed cells in order to determine if the transformed cells can acquire the ability to organize stress fibers. Using a rabbit polyclonal antibody that binds to the 35, 37, and 40\kilodalton isoforms but not to the 30\kilodalton isoforms, we have found them to be localized in stress fibers in fully spread cells. In freshly plated cells, we have found that the antibody stains the ruffles at the peripheries of the cells, in contrast to previous reports that ruffles lacked tropomyosin. The same antibody also stains ruffles at the edges of transformed cells, which never contain stress fibers. Injection of an affinity-purified derivative of this antibody into normal cells disrupts stress fibers in the cells and alters cell shape. Another antibody has been raised in rabbits to a 30\kilodalton protein from transformed cells that appears to be an isoform of tropomyosin. This antibody stains a reticular network just beneath the plasmalemma of normal and transformed cells but does not stain stress fibers in normal cells. The possibility that this is a membrane-associated isoform of tropomyosin is being investigated. Monoclonal antibodies to individual tropomyosin isoforms are now being raised. Actin binding studies indicate that the 30\kilodalton tropomyosin isoforms bind less tightly to actin than the other three isoforms. (L)


The main aim of this proposal is to allow the principle investigator to become more independent and to perform a pilot study in the area of cell biology with an emphasis on the membrane targeting and trafficking of vitamin C transporter and to generate additional publications and data for future grants. Vitamin C (ascorbic acid) is essential for normal human growth and development. Human intestine and other tissues express two sodium-dependent vitamin C transporters (hSVCT1 and -2). Recent studies have shown the involvement of specific Rab proteins in the membrane targeting/localization, trafficking and function of membrane proteins in intestinal cells. To date, nothing is known about the role of Rab proteins in cell biology and transport physiological aspects of hSVCT1 with specific regards to membrane targeting/localization, trafficking and function in human enterocytes. Recent studies have shown that the alteration in membrane targeting and trafficking events may affect the polarized expression of the given transporters and lead to overall impaired absorption/secretion in epithelia. Further, these cellular events were found to play an important role in the regulation of certain transport processes via insertion/retrieval of the involved transporters to and from the cell membrane. Therefore, studies aimed at regulators such as Rab proteins in membrane targeting and trafficking of transport proteins are of great significance and of cell biological/physiological importance. Recent studies have shown the involvement of Rab8a and 11a in the membrane targeting and trafficking of transport proteins in intestinal epithelial cells and also linked the proteins to human intestinal microvillus inclusion disease. Our specific aim is to determine the role of Rab8a and 11a proteins in polarized membrane targeting/localization, trafficking and function of hSVCT1 in vitro using human enterocytes and confirm results in vivo using Rab8a knockout mice. In these investigations we will use a combination of cell/biochemical/molecular biological and imaging methods. Outcome of these investigations will not only provide the applicant an opportunity to reach his career goals but also contribute to enhance the understanding of the cell biology of hSVCT1 and also other micronutrient transporters. PUBLIC HEALTH RELEVANCE: Vitamin C is an essential nutrient for human well-being and cannot be synthesized, obtains it from diet via intestinal absorption. Human intestine plays a role in regulating and maintaining body vitamin C homeostasis, studies on the cell biology/physiology of its transporters in intestinal cells are important. Outcome of these investigations will assist us to optimize intestinal vitamin C levels in deficiency and sub-optimal levels.


Plasmid-directed gene expression is now near the efficacy barrier that to date has prevented commercialization of plasmid-based therapies for human health applications. Previous innovations such as electroporation (EP) increased transgene expression through more effective gene delivery. In Phase I we developed potent minimalized antibiotic-free expression vectors, and demonstrated dramatic improvements in transgene expression may be obtained through vector design innovations. Two platform technologies were developed. " Novel compositions that prevent vector-backbone mediated transgene silencing from plasmid vectors (Anti-Silencing Elements: ASE platform) " Novel vector backbone functionalities that improve transgene expression from plasmid vectors after transient transfection (transient expression enhancers: TEE platform) In Phase II we hypothesize that combining ASE and TEE vectors with state of the art plasmid delivery will create enabling vector-delivery platforms for gene therapy. In Specific Aims 1 and 2 optimal ASE/TEE antibiotic-free vector - EP delivery platforms for skeletal muscle and cutaneous gene therapy, respectively, are identified. In Specific Aim 3 the cutaneous gene therapy platform is applied to create a hypoxia-inducible factor 11 (HIF-11) based gene medicine for diabetic foot ulcer treatment. In Specific Aim 4 a dermatological gene therapy to treat skin aging is developed using a keratinocyte growth factor (KGF) vector- microdermabrasion delivery combination. Specific Aims 3 and 4 are performed in collaboration with wound healing gene therapy expert Dr. John Harmon at Johns Hopkins University and dermatology gene therapy expert Dr Aaron Tabor at Gene Facelift, LLC. The vector-delivery platforms developed herein will further improve gene expression to levels that will enable gene medicine licensure for multiple applications for unmet public health needs. In Phase III the gene therapies for diabetic foot ulcers and skin cosmetics will undergo clinical development. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to validate a novel antibiotic-free non-viral gene therapy platform, and as such is responsive to NIGMS SBIR high-priority area of interest in development of improved vectors for gene transfer. The vectors contain transient expression enhancers that improve transgene expression level and duration after gene delivery to skin or muscle. The platform will be applied to create gene therapy products to treat diabetic neuropathic foot ulcers and skin aging.


ISS Inc., in collaboration with Dr. Franceschini of the Massachusetts General Hospital and Dr. Yodh of the University of Pennsylvania, proposes to develop and construct the prototype of a novel instrument to monitor cerebral oxygen metabolism (CMRO2) that combines a frequency-domain near-infrared tissue oximeter (FDNIRS) and a diffusion correlation spectroscopy (DCS) device. Combining FDNIRS and DCS will enable us to measure cerebral hemoglobin oxygenation (SO2) and an index (BFi) of cerebral blood flow (CBF) to estimate CMRO2. Our clinical collaborators Dr. Ellen Grant of Children's Hospitals in Boston, and Dr. Daniel Licht of Children's Hospital of Philadelphia will advise us on optimizing the device for the neonatal ICU clinical setting. The ability to rapidly an accurately assess cerebral metabolism at bedside in neonates is critical to improving patient management in neonatology. Currently there is no bedside tool that can accurately screen for brain injury, monitor injury evolution or assess response to therapy. Head ultrasounds are notoriously insensitive and nonspecific, EEG serves a complementary role, and MRI is too expensive and time-consuming to be used for screening or monitoring, and involves unnecessary risks of destabilizing critically ill infants who must be transported to the MRI suite. Conventional NIRS oximetry has failed to become a routine clinical tool in neonatology due to the poor sensitivity of SO2 in detecting brain injury hours after the insult, when equilibrium between oxygen delivery and consumption is reestablished. The direct measure of cerebral oxygen utilization we propose to measure with this novel instrument will provide higher sensitivity to detect brain injury and the ability to monitor normal and abnormal brain development. If successful, this device will allow for real-time interventions and thus could improve clinical outcome. Our long-term plans encompass the commercialization of the first FDNIRS/DCS cerebral metabolism monitoring system. In phase I we will build the first commercial prototype, start to develop unified acquisition and data analysis software, and test this new system in phantoms and adult human subjects. We will then perform a pilot study in newborns and compare infants with seizure activity confirmed by EEG with healthy controls, to validate the hypothesis that a measure of oxygen consumption is better able to diagnose brain damage than measure of hemoglobin oxygenation. While CMRO2 monitoring has been performed previously in a research setting, by combining ISS FDNIRS systems with lab-built DCS instrumentation, these measurements have required highly trained operators and data analysis by optical imaging experts. Our goal is to transform these cumbersome research systems into a more robust turnkey device that will enable medical staff to obtain clinically and relevant measurements at the bedside. After completion of the Phase I portion of the project, we will proceed with Phase II, during which we plan to refine the system and start demonstrating the clinical relevance of our CMRO2 measurements in three hospitals (Brigham and Women's and Children's Hospitals in Boston, and Children's Hospital of Philadelphia with Dr. Daniel Licht). If the plan is accepted in its entirety, we expect to complete development of the FDNIRS/DCS system, which we call MetaOx, within the projected four-year period.


This project attempts to delineate the nature, frequency, and natural history of clinical manifestations among women heterozygotes for adrenoleukodystrophy.


Although dialectical behavior therapy (DBT) has been efficacious in controlled studies, its effectiveness in community settings, and for patients with borderline personality disorder (BPD) and comorbid depression, remain to be determined. My research will address these questions within the framework of effectiveness research, by comparing DBT with treatment as usual. Factors that contribute to patient attrition and outcome from DBT will also be identified, including universal change processes and patient characteristics. Individuals with comorbid BPD and major depressive disorder will be randomly assigned to either DBT or a treatment as usual condition. Assessments will be obtained prior to, during, and at posttreatment (12 months). DBT treatment adherence will be assessed and treatment groups will be compared on a number of outcome variables including severity of depression and number and severity of parasuicidal acts.


This component describes plans for the support and management of a series of pilot projects, including procedures for scientific and technical review. Pilot studies in the UConn ARC have served as the basis for the development of programs of research that are integral to the Center, but which also extend beyond it through a variety of other funding mechanisms. During the current grant cycle, pilot studies have resulted in a total of 14 peer-review publications and have contributed to five successful grant applications, which have been funded through R01, R03, or R21 mechanisms. In these ways, the pilot studies program has played a crucial role in the vitality and growth of the UConn ARC. Seven separate projects are described, of which three will be initiated during the first two years of the grant period (Phase I). The remaining four projects are scheduled for implementation during the final three years of the grant (Phase II). Based upon the success of the pilot studies program during the current and previous grant periods, a tripartite approach is employed that focuses on three main investigational areas: neuropharmacology/neurobiology, electrophysiology/brain imaging, and treatment trials. This approach addresses the major research foci of the UConn ARC: vulnerability to alcohol-related problems; basic mechanisms of problem drinking, dependence, and relapse; and the treatment of alcohol dependence. During Phase I of the new grant period, proposed studies include the generation and phenotyping of transgenic mouse lines with altered expression of the GABAA receptor a6 subunit gene, a study of the electrophysiologic effects of hazardous drinking and depression in women, and a treatment trial that examines the safety and feasibility of using the opioid antagonist naltrexone to reduce problem drinking in adolescents. Studies to be conducted during Phase II of the grant cycle include a study of alcohol and neurosteroid modulation of GABA function and a study of the impact of purified antibodies directed against dopamine receptor subtypes on voluntary ethanol intake. This phase will also include a study of the effects of alcohol, cocaine, and antisocial personality disorder on cerebral blood flow, measured using functional MRI. Finally, Phase II will include a trial evaluating the effects of a low-cost, contingent reinforcement approach on ambulatory treatment attendance and retention, and on alcohol and drug use in alcohol-dependent patients.


The purpose of this program is to implement and evaluate a comprehensive educational program in the schools to alter the cardiovascular "at-risk" behavior of children and their parents. The overall goals are threefold. First, the program is designed to prevent children from developing behavior patterns which might result in heart disease as adults. Second, the program is designed to alter the current behaviors of children and adults which are believed to result in heart disease. Lastly, it is felt that a comprehensive risk factor program aimed at both parents and children is likely to have an impact beyond the initial point of intervention, that is, that changes will be sustained over time. The risk factors to be addressed include smoking, nutrition, and blood pressure management. All interventions are based on social learning theory and are designed to be delivered by older children who should be good role models for the children receiving the program. The programs to be conducted are adaptations of programs of known efficacy and will include additional evaluation and modification of the programs to be delivered by older peers, direct instruction of parents, and training of children to talk with their parents about their health behavior.


The rate of cholesterol nucleation and crystal growth from metastably supersaturate model and native human gallbladder biles has been shown to importantly affected by factors present in biliary proteins. These kinetic factors are both inhibiting and promoting in activity. Our recent studies show that they are both glycoproteins, a well-defined 63 kDa inhibitor and most likely @ 140 kDa promotor. These glycoproteins have been found in different lectin chromatography-bound fractions with differing sugar specificities. We have developed a sensitive and reproducible new spectrophotometric assay system that facilitates not only their detection but permits estimates of their relative potency. Using this assay, we have found that both glycoproteins are present in normal biles. This suggests that in Health a balance of the kinetic factors is present and that an undefined imbalance may play a significant role in cholesterol gallstone pathogenesis. Given our present findings, with progress in coming months we expect to complete the Isolation and partial characterization of the two glycoproteins with optimal "scaling-up" of production. The following logical objectives have therefore been established. Firstly, we wish to accomplish quantitation of the two effector glycoproteins. Radioimmunoassay (RIA) (and related immunoblotting for final purity assessment) seems the only reasonable methodologic approach. Secondly, a biophysicochemical characterization will be undertaken. This will consist of an assessment of the magnitude and functional importance of the sugar- moieties, followed by amino acid analysis of both effector glycoproteins. Thirdly,, their individual mechanism(s) of action will be examined. This will be done by Quasi-elastic light-scattering (QELS) analysis of the crystal growth process to assess the normal pattern as altered by the glycoproteins, especially the inhibitor. Analysis of the spectrophotometric crystal growth curve will also be undertaken to define quantifiable parameters. For study of mechanistic aspects of the promotor glycoprotein,k we propose to use Differential Scanning Calorimetry, QELS and related techniques to assess fusogenic vesicle protein-lipid interactions. Comparative surface activity measurements will also be made. Lastly, we propose to use the previously-developed quantitation methods (RIA) to determine the source(s) and natural distribution of the effector glycoprotein.


This is a revised competing continuation of a grant focusing on the biomechanics of traumatic brain injury. In this continuation application, we study how tissue strains translate to cellular strains in the hippocampus, and proceed to concentrate on the NMDA receptor (NMDAR) mechanosensitivity and its role in hippocampal neuronal death. The broad, long term objective is to examine if regional neuronal populations are 'mechanically vulnerable' and susceptible to injury, to test if extrasynpatic and synpatic NMDARs have different mechanosensitivity, and to use the data on NMDAR mechanosensitivity to evaluate both immediate and delayed treatment strategies to reduce neuronal death after mechanical injury. The specific aims of this research plan are as follows: Aim 1: To describe the local micromechanical environment in organotypic hippocampal cultures subjected to realistic, in vivo deformations associated with injury. Aim 2: To measure the mechanoactivation threshold for extrasynaptic and synaptic NMDAR using recombinant NMDAR expressed in HEK293t cells and organotypic hippocampal cultures. Aim 3: To examine the activation and control of both JNK and ERK from mechanosensitive NMDARs, using this information to examine acute and delayed treatment strategies for reducing cell death after mechanical injury. Our overlying hypotheses are (a) at the same level of tissue stretch, neurons in the dentate gyrus and CAS experience higher mechanical deformations than other hippocampal regions, (b) NMDARs are stretch sensitive due to linkages to the cytoskeleton, (c) the mechanoactivation of extrasynaptic NMDA receptors will influence the activation of JNK through STEP, and (d) ERK activation will be preferentially controlled by synaptic NMDARs, with the duration of ERK signaling mediated by the activity of STEP. Relevance: This work will study how neurons in the hippocampus are injured during traumatic brain injury. The work will concentrate on studying the mechanical activation of a receptor known to play a role in memory formation and cell death. Using the information of receptor activation, the investigators will develop and test treatment strategies to reduce neuronal death following traumatic brain injury.


A high-growth PR8 virus for pandemic vaccine production in ST6-Vero cells. In 2009, the traditional means of vaccine manufacturing for the H1N1 influenza pandemic failed to yield sufficient doses during the peak of the crisis. A vaccine production method that can universally accommodate variant strains of virus in much shorter timelines would greaty benefit the interdiction of a growing pandemic threat. Cell-based production systems have been proposed to replace the protracted egg-based processes. Cell culture-based technology is robust, reliable and is a more rapid and efficient alternative to egg-based technology for vaccine producers. For pandemic vaccine production, distinct advantages of cell-based over egg-based vaccine production are speed (12 weeks), capacity (kg scale) and versatility (quick response to new antigens) so that production lots with significantly higher viru yields may be obtained in nearly half of the time. Despite great promise, no cell-based system has been approved for use by the FDA and technical barriers currently exist in the use of cell lines for manufacturing of flu vaccine. These barriers are 1) limited virus production from current cell systems, and 2) reliance on cell lines that spontaneously form tumors in vivo. To overcome these challenging barriers, FluGen Inc. has generated a Vero cell line that stably expresses the human 2,6 sialyltransferase gene I necessary for generation of human-specific influenza receptors (ST6-Vero). ST6-Veros offer greater infection rates with virus isolated from clinical samples than normal Vero cells. ST6-Vero also may be grown to highly packed cell densities in commercial bioreactors thereby providing more host cells for viral replication. The goal of this proposal is to generate a high-growth donor A/Puerto Rico/8/34 (HG-PR8) virus to further enhance viral propagation in FluGen's ST6-Vero cells. The specific aims of the Phase I project are to 1) generate a high-growth PR8 donor virus by serial passaging in ST6-Vero cells; 2) demonstrate increased productivity of ST6-Vero cells by producing HGPR8- H1N1 vaccine virus in scaled-up suspension cultures; and 3) confirm the antigenicity and immunogenicity of the ST6-Vero produced HGPR8-H1N1 pandemic virus by demonstrating protection in mice. Upon completion of these Phase I aims, a HG-PR8/ST6-Vero cell-based vaccine production system will be realized that is capable of yielding extraordinary titers of 109 pfu/ml, and triggering the expected immune response, as evidenced by protection of mice by viral challenge. For Phase II, qualification of the ST6-Vero cell line and method development for scaling up the process of producing H1N1 pandemic vaccine under GMP conditions will be pursued. The intended commercial product is a licensable platform vaccine production system that can rapidly produce pandemic vaccine efficiently and at very high titers. PUBLIC HEALTH RELEVANCE: A high-growth PR8 virus for pandemic vaccine production in ST6-Vero cells. In 2009, the traditional means of vaccine manufacturing in eggs failed to yield sufficient doses during the peak of the H1N1 influenza pandemic crisis. FluGen Inc. proposes to engineer a cell-based vaccine manufacturing system with the highest rate of production in the industry for these Phase I investigations. Our proprietary ST6-Vero cell line stably expresses the human ?2,6 sialyltransferase gene I necessary for generation of human-specific influenza receptors. ST6-Vero cells result in increased virus yields and hence produce greater amounts of hemagglutinin than normal Vero cells. ST6-Vero also may be grown to highly packed cell densities in commercial bioreactors thereby providing more host cells for viral replication. To enhance cell-based productivity even further, we will generate a high-growth A/Puerto Rico/8/34 (HGPR8) donor virus specific for ST6-Vero which will confer high viral growth property in ST6-Vero cells. The combination of the high replication of HGPR8 in highly infectable ST6-Vero cells that may be grown to immense densities will provide a rapid and high yielding vaccine production system.


New, high performance hardware for computing and data storage provided with additional funding from the DOE-OBER is being tested. A four-processor Digital Alpha server running Digital UNIX and a 750 GB storage array are in commissioning phase to serve BL9-2, BL9-1, and BL7-1 in the near future for data storage and analysis. The high readout speeds of the two MAR345 detectors on BL7-1 and BL9-1 and the even higher rate of the ADSC-Q4 CCD detector on BL9-2 require fast and robust access to storage arrays. Only if data can be accessed by multiple computers at speeds greatly exceeding the speed of data collection, geometric corrections and data analysis can be done sufficiently. First tests are also underway with SGI Octane computers, which have dual graphics capability allowing for two simulataneous console logins on two consoles (two monitors, keyboards, mice). The long term plan is to provide a unified interface on the SGI computers to data collection and data processing, while the actual compute jobs are performed on remote servers. The benefits are in user friendliness, robustness and performance.


This award will prepare Theoklis Zaoutis, M.D., for a career as an independent investigator in Pediatric Infectious Disease epidemiology, with a particular focus on fungal infections and the use of antimicrobial agents. He proposes a comprehensive, interdisciplinary program that will provide him with the skills, and experience necessary for independent patient-oriented research. The training component includes advanced education in clinical epidemiology and biostatistics, training in Hospital Epidemiology, and dual mentorship from an internationally recognized pharmacoepidemiologist and a renowned medical mycologist. The research component of the award will focus on candidemia and the role antibiotics may play in the development of candidemia. Infections due to Candida spp. are the most common fungal infections in hospitalized patients and the fourth most common isolate recovered in cases of nosocomial bloodstream infection in the United States. The proposal has three specific aims: Aim 1: to determine the risk factors for candidemia in children. This specific aim hypothesizes that the use of antibiotics with activity against anaerobic bacteria is associated with an increased risk of candidemia. This hypothesis will be tested using two nested case-control studies. The first study will test the hypothesis in a sample of children with cancer. The second study will test the hypothesis in a sample of children without cancer. The primary analysis will compare children with candidemia (cases) to children without candidemia (controls). Cases and controls will be matched on time at risk using incidence-density sampling. Aim 2: to develop a clinical prediction rule for candidemia in children without cancer. This specific aim will be based on the same cohort of hospitalized patients receiving antibiotics as described in SA1. Aim 3: to determine the clinical outcomes of children with candidemia. This specific aim will use a retrospective cohort design to test the hypothesis that children with candidemia caused by C. albicans have poorer clinical outcomes than children with candidemia caused by non-albicans Candida spp. The primary analysis for this aim will utilize survival analysis methods. This work may establish a rational basis for guiding antibiotic selection in high-risk children. Future studies will be planned to evaluate the effect of interventions designed to decrease the risk of developing candidemia as well as the effect of the prediction rule in selecting more effective prophylactic and preemptive therapy for children at high risk for the development of candidemia. [unreadable] [unreadable] [unreadable]


Fibromyalgia (FM) is a rheumatic disorder characterized by the presence of widespread musculoskeletal pain and the presence of tender points. Other symptoms, including fatigue, sleep disturbance, and neuropsychological complaints, contribute significantly to the morbidity associated with FM. One of the most prominent complaints in patients with FM is impaired cognitive ability. The notion that cognitive deficits are fundamental to FM is impaired cognitive ability. The notion that cognitive deficits are fundamental to FM has some credibility, as there is growing evidence that there are subtle but important cognitive deficits associated with Chronic Fatigue Syndrome (CFS), a related disorder, that cannot be explained by psychiatric symptoms. It is possible that cognitive defects in FM patients could result from single or multiple central nervous system perturbations associated with FM. In the present proposal, we will correlate cognitive function of FM patients with measures of neuroendocrine function. A basic thesis advanced is that FM patients may have both cognitive and neuroendocrine function similar to that of controls subjects who are 20 to 30 years older. Indeed, cognitive testing in patients with CFS reveals changes similar to those seen in subjects of advanced chronological age. In two experiments, FM patients will be compared to age-and education- matched controls, as well as to education-matched older adults. Neuroendocrine function will be measured as well, as will depression, pain, fatigue, and beliefs about memory function. This approach permits us to determine whether there are differences in cognitive function of fibromyalgia patients from others, and whether cognitive agins is a good model for understanding the cognitive effects of FM. In addition and perhaps more importantly, the integration of a cognitive approach with a neuroendocrine approach will allow us to determine what mechanisms account for the cognitive differences--neurochemical, psychiatric, or experience pain and fatigue. Knowing the mechanisms underlying observed cognitive deficits, rather than merely demonstrating that there are deficits, has important implications for treatment of the disorder as well as for understanding its etiology.


Operons are an important feature of the C. elegans genome. Their transcripts are polycistronic pre-mRNAs that are processed by 3'end formation and trans-splicing. These two events occur in close proximity between operon genes. Although 3'end formation is generally accompanied by transcription termination, in operons it is not. We have identified the key sequences and many of the key trans-acting proteins and snRNAs responsible for carrying out these events, and we are determining what roles they play and how. The key sequence required for both the trans-splicing and for preventing transcription termination is the Ur element that occurs ~50 bp downstream of the 3'end cleavage site. We will determine what binds there. We have recently succeeded in obtaining several templates from operons that are correctly trans-spliced in vitro in a C. elegans embryo extract. This indicates that correct trans-splicing is a property of proteins that bind to the RNA, rather than requiring co-transcriptional RNA processing. We will exploit the in vitro system to test a wide range of mutant substrates to identify all sequences required for correct trans-splicing. We will then identify the macromolecules that act at these sequences and determine what roles they play. Normally transcription termination accompanies 3'end formation, but in operons this cannot be the case. We will use chromatin IP to analyze what phosphorylation events occur to the RNA polymerase CTD as it traverses an operon. These studies should provide important new insight into how 3'end cleavage can occur without accompanying transcription termination in operons. This work could also provide strong support for, or refutation of, the torpedo model for transcription termination. We are also studying the roles of the protein components of the trans-splicing snRNPs. Functional studies involving mutants of these components and how they interact with mutants in functionally related genes are proposed. We have discovered that two of these proteins are bound to a novel snRNA, Sm Y, currently the only snRNA whose function is not known. We will study the possible role of this RNA in trans-splicing and operon pre-mRNA processing. We have hypothesized that these proteins are required for recycling the Sm proteins from the branched intermediates following trans-splicing, and we will test this model using both in vivo and in vitro experiments. Finally, we have devised a genetic screen for mutants in genes required for proper trans-splicing. PUBLIC HEALTH RELEVANCE This project focuses on basic means of gene expression in nematodes, organisms that are responsible for a huge disease burden in humans, as well as domestic animals and cultivated plants. The process under study here, trans-splicing, occurs in nematodes but not their hosts. It is hoped that if we can come to understand this process in detail it may suggest ways to interfere with it, such that the nematode would be sensitive to a treatment that the host is not. Besides this, the basic knowledge we obtain from this project may be important in understanding mechanisms of gene expression in general, especially processing of mRNAs, and this basic knowledge may provide clues for means to treat mRNA splicing defects responsible for many genetic diseases.


This study of membrane transport is targeted to provide the basic scientific information needed to: determine the mechanisms by which physiological control systems are altered in the aged; and use this new information as a age for the eventual development of appropriate techniques and procedures which will prevent and/or enable the aged to cope effectively with their debilities. Membrane vesicles derived from the luminal brush border segment and the antiluminal basal-lateral region of the renal tubule epithelial cell plasma membrane are used as model systems. Topics investigated include: 1) the role of the membrane potential in the Na ion gradient-dependent uptake of D-glucose by brush border membrane vesicles; 2) the mechanisms and specificities of amino acid transport systems; 3) the mechanism and regulation of phosphate transport; 4) mechanism of the D-glucose dependent Na ion transport; 5) the isolation and characterization of the Na ion gradient-dependent glucose carrier by reconstitution of synthetic membranes; 6) molecular organizaton and orientation of membrane components in the membrane.


One aspect of infant functioning, effectance motivation, the motivation to master problems and to have an impact on his environment, has received considerable theoretical attention, but little empirical study. In this study we have developed methods to measure infants' efforts to secure feedback from the environment and their motivation to solve problems. Data have been collected on 44 one year old infants, 23 boys and 21 girls. We are analyzing the relationships between scores on our effectance motivation tasks and contemporaneous measures of cognitive development and exploratory behavior. We are also looking at the early antecedents of effectance motivation by analyzing relationships between specific components of the environment at 6 months and scores on effectance motivation at one year.


PROJECT SUMMARY/ABSTRACT The loss of glucose-stimulated insulin secretion (GSIS) is a critical pathophysiological event precipitating development of hyperglycemia in Type 2 diabetes mellitus (T2DM). Recent evidence suggests that loss of GSIS in diabetes is associated with metabolic reprogramming toward reduced mitochondrial function; however mechanisms underlying these observations remain largely unknown. Recent single cell transcriptomics studies of human ?-cells identified SLC4A4 as one of few unique genes highly expressed in T2DM ?-cells and repressed in non-diabetic ?-cells. Slc4a4 encodes for Na+-nHCO3- cotransporter, NBCe1B in the pancreas and plays a key role in regulating intracellular pH (pHi). Importantly, increased activation of NBCe1 has been associated with enhanced intracellular glycolysis and impaired mitochondrial function suggesting it may contribute to loss of GSIS and consequent development of T2DM. Preliminary dissertation studies support this hypothesis and demonstrate that inhibition of NBCe1B activity in ?-cells improves GSIS in vitro and enhances glucose tolerance in vivo. These cumulative observations led us to develop a doctoral dissertation direction with an overall objective to characterize the role of NBCe1B as a novel regulator of ?-cell metabolism and dysfunction in T2DM. Accordingly, Specific Aim 1 (F99) will test the hypothesis that ?-cell dysfunction in T2DM is driven by metabolic reprogramming mediated by cellular alkalization through activation of NBCe1B. Given the critical role of NBCe1 in maintaining systemic pH homeostasis, the F99 uniquely positions me to elucidate novel mechanisms associated with dysregulation of acid-base balance in the kidney during the K00 phase. Specifically, the A-isoform of NBCe1 (NBCe1A) functions as the key mechanism of HCO3- reabsorption in the kidney. Deletion of NBCe1A is associated with metabolic acidosis and cortical cysts within the collecting duct (CD). Soluble adenylyl cyclase (sAC) has been identified as a HCO3- sensor within the CD. Previous work demonstrated that impaired NBCe1A-mediated HCO3- reabsorption activates sAC-cAMP/PKA mediated signaling. Interestingly, persistent cAMP/PKA activation within the CD has also been demonstrated to be a key mediator of cyst development and proliferation in polycystic kidney disease (PKD). Therefore, the main objective of my proposed postdoctoral research direction is to characterize the role of NBCe1A as a novel regulator of cystogenesis through activation of sAC-cAMP/PKA signaling pathway. Accordingly, Specific Aim 2 (K00) will test the hypothesis that impaired NBCe1A-mediated HCO3- reabsorption activates a soluble adenylyl cyclase-cAMP/PKA signaling cascade in the collecting duct promoting proliferation and cystogenesis in models of PKD. Together, the F99 and K00 will propel me to achieve my long-term goal to lead an independent research program in nephrology.


Mitochondrial Respiration and Superoxide Production in Healthy and Failing Heart. In cardiac myocytes, mitochondrion plays multi-functional roles in oxidative metabolism, ion homeostasis, signal transduction, and cell fate regulation. Mitochondrial respiration through the electron transport chain (ETC) activity drives ATP synthesis and reactive oxygen species (ROS) generation. In the failing heart, mitochondrial respiration is often compromised, resulting in decreased ATP production and, paradoxically, increased oxidative stress. It is therefore of great interest to determine how mitochondrial respiration and ROS production are regulated in the healthy heart and how they contribute to oxidative stress in the failing heart. Recently, we discovered a transient superoxide production event, named superoxide flash, in individual mitochondria of cardiac myocytes and the myocardium. Preliminary data indicate that the superoxide flash requires intact ETC activity, and its frequency is altered by physiological or pathological treatments. We hypothesize that the superoxide flash is coupled to stochastic acceleration of ETC activity in single mitochondria and modulated by key regulators of mitochondrial bioenergetics, including Ca2+, permeability transition pore (PTP), and fission/fusion. If this hypothesis is true, superoxide flashes may serve as a composite index of single mitochondrion respiration and ROS production. Further, imaging superoxide flashes may help determine whether mitochondrial or cytosolic ROS is responsible for oxidative stress in the failing heart. We propose the following specific aims to determine the mechanistic coupling of mitochondrial respiration and superoxide flash production and their role in oxidative stress in heart failure: Aim 1: To test the hypothesis that superoxide flash arises from transient acceleration of mitochondrial respiration and is modulated by mitochondrial Ca2+, PTP and fission/fusion dynamics. Aim 2: To test the hypothesis that pathological stress inhibits superoxide flash activity at an early stage of heart failure and prior to detection of overt signs f mitochondrial dysfunction. Aim 3: To determine whether increased mitochondrial or cytosolic ROS contributes to oxidative stress during mitochondrial respiratory dysfunction. PUBLIC HEALTH RELEVANCE: This project investigates the mechanistic coupling of mitochondrial respiration and bursting superoxide production. Our goal is to establish that decreased superoxide flash activity is a novel and early biomarker for mitochondrial dysfunction, which leads to oxidative stress in heart failure.


Vibrio cholerae causes the fatal epidemic diarrheal disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. ToxT, an AraC-type regulator, directly activates the promoters of the primary virulence factors. The expression of toxT requires cooperation between two homologous pairs of transmembrane activators, ToxRS and TcpPH. Activation of tcpPH expression initiates the cascade by a unique interaction between two regulatory proteins AphA and AphB. AphA is a member of a new winged-helix transcriptional regulator family and AphB is a LysR-type activator. Transcriptional activation at these various promoter occurs only in response to certain environmental stimuli. The long term goals of this proposal are to understand the molecular basis of this regulation so as to facilitate the development of better strategies to control the infectivity of V. cholerae. Achieving these goals requires an understanding of the molecular mechanisms by which the regulatory proteins function at their cognate promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. One such stimulus, cell density, influences the cascade through the quorum sensing regulator HapR which represses the expression of the aphA promoter. Aim 1 focuses on elucidating the mechanism by which HapR represses aphA expression. Evidence indicates this process involves antagonizing the function of two distinct activators, Lrp, the leucine responsive regulatory protein, and VpsR, the biofilm regulator. Aim 2 sheds new light on the molecular mechanisms by which pH influences the expression of the cascade by the recent discovery that AphB plays a role in the acid tolerance response in V. cholerae and regulates cadC and a number of other genes potentially involved in this response. Elucidating the relationship between AphB and the regulation of acid tolerance will provide new insights into how the activity of the protein is influenced by environmental stimuli. Aim 3 focuses on investigating the unique cooperative mechanism between AphA and AphB that initiates virulence gene expression in V. cholerae. This proposed work will facilitate future efforts to identify new molecules that interfere with the functions of these regulators and which may serve as novel antivirulence drugs. [unreadable] [unreadable] [unreadable]


Surfactant Protein B (SP-B) plays a central role in alveolar type II cell physiology. As a component of pulmonary surfactant, it facilitates transfer of phospholipids to the air-liquid interface, lowering of monolayer surface tension, and altering of monolayer composition. SP-B is critical to the differentiation of type II cells, specifically lamellar body genesis. We have been exploring the post-translational regulation of SP-B production. This stems from observations that despite increasing SP-B RNA, mature SP-B protein does not accumulate in the fetal lung until late in gestation, thereby allowing for a burst of SP-B production that facilitates LB genesis and surfactant secretion into fetal lung fluid. We have shown that post-translational events in SP-B biogenesis are important in regulating the production and transit of SP-B in the alveolar type II cell. These events are especially critical when de novo synthesis of surfactant is essential, as in the final phase of lung maturation for the transition to air breathing (as in RDS), and when extracellular and/or intracellular surfactant stores are depleted (as in ARDS). Work from the previous funding period demonstrated that enzymes involved in proSP-B post-translational processing are important to type II cell physiology. Based on new preliminary data, we hypothesize now that post-translational events involving the protease Pepsinogen C (PGC) and ER chaperones such as ERp29 are critical for regulating SP-B production, and consequently lamellar body genesis and type II cell physiology. We will test this hypothesis in 3 specific aims: 1) Characterize the role of PGC in SP-B proteolytic processing, 2) Establish key transcriptional elements directing the developmental and cell-type specificity of PGC expression, and 3) Demonstrate that chaperone interactions, specifically ERp29:proSP-B interactions, facilitate export of proSP-B from the ER. We will use an in vitro system of human type II cell differentiation to examine the process of lamellar body genesis as a result of disrupted SP-B processing/trafficking. Project Narrative: Surfactant therapy has been a success for premature infants with respiratory distress, but has not been as successful in adults. Adult respiratory distress syndrome is associated with dysfunction or decreased Surfactant Protein B (SP-B). These studies will improve our understanding of factors that control SP-B production and will provide a foundation for novel therapeutic strategies to enhance SP-B production and surfactant function. [unreadable] [unreadable] [unreadable]


The objective of this research is to study the biochemical controls involved in the development and function of lung tissue. Both hormonal and genetic factors will be analyzed using isotopic and immunochemical techniques. Since corticoids are apparently involved in maturation of the lung's ability to produce surfactant material, efforts will be made to identify and characterize a specific binding protein or "receptor" for these hormones. The properties of this receptor will be examined during perinatal development of the lungs in rats and sheep. This binding protein will also be elucidated in human autopsies to assess the fate of the receptor in lungs of children suffering from hyaline membrane disease. The properties of lung chromatin and its components (nonhistone proteins) during ontogeny will be studied, utilizing immunochemical method developed in this laboratory. This method can measure possible changes in the structure and function of chromatin, and will also be used to assess chromatin during lung deficiency as hyaline membrane disease.


DESCRIPTION: (Applicant's Abstract) This application is a request for a Mentored Research Scientist Development Award (KO1) from the National Institute Drug Abuse (NIDA) for Lisa R. Metsch, Ph.D. Her commitment to a research career in drug abuse combined with her training and research interests in HIV/AIDS and health services research and the opportunities offered by the proposed mentor, Clyde B. McCoy, Ph.D. and the interdisciplinary research team at the Comprehensive Drug Research Center and the newly NIDA funded Health Services Research Center at the University of Miami make the KO1 from NIDA an ideal funding mechanism for her at this stage of her career. For this award, she will participate in a rigorous interdisciplinary, collaborative, applied drug abuse research program that will lead to her gaining the needed skills of research methodology, project management, grantsmanship, and publication essential to the success and survival of independent research scientists. The proposed research is a theoretically-driven 5-year multidisciplinary project to study access and utilization of health services among women drug users living with HIV infection. A total sample of 600 HIV seropositive African American women injecting drug users, non-injecting drug users, and non-drug users will be selected from previous Miami studies to be enrolled i the proposed project. These women will be administered a comprehensive health interview schedule that is guided theoretically and focuses on personal background factors, individual, social, and environmental factors, and evaluated and perceived need as it relates to health care utilization. The proposed research is the first stage of a two stage process. Using the proposed theoretical framework, data gathered from this study will be used to develop intervention strategies. Stage 1 (proposed study) will provide the theoretical methodological, and empirical basis for the development of an intervention which be carried out in a future Stage 2 study.


The tetradecapeptide somatostatin (SS-14) is synthesized as part of a large precursor, preprosomatostatin (PPSS). Several different forms of PPSS have been characterized in various species. After cotranslational removal of the signal peptide from PPSS, the resulting prosomatostatins (PSSs) are subjected to specific posttranslational cleavages to release SS-14, somatostatin-28 (SS-28) and other peptides derived from the precursors. The specific aims of this application include: Investigation of the bioactivity of a newly discovered hydroxylysine-containing form of SS-28, aSS-28, which is a demonstrated metabolic cleavage product of one form of PSS in anglerfish islets; Determination whether a single or separate prohormone converting endopeptidases (PCEs) cleave SS-14 and aSS-28 from anglerfish islet PSSs; Isolation and elucidation of the complete primary structures of PSS processing PCEs; Isolation and structure determination of a carboxypeptidase B-like (CPB) exopeptidase which is involved in PSS processing. Effects of aSS-28 on islets and dispersed islet cells will be examined by monitoring hormone release with RIAs. The PSS processing PCEs and CPB can be isolated by employing ion exchange and hydrophobic chromatography. Experiments will be performed to determine whether the anglerfish enzymes can cleave mammalian PSSs. Microsequencing procedures will be employed to obtain partial sequence information. Complete sequences of the PCEs and CPB will be obtained by applying recombinant DNA methodologies. The long term objectives are to determine the characteristics of PSS converting enzymes which render them specific. The process of characterizing the anglerfish islet converting enzymes will involve generation of probes which will be useful in screening mammalian tissues for similar enzymes. The results obtained from these studies will provide an informational base for further investigation of the mechanisms controlling PSS processing. Perturbations in these mechanisms could play a role in the etiology of some types of diabetes mellitus or other disease states. Once the normal mechanisms are understood, aberrations which lead to pathology could be more readily identified.


The objective of the proposed program is to offer basic science training for investigation of growth and development by supporting students in a joint M.D./Ph.D. program. This is the continuation of a program which has been successfully pursued for over 40 years. The trainees are selected from students already admitted to the Pritzker School of Medicine. At a minimum, they have a Bachelor of Science degree and usually two years of medical school training. They are selected on the basis of demonstrated ability in basic science, indication of devotion to pursuit of a research and teaching career in medicine, and outstanding academic credentials. Selection is carried out by a faculty committee and is based on academic records, recommendations, prior accomplishments, and personal interviews. All students must fulfill the Ph.D. requirements of a particular basic science department or committee of their choosing. A schedule of training is determined for each student so that basic science coursework and research training is integrated between the pre-clinical and clinical phases of medical school. Often, certain of the Ph.D. requirements may be completed during elective periods of the pre-clinical curriculum. Completion of research training occurs before or during the final clinical phase of the M.D. program. Thus the M.D./Ph.D. program is usually completed in five or six years, although the training period supported by this grant is often less than five years. Any intermediate funding is provided by the trainee's mentor, private awards, or institutional funds. Fifteen to eighteen trainees will typically be in the program at any given time, usually allowing admission of two to three new trainees annually. This program attracts outstanding candidates, has had no attrition over a 30-year period, and produces exceptional M.D./Ph.D. graduates who receive acceptance to outstanding residency programs and research fellowship programs. The training of dual-degree professionals contributes in a substantial way to the quality of health care in this country. Since these individuals are trained as both clinicians and researchers, they have a broad perspective and can generate novel approaches to medical issues. Graduates of this program hold major positions in universities, research institutes, the NIH, and pharmaceutical companies.


Hyperglycemia can lead to irreversible tissue damage by mediating a number of biochemical and structural protein alterations. One of these known changes involves the formation of advanced glycated end products or age's which may interact with excessive levels of blood glucose (hyperglycemia) that eventually cause the complications associated with diabetes mellitus. Pharmacologic inhibition of age-mediated protein crosslinking by aminoguanidine hydrochloride (pimagedine) may prevent and/or stabilize the development and progression of complications of diabetes, specifically retinopathy and nephropathy.


The long-term goal of this work is to implement novel gene-based therapeutics to reverse contractile performance deficits in the failing human heart in vivo. This proposal is focused on troponin I (TnI), the inhibitory subunit of the troponin complex and molecular switch to cardiac contraction. The focus of this proposal on advances in troponin function in the diseased heart has considerable health relevance and, accordingly, well serves the mission of the NIH. Novel preliminary evidence is presented on troponin-modified myofilaments dictating myocyte Ca2+ handling through a direct effect on sarcoplasmic reticulum (SR) Ca2+ load. The overarching hypothesis that guides this proposal is myofilament function, by virtue of unique properties of histidine modified TnI at codon 164, uniquely and directly tunes SR Ca2+ content independent of effects on canonical regulators of SR Ca2+ load, including SERCA2a -dependent rapid Ca2+ sequestration. We further posit that novel bio-engineered adeno-associated viral vectors, together with our new high fidelity hypoxia responsive gene switch system, will permit translation of this proposal's molecular/cellular advances (Aim1) directly to the beating heart in vivo in pre-clinical studies in living mammals (Aim 2). The Specific Aims are: Aim 1. To determine the molecular mechanism underlying optimization of global cardiac myocyte Ca2+ handling between the myofilaments and sarcoplasmic reticulum in vitro and in vivo by single histidine-modified troponin. Hypothesis: Real time sensing of the patho-physiological biochemical milieu of the healthy and diseased myocyte by the troponin modified sarcomeres will have a dominant effect to directly decrease the Ca2+ load requirements of the SR, including conditions of accelerated SR Ca2+ influx by an increased SERCA2a/PLN ratio. Aim 2. To implement a novel evolutionary-directed bio-engineered AAV vector and double oxygen genetic sensing system for optimization of efficient systemic gene delivery of modified troponins in the diseased heart in vivo. Hypothesis: Compared to wild-type striated muscle tropic rAAV serotypes AAV1, pseudo-typed AAV6, AAV8 and AAV9, the novel bio-engineered rAAV capsid M41 with O2 sensing genetic elements will lead to higher cardiac tropism and greater efficiency resulting in hypoxia/ischemia sensitive long-term constitutive expression of modified TnIs in the cardiac sarcomeres of small mammals.


It is proposed to investigate pitch recognition judgments where other tones are interpolated during the retention interval. Certain systematic interactive effects which appear to be based on lateral inhibitory interactions will be studied as a function of serial position. These specific effects will also be investigated as a function of ear of input. The influence of concurrent relational information will be studied as a function of retention interval. In sequences where the tones to be compared are simultaneously accompanied by other tones of lower pitch, the effects will be investigated of interpolating simultaneous tonal combinations as a function of their relationship to the standard and comparison combinations. The effects on pitch recognition of varying other tonal attributes (timbre, duration) will also be explored.


Methionine (Met) dependence, the inability of cells to grow when Met is replaced by its immediate precursor homocysteine (Hcy) (Met-Hey+ medium) is a metabolic defect that we have found to be highly prevalent in human cancer cells. In contrast, normal cells are Met independent and grow well in Met-Hey+ medium. Our laboratory, which has defined many of the basic characteristics of Met dependence, has recently shown that despite high levels of Met synthesis from homocysteine, Met-dependent cells maintain low free Met pools and resulting low S-adenosylmethionine (AdoMet) pools but high levels of S-adenosylhomocysteine (AdoHcy) in Met-Hcy+ medium. The resulting AdoMet/AdoHcy ratio, which is the global regulator of transmethylation, is thus very low in Metdependent cells in Met-Hcy+ medium. In this light, we have found that Met-dependent human cancer cells often have DNA that is highly undermethylated and also have altered methylation patterns of the sis and myc oncogenes. On the basis of these results, we propose to further elucidate the biochemical basis of low freeMet pools in Met-dependent cells by measuring Met flux into Met tRNA and efflux of Met out of the cell. We will also determine if the enzymes adenosine deaminase or AdoHcy hydrolase (synthetase) are responsible for the high AdoHcy levels in Met-dependent cells in Met-Hcy+ medium. We will determine, for possible therapeutic purposes, if actual human tumor tissue is Met-dependent by measuring Met, AdoMet and AdoHcy levels when the tissue is incubated in Met-Hcy+ medium labeled with [35S]homocysteine synthesized in our laboratory by a new procedure such that it is Met free. To understand the relationship of Met dependence to DNA hypomethylation, altered oncogene methylation and oncogenesis itself, we will select for Met-independent revertants, which we have previously shown selects for more "normal" cells, to see if in the revertants: (1)\there are concomitant changes toward normal in total DNA methylation and sis and myc oncogene methylation; (2)\there are decreases in the activity of these oncogenes; and (3)\there is a loss of tumorigenicity.


Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a major advance in the field of biological mass spectrometry, providing high ion yields for low quantities of biopolymers, even in the presence of buffer and other biochemical components that would preclude analysis by other mass spectral techniques. Much of the research in the field to date has concentrated on peptides, proteins, and oligonucleotides, yet the technique also holds considerable promise for oligosaccharide analyses beyond the limited work that has already been demonstrated. This application seeks to develop methods to increase the applicability of MALDI-MS to challenging problems in glycobiology. The research will be directed towards four goals: (1) maximizing the sensitivity for biopolymers, particularly oligosaccharides and glycoconjugates and their derivatives; (2) increasing the information content of the spectra through sample pretreatments that induce mass shifts; (3) developing experimental methods to enhance fragmentation and obtain systematics to help determine the rules of post-source decay fragmentation; and (4) exploring approaches to the analysis of two-dimensional surfaces, both those that occur in biological or biomimetic systems, and those that result from analytical separations or the generation of synthetic product arrays.


DESCRIPTION (adapted from the application) The prevalence of end-stage renal disease (ESRD) continues to increase, and mortality among ESRD patients remains high. The principal hypothesis of this proposal is that care of patients with chronic renal failure prior to ESRD (pre-ESRD) is sub-optimal, and this adversely influences outcomes after initiation of ESRD therapy, such as morbidity, mortality and cost. The research will include a macro viewpoint using data from large databases such as the United States Renal Data System (USRDS) and Health Care Financing Administration (HCFA), and a micro viewpoint using detailed primary data collection at a single tertiary care hospital--New England Medical Center (NEMC). USRDS and HCFA files will be used to determine the prevalence and predictors of malnutrition, anemia, late initiation of dialysis and delayed referral to the nephrologist, and their impact on subsequent clinical variables. Separate analyses of pediatric and transplant patients will be undertaken, and compared with dialysis patients. NEMC data will be used to study the impact of delayed nephrology referral and delayed vascular access placement on hospitalization and cost. The results of this proposal are expected to provide important information regarding pre-ESRD care, and for the development of strategies to improve ESRD outcomes. The principal investigator has designed a comprehensive series of studies to evaluate the hypotheses enunciated in this proposal, and is well qualified to carry them through to completion. In addition to a combined fellowship in Adult and Pediatric Nephrology, she holds a Master's degree in Epidemiology. She is mentored by investigators with extensive experience in clinical investigation and advised by a panel of world leaders in epidemiology, clinical trials and health services research. Within a limited time, she and her mentor have put together an infrastructure tailored to achieve the goals of this proposal. The practical experience from the research proposed, and the comprehensive education program outlined are expected to build on the candidate's current skills and experience, and facilitate her transition to an independent investigator in Pediatric and Adult Renal epidemiology.


The success of the DEARC depends upon the ability of the ADMINISTRATIVE CORE to facilitate interactions and attain the highest quality research among the projects. These goals will be achieved through multiple mechanisms. (1) The quality, progress, and fiscal integrity of research components (i.e., MAIN and PILOT PROJECTS) will be assured through both internal and external guidance systems, (a) Two internal systems will be an Executive Committee (formed by the Center Director and Scientific Director) and a Coordinating Committee (comprised of the Pis of the Main Projects and the Cores). Pis will report to these committees through regular written reports and orally at monthly meetings and at the annual DEARC Retreat, (b) Two extra-DEARC bodies will oversee the activities of the DEARC: an Internal Advisory Board comprised of administrators at Binghamton Univ., SUNY- Cortland, Syracuse Univ., and Upstate Med. Univ. and an External Scientific Advisory Board comprised of nationally prominent, senior alcohol and developmental biology researchers. (2) A critical facet of the DEARC will be the three Scientific Cores (ANIMAL, CELL/MOLECULAR, and NEUROANATOMY CORES). The two internal systems described above will oversee the CORES, assuring the quality of the data generated by the CORES and the productivity of the CORES. (3) DEARC researchers are located at three SUNY campuses- in Binghamton, Cortland, and Syracuse. The ADMINISTRATIVE CORE will assure that handling of samples and data is efficient and that communication between these campuses is seamless. These will be achieved using well established inter-campus courier services, electronic communications, and sophisticated teleconferencing systems. (4) The PILOT PROJECT PROGRAM is designed to generate new ideas and new programs. The ADMINISTRATIVE CORE will guide this program. This involves soliciting and reviewing applications and monitoring the progress of the funded projects. (5) The ADMINISTRATIVE CORE will support the continuing education of DEARC personnel. This will occur through journal clubs, team-taught courses, and seminar series. These offerings will be made available to more people through teleconferencing and videotaping (archiving). Other communications by DEARC personnel (e.g., poster, manuscript, and grant preparation) will be supported by the Administrative Core. Together, the ADMINISTRATIVE CORE will serve as a system for maintaining quality and progress of the DEARC and supporting the communication within and beyond the DEARC.


The general goal of this program is the development of new techniques to study macromolecules and assemblies. The current specific objectives are to develop methods for handling and analysis of chromosome-sized DNA molecules and methods for producing labeled proteins in vivo. Pulsed field gradient gel electrophoresis allows high resolution separations of DNA molecules as large as 5000 kb. This will be used to develop rapid gene isolataion and mapping methods. Refinements of current pulsed field techniques and methods for specific fragmentation of larger DNA will be developed so that the rapid mapping methods can be extended to human genes. Among the proposed applications are electrophoretic karyotypes and genome finger prints, techniques for exploring genome structure adjacent to a cloned marker, and methods for exploring the dynamics of mobile genetic elements. In other studies the genes for strepavidin and aequorin will be cloned and expressed in mammalian cells. Vectors will be constructed that produce fusions between these proteins and normal cellular proteins. This will allow specific in vivo labeling of any gene product.


DESCRIPTION: The proposed experiments are designed to utilize a vertebrate genetic system capable of identifying dominant mutations affecting the zebrafish retina, with particular emphasis on mutations involved in rhodopsin trafficking. The long-term objectives of the proposed research include the identification of genes associated with blindness caused by age-related retinal degeneration and the characterization of the cellular and molecular mechanisms underlying retinal degeneration. Mutations causing dominantly inherited night blindness will be isolated by a behavioral assay and confirmed by histological and physiological methods. Proper rhodopsin localization and transport will be evaluated by breeding mutant zebrafish with transgenic zebrafish that express a transgene encoding a rhodopsin-green fluorescent protein (GFP) fusion protein and examining the progeny by confocal fluorescent microscopy. Additional characterization of other transport systems within the photoreceptors of mutant fish will elucidate possible interactions between cellular transport mechanisms. Finally, identification of the genes responsible for these mutations will be achieved by positional cloning strategies. The proposed work will provide insight to the mechanisms underlying photoreceptor cell death that ultimately lead to retinal degeneration and blindness associated with aging.


The ultimate goal of this project is to provide insight into the functioning of cellular machines composed of RNA and protein subunits. As a model system, we are studying the introns of the yeast mitochondrial cytochrome b pre-mRNA and the proteins that assist in their splicing. In these simple systems, the RNA components contain the active site for the splicing reaction while the protein components enhance the rate of splicing by holding the RNA in its active conformation. As a first step toward understanding the functioning of this system, we have determined the crystal structure of the bI3 maturase, an intron-encoded protein that facilitates splicing of the intron that encodes it. This crystal structure revealed a conserved nucleic acid binding surface with other members of the LAGLIDADG endonuclease family. Furthermore it revealed how this protein has been inactivated as a functioning DNA endonuclease while maintaining the core structure. Biochemical experiments performed in our collaborator's laboratory showed that this protein binds to the folded self-splicing RNA intron in a peripheral region distant from the splicing active site. Thus, the protein acts at a distance to facilitate folding of the self-splicing RNA. The protein also was shown to bind to the minor groove of the RNA versus typical binding of the LAGLIDADG endonucleases to the major groove of DNA substrates. This suggests that the protein is not changed structurally from DNA-binding homologues, but instead takes advantage of the RNA's wider and more accessible minor groove.


The Administration/Biostatistics Core has two major functions. First, it will provide centralized administrative and fiscal management for all research projects and cores associated with the Program Project. These services will involve coordinating the programmatic aspects of the research activities of the participants and include fiscal overview, preparing and assembling yearly Progress Reports, manuscript and report typing, organizing and scheduling meetings and seminar speakers, and the general allocation of resources. The Biostatistics component will provide the bioinformatics necessary for the design and analysis of carcinogenesis experiments, analysis of gene expression array data and dual labeling results as well as general statistical tests. The Administration Core will be headed by Dr. Medina and assisted by Dr. Rosen, and staffed by a full time administrative secretary, Kathy Key. The Biostatistics component will be headed by Dr. Susan Hilsenbeck.


Accumulate and assess clinical oncologic data for prostatic cancer with a solid phase radioimmunoassay technique for prostatic acid phosphatase (TIA-PAP). Continue development, of the RIA-PAP technique as a screening technique in the early diagnosis of prostatic cancer utilizing a large random population of elderly males.


Yeast cells respond to nutrients and other signals in the environment both by adjusting their transcriptional and metabolic profiles to make optimum use of the available nutrients and by selecting a developmental program that maximizes their potential for survival. Our recent studies, fueled by genomic tools, have refined our knowledge of the components and connections within individual pathways and the interconnections between pathways. We propose to continue these studies, focusing on how the cell coordinates input from several signaling pathways to yield an appropriate and coherent response in its transcriptional and developmental programs. We propose to develop a comprehensive topology of the network around the Ras and TOR pathways through which the cell transmits information regarding nutrient availability. We will accomplish this by further measurements of global transcriptional changes in response to nutrient shifts of mutant strains with increased or diminished activities of key signaling components. We will also apply new computation tools to extract identities of additional transcription factors and define the role of chromatin structure in mediating nutrition induced transcriptional changes. We then plan to address the means by which the cell integrates information from different signaling pathways, focusing on dissection of specific transcription factors and the yeast homolog of the mammalian Akt kinase, which our work has highlighted as the loci at which the TOR and Ras pathways converge. We anticipate that these studies will provide a complete topology of the nutrient signaling network in yeast cells as well as provide means of defining comprehensive signaling networks in larger eukaryotes. Such detailed signaling network representations should enormously facilitate assessing the consequences of intervention at specific steps in a signaling pathway to achieve a therapeutic treatment of diseases, such as cancer or diabetes, which often result from the cell's failure to properly balance input from multiple signals.


The development of new synthetic methods for the enantio- and regiospecific construction of molecules of medicinal interest for the treatment of Acquired Immunodeficiency Syndrome (AIDS) is important to the overall mission of the NIH. The specific aims of this project encompass the development of new synthetic methods for the preparation of the HIV-inhibitor, conocurvone. Conocurvone is a naturally occurring trimeric quinone that was isolated from an Australian shrub by researchers at the National Cancer Institute. The key structural feature of the molecule is a quinone central core to which is attached to hydroxyquinone subunits called teretifolione B. Conocurvone has been shown to possess potent anti- HIV-1 activity in the in vitro HIV-1/CEM-SS assay model. An important feature of the biological activity of conocurvone is its wide therapeutic index of approximately 2500! The high potency and low toxicity of conocurvone makes it an ideal candidate for further drug development.The first phase of the project involves the development of regiospecific routes to conocurvone and related trimeric quinones. An underlying theme of the proposed chemistry is the use of palladium metal to facilitate the formation of the carbon-carbon bonds that link the hydroxyquinone subunits to the quinone central core. The second phase of the project will involve synthesis and biological testing of conocurvone analogs.


Craniofacial skeletal muscle defects occur in certain myopathies, such as muscular dystrophy; in congenital deformities, such as hemifacial microsomia and facial/palatal clefts; and as a result of surgical procedures for oral cancer or trauma. Success in the repair or replacement of muscle defects is limited by difficulty in transplantation and survival of muscle tissue. An important structure of muscle is the myotendinous junction (MTJ), which transduces force generated by muscle to its connective tissue attachment site. How this complex structure is formed in developing muscle or repaired after injury or disease is poorly understood. Attachment of the muscle fiber to the connective tissue appears to involve adhesion receptors, including the alpha7-beta1 integrin. In addition, during muscle development and repair, alpha7 integrin seems important for myoblast adhesion and motility. The long- term objective of the proposed studies is to further define the molecular mechanisms by which the laminin-binding alpha7 integrin organizes the MTJ in developing and regenerating skeletal muscle. The hypothesis is that the alternatively spliced isoforms of alpha7 not only regulate transient adhesion during myoblast motility but also form the long-lived MTJ. The specific aims are 1) to determine the expression levels and distribution of laminin-binding integrins during skeletal muscle development, 2) to analyze the functionality of alpha7,beta1 alternatively spliced isoforms, and 3) to determine the role of the alternatively spliced extracellular domain in regulating alpha7 activation. The experimental approach is first to define the developmentally complex expression patterns of the alpha7 splice variants and their ligands. Next, alpha7 isoforms will be analyzed for their role in cell motility and assembly of MTJ-like structures. Finally, the role of the extracellular domain splice variants in regulating alpha7 activity will be addressed using molecular approaches. These studies will enhance understanding of the structure and assembly of the MTJ and suggest new approaches in tissue engineering to promote reconstruction of craniofacial skeletal muscle defects caused by disease, trauma, or surgical procedures.


We are interested in the structure and mechanism of reverse transcriptase. Towards this goal we plan to study the mechanism of synthesis of plus strand of DNA, isolation of active DNA polymerase free of RNase H activity, fractionation of mammalian reverse transcriptase to generate RNase H and DNA polymerase activities, characterization of gag-pol precursor proteins encapsidated in the mature virions, localization of lesions in reverse transcriptase obtained from temperature-sensitive mutants by tryptic peptide mapping, study of chain elongation by electron microscopy, and use of chemically-synthesized restriction enzyme sites on primers to synthesize cDNA. We are also exploring the use of reverse transcriptase to fill in gaps in restriction endonuclease generated termini for DNA sequencing and S1-mapping.


The aim of our research program is to understand the causes of autoimmune thyroid disease, the mechanism of the immune response and, if possible, to develop preventative or therapeutic immunomodulatory measures. We will identify dominant and pathogenic epitopes of TSH receptor (hTSH-R), determine genetic factors promoting the disease, determine the relationship of epitope recognition to MHG genotypes, and study potential immunotherapeutic measures in vitro and in vivo in a SCID mouse model. Epitopes will be identified using recombinant antigen and synthetic peptides reacting with PBMG, T cell lines and T cell clones derived from patients with GD and controls. We will study populations, families with a high incidence of GD, especially homozygous HLA-DQA1 *0501 patients, and young individuals in such families in the early phase of disease. To establish biologic importance of the epitopes we will consider 1) differential reactivity comparing patients with GD and controls, 2) concordance of reactivity by PBMC, and derived T cell lines and clones, 3) correlation of epitope recognition with disease and MHC genotype within families, 4) recognition of epitopes in young family members genetically at risk for GD, 5) changes in lymphocyte reactivity following antigen deprivation, and 6) relation of specific epitopes to MHG genes. Epitope reactivity will be analyzed following mutation of the peptide. Peptides which may be able to act as antagonists to the natural epitope, or to induce anergy in T cell lines or clones in vitro will be investigated. We will study, in SCID mice, the ability of T cell lines, reacting to specific epitopes, to reconstitute thyroiditis and produce thyroid stimulating antibodies in animals also xeno- transplanted with isologous thyroid tissue. In this model we can also test peptide antagonists, epitope induced T cell anergy, and induction of tolerance by antigen. Class II MHC genotypes will be determined by a PCR/site specific oligonucleotide methodology in Caucasian populations, Black populations, and Caucasian and Black family groups, to determine linkage of HLA with disease. Since it is clear that HLA associations explain only part of the genetic influence, we will proceed to type, in specific highly informative families, a group of at least eight candidate immune-associated genes for linkage to GD.


During terminal differentiation epidermal keratinocytes manifest a programmed set of morphological and biochemical changes that result in the production of two major structures: 1) an envelope of covalently linked protein enclosing 2) a constellation of keratin intermediate filaments. A major precursor of the envelope is a soluble protein called involucrin (hINV) which is incorporated into the envelope by a calcium- dependent transglutaminase. hINV is likely to account for the majority of glutamyl-lysine linkages that hold the envelope together. In spite of its importance, inadequate information is available regarding which glutamines within involucrin are targeted for crosslinking by transglutaminase or which sections of the hINV molecule are essential for high strength envelope formation. Active envelope formation is essential for survival and abnormal envelope formation is a feature of several epidermal diseases. The ultimate aim of the experiments described in this proposal is to understand the role of hINV in the envelope assembly process and how this impacts on the disease state. To provide tools for these studies, we cloned and sequenced the complete hINV gene, structurally characterized the protein and produced the normal and mutant hINV proteins in bacteria. Our results show that the molecule is an extended alpha-helix composed of highly similar, tandemly linked repeats of ten amino acids. Each repeat contains three glutamine residues, each of which is a potential crosslink site. The proposed studies are designed to gain a better understanding of cornified envelope structure and the role of hINV in envelope formation and are a logical extension of the studies completed during the initial two years of grant support. In the present experiments we propose to 1) identify which proteins become crosslinked to hINV during cornified envelope formation, 2) construct a series of hINV mutants to determine why GLN496, among the more than 100 glutamine residues present in the hINV protein, is the preferred site for initial crosslink formation and 3) determine the effects of expression of selected mutant hINV proteins on epidermal function in transgenic mice.


Breast cancer is the second leading cause of cancer death among women in the United States. About 80% of such deaths occur in women age 65 years or older. These women also have a lower survival rate and are less likely to receive recommended treatment. Identifying geographic areas where survival from breast cancer among women 65 years of age or older is lower and the underlying factors responsible is important to ensure that differences in treatment are addressed and that interventions can be designed at a level where they can be implemented. The aims of this revised application are to: 1) examine small-area geographic clustering of breast cancer survival among women 66 years of age and older; 2) Determine the extent to which geographic variation of survival can be explained by the geographic variation in area social and economic deprivation among women age 66 and older; and 3) identify potential pathways by which area social and economic deprivation explains any geographic variation of breast cancer survival among women age 66 and older. Based on the reviewers' comments, we have refocused the conceptual model and associated hypotheses on delineating the geographic variation of area social and economic deprivation on the geographical variation of breast cancer survival and the pathways by which area deprivation influences this outcome. The analytic models have also been refocused and are described in more statistical detail. To address the specific aims, the following data sources will be linked: 1) 1992-1999 data from the Surveillance, Epidemiology, and End Results (SEER) program (survival, demographics, stage at diagnosis, treatment, tumor biology); 2) 1991- 1999 Medicare data and its linkage to the SEER data (comorbidity, treatment); 3) 1992-1999 data from the Centers for Medicare and Medicaid (availability of and proximity to medical care) and 4) the 1992-1999 intercensal estimates (area deprivation measures). Advanced Bayesian analyses of breast cancer survival will be performed. The findings of the proposed study will identify geographic disparities in survival and factors responsible, which can be used to develop and implement future interventions to reduce these disparities. [unreadable] [unreadable]


The objectives of this study are to determine whether persistent alterations in the GABAA receptor complex (GABAR) can provide a molecular explanation for the development of physical dependence on ethanol in an animal model of alcoholism. Chronic intermittent ethanol (CIE) administration to rats has many features resembling human alcohol abuse behavior, including long-lasting susceptibility to readdiction. The numerous episodes of ethanol (EtOH)-induced depression of the nervous system and the following rebound hyperexcitability (withdrawal) have been shown to exert a kindling-like effect, i.e., a persistent increased severity of the hyperexcitable withdrawal symptoms. Rats treated in this manner become EtOH-dependent, one measure being a decreased seizure threshold to the convulsant drug pentylenetetrazol (PTZ), a blocker of the GABAR. The hyperexcitability to PTZ (kindling) lasts at least 40 days after cessation of EtOH. The CIE rats exhibit elevated anxiety, show tolerance to the sedative action of EtOH and cross-tolerance to other sedatives, and impaired hippocampal memory. Neurochemical and electrophysiological studies have been focused on whether this ethanol withdrawal syndrome can be associated with alterations in GABAR, and have demonstrated a significant reduction, specifically in the hippocampal formation, in GABAR function, as well as multiple alterations in the molecular properties of GABAR. We showed a restructuring of GABAR subunit composition consistent with changes in electropharmacology of GABAR-mediated synaptic and extrasynaptic tonic currents. These biochemical and physiological changes appear relevant to the altered behaviors. The same persistent alterations seen in CIE are also observed transiently after a single administration of an intoxicating dose of EtOH. In future we propose to study the molecular and cellular mechanisms whereby this GABAR plasticity develops and how it becomes persistent. In addition to acute and chronically EtOH-treated rats we will extend the model to mice to allow studies on genetically engineered animals with altered GABAR to help determine their role in developing dependence. We suggest that reduced GABAR function in ethanol-dependent individuals has profound effects on various emotional and intellectual aspects of brain activity. Finding the molecular mechanisms responsible may help in treatment of withdrawal symptoms and hopefully in reduction of ethanol dependence. This type of mammalian animal model seems to have great potential for uncovering important insights into abuse mechanisms. In addition, our studies on animal models of alcoholism will allow families and health professionals' better understanding of what environmental and genetic factors contribute to the susceptibility for alcohol abuse, of the behavioral changes of the alcohol abuser, and of possible behavioral modification and medications to consider in treating the disorder. PUBLIC HEALTH RELEVANCE: This project studies the cellular and molecular mechanisms of alcohol dependence in a rodent model in hopes of developing therapeutics for prevention and treatment of alcoholism. Rats and mice are given chronic intermittent ethanol (CIE) and studied for changes in inhibitory neurotransmission in brain involving receptors for the neurotransmitter 3-aminobutyric acid (GABA). The amounts, locations, and functions of the GABA receptors are related to the behavioral changes seen in alcohol dependence such as hyperexcitability, increased anxiety, sleep disturbances, and seizure susceptibility.


In Vivo Raman spectroscopy requires use of optical fiber probes for laser light delivery and collection of return Raman signals. Fluorescence and Raman background generated in the silica fibers hinder measurement of Raman spectra from biological tissue samples in vivo. We are developing and evaluating various optical fiber probes in which laser band pass filter and cut-off filters are incorporated. Dielectric band-pass filter incorporated in the excitation fiber eliminates background generated in the fiber and allow only the excitation light to illuminate the tissues. A dielectric cut-off filter incorporated in the collection fibers blocks the laser light from entering the fiber and passes only the Raman scattered light emitted by tissues. We are working closely with Visionex and currently evaluating the performance of their probes. Parameters such as the transmission wavelength, efficiency of the filters, the extent to which the fiber background is suppressed, and also the compatibility of these for clinical measurements are being evaluated.


PROJECT SUMMARY/ABSTRACT The Society of Surgical Oncology (SSO) and its members are dedicated to advancing and promoting the science and treatment of cancer worldwide. The SSO provides leadership to US and international health professionals on the importance of surgery in multidisciplinary cancer care, with a commitment to foster the research careers of young investigators and clinicians to propel discovery and ultimately improve patient outcomes. SSO and its annual educational symposium focus on current scientific advances in basic and translational science, controversies, clinical trials, and novel techniques in the following specific disease and tumor types: breast cancer, melanoma, sarcoma, pancreas and hepatobiliary malignancies, esophageal cancer, thyroid and adrenal cancer, gastric cancer, colorectal cancer, neuroendocrine tumors, and peritoneal surface malignancies. The meeting convenes more than 1900 surgical oncologists, general surgeons, and oncology health professionals from 45 countries to engage in peer-to-peer and mentor-to-trainee dialogue, debate, and didactic learning through a variety of mediums for an educational offering that has no clear parallel. The symposium offers more than 500 scientific abstracts covering novel science and clinical trials, video sessions and over 400 poster presentations. The Scientific Program Committee (SPC) collaborates with SSO?s seven Disease Site Work Groups, panels of experts who ensure faculty selection and content development is by those with nationally recognized expertise in the tumor area, peer-reviewed publications in the educational topic area, and skill and experience in speaking and teaching. Meeting attendance is diverse. Two-thirds of physician attendees are US-based from academic and community-practice settings and represent all stages of a surgical oncology career. The SSO Annual Cancer Symposium provides networking opportunities to help augment careers and to cultivate clinical collaborations. As time passes, the line between what is considered a community hospital and an academic setting continues to be blurred in the US. As many academic centers become more clinically focused, community hospitals are becoming larger and increasing their own focus on research infrastructure and clinical trials. As such, both ?traditional? academic and community surgical oncologists have more and more in common. The conference presents an opportunity not only for community surgeons to gain access to research being conducted in academic medical centers and engage with senior faculty, but also for academic physicians to learn how community hospitals currently deliver care. Younger surgical oncologists and surgeons gain experience presenting on a national level and interacting with esteemed senior experts and faculty. This opportunity for engagement between renowned experts and junior faculty is uniquely availalble at this meeting.